Effect of dietary supplement from mono‐culture fermentation of Moringa oleifera seeds by Rhizopus stolonifer on hematology and markers linked to hypercholesterolemia in rat model

Abstract Mono‐culture fermentation by Rhizopus stolonifer could promote the healthiness of immune systems and cholesterol levels. Hence, we examined the effect of diet from mono‐culture fermentation of Moringa oleifera seeds by R. stolonifer (MCF‐MORS) on hematological parameters and fundamental indicators of hypercholesterolemia in rat. The animals were divided into six groups (n = 6). Group 1 was placed on basal diet. Group II, III, IV and V were placed on a basal diets supplemented with 7.5%, 15%, 22.5% and 30%, respectively, of MCF‐MORS. Group VI was placed on basal diet fed with unfermented M. oleifera seeds (UF‐MOS). The experiment lasted for eight weeks. The results revealed 7.5% MCF‐MORS as better biological method to augment PCV, RBC and Hb count in animal model. Also, 7.5% and/or 15% MCF‐MORS demonstrated highest levels in centrophils, neutrophils and eosinophils, whereas the levels of lymphocytes, basophils and monocytes showed no significant difference. Similarly, 7.5% and 15% MCF‐MORS modulated LDL and HDL, respectively, better than UF‐MOS; but showing no difference in cholesterol level. MCF‐MORS also maintained architectural integrity of villi and splenocytes better than UF‐MOS. We therefore concluded that diet from MCF‐MORS at 7.5% and 15% modulates HDL, LDL, cholesterol and immune system‐related disorders better than UF‐MOS in rat model.


| INTRODUC TI ON
Moringa oleifera (MO) is regarded as miracle plant due to its multiple uses. Virtually, all the parts of the plant are edible and utilized as traditional diets in many countries of the tropics and subtropics (Farooq & Umer, 2007). The stem bark, root, bark, fruit, flowers, leaves, seeds, and gum are widely used in India folk medicine (Compaoré et al., 2011), while the powdered seeds and roots are applied as spices and curries (Abdulkarim, Long, Lai, Muhammad, & Ghazali, 2005) and feed supplement for livestock. Also, recent studies described its nutritional and medicinal properties as a nonfood product (Anjorin, Ikokoh, & Okolo, 2010;Anwar, Latif, Ashraf, & Gilani, 2007).
They also reported that the leaves contain both vitamins and essential minerals such as vitamin A, vitamin B, vitamin C, calcium, iron, potassium, essential amino acids, and high protein content (Anjorin et al., 2010).
Experimentally, some findings reported the efficacy of MO seeds against arsenic-induced hepatocellular degeneration in female albino rats (Chattopadhyay et al., 2011). Also, M. oleifera showed a notable array of medicinal applications with high dietary value (Anwar et al., 2007). Anti-inflammatory efficiency and hypotensive property of M. oleifera have been documented (Ndiaye et al., 2002). It was similarly demonstrated that M. oleifera leaves inhibited hyperlipidemia and hepatocytic disarrays when initiated by dietary iron shortage (Ndong, Uehara, Katsumata, Sato, & Suzuki, 2007), while its seed extract protected hepatic cells from necrotic damage and fibrosis in rat model (Fakurazi, Hairuszah, & Nanthini, 2008;Hamza, 2010).
Further result also suggested that edible oil from MO seeds potentially protected rats in chemical-induced hepatitis (Mansour et al., 2012). Another study similarly indicated that co-administration of monoisoamyl dimercaptosuccinic acid (MDA) and MO seeds powder protected arsenic-induced oxidative stress and metals distribution in mice (Mansour et al., 2012). Following these preventive effects of MO seed, recent study proved that fermentation of MO seeds increased the protein content, essential amino acid, and polyunsaturated fatty acid profiles with concomitant reduction in its antinutrient compositions (Oluwole, Oluwole, & Oluwaseun, 2013).They then advocated that its fermented products should be consumed rather than the germinated and raw M oleifera seeds because of its high contents in essential minerals (Oluwole et al., 2013).
Fundamentally, fermentation is one of the biotechnological methods employed to potentiate the nutritional quality of legumes, cereals, and all form of seeds (Zhang, Xu, & Wang, 2007).
Also, it is widely used for producing and preserving foods on local and industrial levels (Ndams, Tegbe, Ogundipe, & Sheyin, 2011).
Strong evidence has shown that fermentation enhances nutritional value for several fermented products, especially yoghurt and wine (Zhao, Zhang, & Zhang, 2010). Recent finding reported that microorganisms have been found to be highly diverse in biochemistry, physiology, and nutritional modes (Vogel, Sarath, Saathoff, & Mitchell, 2011). But its selection for industrial application should be based on its ability to produce high yields of the desired product (Vogel et al., 2011;Zhang et al., 2007). Although, the nutritional value of fermented foods had long been recognized, but the scientific bases for many of the nutritional claims have been scantly studied (Kavanagh, 2005). It was reported that protein of plant source has more nutritional benefits than animal origin without health risks to livestock and humans (Melanson, MacLean, & Hill, 2009). Also, the fermentation of crop residues or seeds improves the digestibility of feedstuff (Yu, Guo, Zhang, Yan, & Xu, 2009) and the nutritional value particularly with the use of fungi to disrupt plant cell wall for promoting fermentable energy to the intestinal microbes. Specifically, mono-culture fermentation of MO seeds by fungus to increase protein availability, digestibility, and reduction of its anti-nutrient contents is still current (Nuha, Isam, & Elfadil, 2010) as there is little or no information on immuno-protective action of fermented M. oleifera (FMO) seeds. However, we speculated that mono-culture fermentation by Rhizopus stolonifer could promote the healthiness of immune systems and cholesterol levels in mammals. We also hypothesized whether dietary FMO seeds could modulate metabolic toxicity than dietary raw M. oleifera (RMO) in rattus novengicus model. For this reason, this study examined the effect of diet from mono-culture fermentation of MO seeds by R. stolonifer on hematological parameters and markers linked to hypercholesterolemia in rat model.

| Sample selection and preparation
The fresh sample was collected from the premises of Nigerian open manually to release the dry seeds. The seeds were dried at 60°C in the oven to maintain the moisture content. Thereafter, seeds were powdered with an electrical grinder to particle size of about 2 mm and stored in air tight container for further use.

| Quantification of compounds by HPLC-DAD
Analysis of phenolic compounds by HPLC-DAD Reverse phase chromatographic analyses was carried out under gradient conditions by the method of Boligon et al. (2015) and Reis et al. (2014).

| Natural fermentation of Moringa oleifera powder
The fermentation of the powdered M. Oleifera seeds was performed according to the method described by Kayode and Sani (2008).
Briefly, 5 g of powdered seeds was added to 5 ml of sterile distilled water. The mixture was stirred properly for a uniform mash to be obtained. The fermenter was covered, and the content was allowed to ferment at room temperature (28 ± 2°C) for 7 days. Also, the anti-nutrients for both fermented and nonfermented MO seeds were determined. In addition, macro-and micro-elements for both fermented and nonfermented MO seeds were determined by flame atomic absorption spectrophotometer.

| Isolation of fungal organisms from naturally fermented Moringa oleifera seeds
Fungi were isolated from the naturally fermented seeds through serial dilution and pour plate methods using Potato Dextrose Agar (PDA) into which 10% streptomycin solution was added to inhibit bacteria growth. Culturing was performed in duplicates. The plates were incubated at room temperature (28 ± 2°C) for 48 hr. Isolated visible fungal colonies were further subcultured to obtain pure cultures. Morphological and microscopic analysis to identify the isolates were carried out and compared with literature (Stanley, Elizabeth, & Holt, 2001).The pure isolates were maintained on agar slant and kept in the refrigerator at 4°C for further use.

| Macroscopic characterization of fungi isolates from Moringa oleifera seeds
The plates were examined macroscopically for morphological characteristics. Characteristics considered were color of the colony, production and release of pigment into the medium, rate of growth (fast or slow) during incubation, diameter of colony, spreading of the colony and the mycelia. Characteristics observed were in line with literature for the identification of the isolates (Stanley, 2001).

| Microscopic characterization of fungi isolates from Moringa oleifera seeds
The isolates were observed in wet mount preparation under X10 and under X40 objective lens. They were observed before staining with cotton-blue in Lactophenol and observed again under X10 and under X40 objective lens. Indexes of observation were hyphae length, type (sporangiophore or conidiophores), breath, size, color, presence or absence of septa, rising or spreading, branching type, spore shape, and texture of the cell wall (smooth or rough). The observations were identical with literature (Stanley, 2001).

| Mono-culture fermentation of Moringa oleifera seeds by Rhizopus stolonifer (MCF-MORS)
Rhizopus stolonifer spore suspension of actively growing mid log phase culture was prepared according to the method described by Sani, Awe, and Akinyanju (1992). Briefly, an agar slant of four day old pure culture of R. stolonifer was used. Ten milliliters sterile distilled water was added to the slant and shaken to wash the spores.
The spore suspension was counted using the Neubauer counting chamber. A spore suspension of about 5 × 10 4 spore suspension/ ml was used for inoculation. Exactly, 20 g of powdered M. oleifera seed sample was added into 250 ml Erlenmeyer flask, plugged with cotton wool, wrapped with aluminum foil, and sterilized in the autoclave at 121°C for 15 min. The sterile sample was mixed with 20 ml of sterile distilled water and stirred properly until uniform mashes were obtained. Thereafter, 2 ml of R. stolonifer mono-culture suspension was used as fermentation starter to inoculate M. oleifera sample in the fermenter. The mixture was allowed to ferment for 72 hr at 28 ± 2°C (Kayode & Sani, 2008;Lawal, Iyayi, & Aderemi, 2005). Fermented sample was dried at 60°C in the oven for 4 hr.
The residue was used for diet formulation, and the proximate composition was analyzed.

| Diet formulation
Experimental diets supplemented with four varied doses (7.5%, 15%, 22.5% and 30%) of mono-culture fermentation of M. oleifera seeds by R. stolonifer (MCF-MORS) for 72 hr were formulated according to a modified method of Yusuf, Bamgbose, Oso, Fafiolu, and Oni (2008) ( Table 1). Soybean was used as the source of protein. This was prepared by adding 30%, 22.5%, 15%, and 7.5% of soybean meal to obtain 100% formulation (Table 2). Thereafter, the proximate quantity of the diet used to feed the animals in each group was measured as reported in Table 2. The unfermented M. oleifera seed was designated as UF-MOS.

Treatment
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Corn meal 50.6 50.6 50.6 50.6 50.6 50.6 The vitamin premix (mg or IU/g) has the following composition; 3200 IU vitamin A, 600 IU vitamin D3, 2.8 mg vitamin E, 0.6 mg vitamin K3, 0.8 mg vitamin B1, 1 mg vitamin B2, 6 mg niacin, 2.2 mg pantothenic acid, 0.8 mg vitamin B6, 0.004 mg vitamin B12, 0.2 mg folic acid, 0.1 mg biotin H2, 70 mg choline chloride, 0.08 mg cobalt, 1.2 mg copper, 0.4 mg iodine, 8.4 mg iron, 16 mg manganese, 0.08 mg selenium, 12.4 mg zinc, 0.5 mg antioxidant. Group 1: serve as the control group placed on a basal diet; Group 2: serve as the group placed on a basal diet supplemented with 7.5% of monoculture fermented pulverized seeds of Moringa oleifera Group 3: serve as the group placed on a basal diet supplemented with 15% of mono-culture fermented pulverized seeds of M. oleifera; Group 4: serve as the group placed on a basal diet supplemented with 22.5% of mono-culture fermented pulverized seeds of M. oleifera; Group 5: serve as the group placed on a basal diet supplemented with 30% of mono-culture fermented pulverized seeds of M. oleifera; Group 6: (Negative control) serve as the normal group placed on a basal diet supplemented with 30% of unfermented pulverized seeds of M. oleifera.
TA B L E 1 Diet formulation for basal and supplemented diets in control and test groups

| Animal handling
Weaned Wistar rats (43.3 ± 2.5 g) from the Central Animal House of the University of Ilorin, Ilorin were used in this experiment. The animals were maintained at a constant temperature (22 ± 2°C) on a 12-hr light/dark cycle with free access to food and water.

| Experimental protocol
The weaned rats were acclimatized for 2 weeks and randomly di- and euthanized 24 hr after the last treatment, and blood was collected by cardiac puncture. The blood was allowed to clot and centrifuged at low speed (3,000 g) at room temperature for 15 min.
The supernatant (plasma) was removed and used for the determination of biochemical parameters.

| Chemicals and reagents
All the kits used for the bioassay were sourced from Randox Laboratories Ltd. (Crumlin, Dublin, Northern Ireland, UK). All other reagents used were in the purest form (analytical grade) available commercially.

| Hematological and immunological estimation
Hemoglobin concentration (Hb %) was measured by Drabkin's colorimetric method (cyanomethemolgobin formation), and packed cell volume (PCV) was estimated by scale of microhematocrit reader (Duncan, Prasse, & Mahaffey, 1994). The concentration of red blood cells (RBC) and white blood cells (WBC) was measured in the given blood sample and was expressed as volume of cells (Coles, 1986).

| Determination of HDL, LDL, and cholesterol
The key lipid profiles: high-density lipoprotein (HDL), low-density lipoprotein (LDL), and cholesterol were measured using commercially available kits (Randox Laboratories Kits, St Louis, MO, USA) according to the manufacturer's guideline.

| Histological examination
Small intestine and spleen specimen were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned. After deparaffinization and dehydration, the paraffin blocks were stained with hematoxylin and eosin for microscopic examination.

| Statistical analysis
The data in each group were expressed as mean ± standard deviation. A one-way analysis of variance (ANOVA) was used to analyze the results and Duncan multiple test was used for the post hoc (Zar, 1984). Statistical package for Social Science (SPSS) 17.0 for windows was used for the analysis, and the least significance difference (LSD) was accepted at p < 0.05. TA B L E 2 Proximate composition (%) of the diets used to feed the control and test groups (Oluwole et al., 2013) and Pakia biglobosa (Compaoré et al., 2011).

| RE SULTS AND D ISCUSS I ON
Also, the fermented MO seeds by R. stolonifer decreased the levels of anti-nutrients such as saponin, oxalate, tannin, and phytate (Table 4) in relation to nonfermented MO seeds. This indicates that UF-MOS contain high levels of anti-nutrients. Studies had reported that excess intake of anti-nutrients particularly saponin, tannin, oxalate, and lectin could cause severe intestinal damage disrupting digestion and nutrients shortages (Fereidoon, 2014;Habtamu & Negussie, 2014). These secondary compounds could trigger IgG and IgM antibodies to elicit abnormal immune responses and concurrently bind to erythrocytes to produce hemagglutination and anemia (Sano & Ogawa, 2014). Furthermore, essential elements such as potassium, magnesium, iron, and copper were significantly (p < 0.05) increased during fermentation by R. stolonifer with concomitant reduction in the levels of sodium, calcium, manganese, zinc, cadmium, and lead (  (Rachel, Thomas, Miquel, Ingvar, & Domenico, 2013). Cd 2+ metabolite in the blood of occupationally exposed workers was also connected to erythrocyte cancer and lymphocyte impairment (Amati et al., 2008). It has similarly been reported that Ca overload had a correlative effect on hyperlipidemia, cardiovascular dysfunction, and hypertension when triggered by oxidative stress (Nisha, Keshav, & Kalpana, 2016). Further report also showed that exposure to Pb significantly (p < 0.05) decreased human hemoglobin (Hb) with evidence of prolonged hemoglobinopathies (Bernard & Franklin, 2013).
Lastly, exposure to surplus inorganic sodium could initiate high blood pressure with severe thrombosis (Kalaitzis, Pasadakis, Bantis, Giannakopoulos, & Touloupidis, 2010). However, these abnormalities may not be propounded in vivo following the mono-culture fermentation of MO seeds by R. stolonifer.
The percentage body weight gain of the group of animals treated with UF-MOS was lower than MCF-MORS (7.5%, 15%, 22.5% and 30%) and the control (Table 6). This study explains that the body weight may depend on PCV amounts, erythrocyte counts, and immune systems. Our opinion corroborates the former result which reported that body weight and leukocyte counts were significantly (p < 0.05) lowered in the anemic piglets than the normal group (Pu et al., 2015). Also, the blood quantity and quality of packed cell volume (PCV) were significantly (p < 0.05) elevated in animals fed with MCF-MORS when compared to UF-MOS group (Figure 1).
Specifically, PCV count was considerably (p < 0.05) lowered by 84.6% in group of animal supplemented with UF-MOS in relation to the corresponding control (Figure 1). The significant increase of PCV count (i.e., MCF-MORS) indicates optimum production in total blood counts by the bone marrow due to the high contents of crude protein and magnesium particularly dietary iron from plant origin.
This agrees with the previous study which reported that abnormal or irregular supply of nutrient-rich iron caused the red blood cells to be malformed or die off at a faster rate than the body can replace them, leading to declined PCV counts (Pu et al., 2015). Essentially, reduced iron is the nutrient commonly associated with anemia because the body uses iron to make the hemoglobin to transport oxygen in the blood cells. However, increased PCV counts and the regeneration of red blood cells had been connected to the wellness of the bone marrow cells potentiated by dietary magnesium, iron, and protein (Gregory, 2006).  are also redox-active elements essential as catalytically active cofactors in enzymes and structurally stabilizing protein production as well as enhancing immune systems (Friedenberg et al., 2016;James & Eric, 2013). Additionally, animal fed with 7.5%, 15%, 22.5%, and 30% MCF-MORS showed no significant (p < 0.05) difference in WBC count in relation to the control (Figure 4), whereas animals fed with UF-MOS were significantly (p < 0.05) reduced the level of WBC when compared to corresponding control ( Figure 6).
Furthermore, lymphocytes, neutrophils and centrophils are the subtypes of white blood cells in a vertebrates' immune system. Figures 5 and 6 Table 7). This is because studies had documented that flavonoids and phenolic acids act as antioxidants to maintain strong T and B lymphocytes (phagocytes) and reduce vulnerability of the body to infectious diseases as well as preventing angiogenesis in cancer cells (Burchi et al., 2011;Tanya, Xavier, & Clifford, 2014). Centrophils produced from pluripotent hemopoietic stem cells further suggest that MCF-MORS provokes the production of growth factors (granulocyte-colony-stimulating factor [G-CSF], granulocyte-macrophage-colony stimulating factor [GM-CSF], interleukin 3 and macrophage-colony-stimulating factor [M-CSF]) better than UF-MOS; as such, prolonged the survival of T-lymphocytes and natural killer cells (NKs) (Mendy, Yonglian, & Yang-Xin, 2009) by upregulating the immune response via cytokine release (Jamila, Bharti, & Kanti, 2003;Michael, Sarah, Neda, Wood, & Shayan, 2013). Moreover, recent study had reported that eosinophils possess substantial pro-inflammatory and cytotoxic activity and responsible for the pathogenesis of various disease processes (Ilesanmi et al., 2016). However, eosinophils were not produced in animals fed with 7.5%, 15%, and 22.5% MCF-MORS as well as the control group (Table 8), signifying its nontoxic effect. Conversely, animal fed with UF-MOS significantly (p < 0.05) increased the level of eosinophils (Table 8). This hike level may be attributed to some quantities of heavy metals in UF-MOS particularly Cd 2+ and Pb 2+ . Recent study had implicated high level of eosinophils in the blood among workers exposed to Pb 2+ (Patricia & Marc, 2013). Basophils were not detected in all the groups (Table 8) (Gianni, Francesco, Gilda, Arturo, & Francescopaolo, 2014). Lastly, groups of animals fed with 7.5%, 15%, and 22.5% MCF-MORS showed no significant (p < 0.05) increase in the level of monocytes when compared to the control and UF-MOS group (Table 8). This indicates that level of monocytes produced is still within the tolerable limit for mammals. Our finding supports the earlier finding which stated that uncontrollable high content of monocyte counts greater than 0·9 × 10 9 /L was associated with the development of monocytosis, pericarditis, or joint effusions and ascites (Papageorgiou et al., 2013). F I G U R E 8 Representative high performance liquid chromatography profile of black seed aqueous extract. Gallic acid (peak 1), caffeic acid (peak 2), p-coumaric acid (peak 3), Rutin (peak 4), quercetin (peak 5), luteolin (peak 6), and apigenin (peak 7)   This suggests that 15% of dietary mono-culture fermentation by R. stolonifer for 72 hr showed the best potency to picks up excess cholesterol in the blood to inhibit hyperlipidemia and complications of atherosclerosis (Mattar & Obeid, 2009;Shanmugma, Román-Rego, Ong, & Kaski, 2010). Secondly, low-density lipoprotein (LDL) was significantly (p < 0.05) depleted in animal supplemented with 7.5% of MCF-MORS when compared to UF-MOS and the control ( Figure 10). This result indicated that 7.5% mono-culture fermentation for 72 hr of MO by R. stolonifer showed the most therapy to prevent the build-up of (bad) LDL-cholesterol within the walls of the blood vessels. Lastly, animals fed with MCF-MORS in all treated groups showed no significant (p < 0.05) difference in cholesterol levels ( Figure 11). No significant difference advocates that MCF-MORS and UF-MOS correspondingly act as hypolipidemia, hypolipoproteinmia, and hypocholesterolemia agents. This was corroborated by the previous finding which reported that crude extract of MO lowered the principal markers linked with coronary artery disease and acute myocardial infarction in high-fat diet rats (UNdong et al., 2007). Also, recent studies showed that MO seeds elevated red blood cells and immune system in cholesterol high fed rat (Frank, Henrietta, Ifeanyi, in the blood and complicated atherosclerosis (Shanmugma et al., 2010). Additionally, the spleen synthesizes antibodies in its white pulp, destroys old, and damaged cells. It responds actively to immune systems through humoral and cell-mediated pathways.

TA B L E 7 Composition of phenolic compounds in MO seeds
Hence, spleen dysfunctions such as splenomegaly, blood-based leukemias, and asplenia have been conventionally identified (Shanmugma et al., 2010). In the present study, animal fed with MCF-MORS showed no visible lesions to the spleen cells, that is, normal visible splenocytes ( Figure 13). This asserts a better physiological wellness of T and B lymphocytes, granulocytes, dendritic cells, macrophages, and other immune cells (Friedenberg et al., 2016) than UF-MOS.

| CON CLUS ION
This study discovered 7.5% MCF-MORS as better biological method to augment PCV count, RBC count, and Hb in animal model. Also, 7.5% and/or 15% MCF-MORS demonstrated highest levels in centrophils, neutrophils, and eosinophils, whereas the levels of lymphocytes, basophils, and monocytes showed no significant difference. Similarly, 7.5% and 15% MCF-MORS modulated LDL and HDL, respectively, better than UF-MOS; but showing no difference in cholesterol levels.
MCF-MORS also maintained architectural integrity of villi and splenocytes better than UF-MOS. We therefore concluded that diet from Group 4 Group 2 NVSA NVSA NVSA NVSA MDSC NCDC MCF-MORS for 72 hr at 7.5% and 15% modulates HDL, LDL, cholesterol, and immune system-related disorders better than UF-MOS in rat model.

E TH I C A L S TATEM ENTS
All the animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institute of Health. Ethic regulations have been followed in accordance with National and institutional guidelines for the protection of animal welfare during experiments. The study's protocols and procedures were ethically reviewed and approved by the ethical committee of the University of Ilorin-Nigeria by the number UIH 1011.

CO N FLI C T O F I NTE R E S T
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.