Efficacy of Selected Botanicals on Fungi Rot Eefect on Proximate and Phytochemical Composition of Walnut Seeds (TetracarpidiumConophorum).

Obani, F.T. , Onyebuchi, E , Agbo E. A. , Onwuka, O. , Nwadinobi, C. A

Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria

Corresponding Author Email: tochyfloxy@gmail.com

DOI : https://doi.org/10.51470/ABP.2025.04.01.33

Abstract

Walnut (Tetracarpidiumconophorum) is a tropical croprich in nutrients. Fungicause rot ofnuts, and alter their appearance and nutrient composition. Fungi nutrientdeterioration and rot impactmanagement is vital research. Efficacy of cloveand West African pepper(WAP)extracts on fungi effects onthe proximate and phytochemical composition of walnut seeds were accessed.Fusariumsolani,F. oxysporium, Rhizopusstolonifer, Lasidioplodiatheobromae, A. nigerand A. flavusused were isolated from walnut seeds in a previous study. Seeds weretreated with clove,WAP, and clove+WAPprior to fungiinoculation, incubation andcomposition analysiswere done using AOAC methods. Experimentaldesign was CRD in triplicates and data were analyzedusing ANOVA at α0.05. Crude extracts influenced fungi effects on the proximate, alkaloid and trypsin content of seeds. Clove recordedhigher crude protein contentforA. flavus(3.06%), A. niger(6.73%), Lasidioplodiasp (6.31%), Rhizopussp (2.86%), and F. solani(3.95%)than controls.Similarly, A. flavus, A. niger, Lasidioplodiasp and Fusariumsolani had higher crude fibre, fat and ash for clove+WAPthan controls.Aspergillusflavus, A. niger,Lasidioplodiasp, RhizopusspandF. Solanihad higher ash and fat than controls for WAP. The energy value was higher than acontrol for all treatments inoculated with F. solani. Alkaloid was higher in treated walnuts;Rhizopussp (0.24-0.37 mg/100g), F. oxysporium(0.27-.45 mg/100g),L. theobromea(0.48-0.72 mg/100g) and A. niger(0.33-0.46 mg/100g)than controls and was similar for trypsin inhibitors.Botanical considerably reduced effects of fungi on nutrients, alkaloid and trypsin inhibitor content of walnut seeds. These botanicals can also be usefully exploited for the management of fungi rots and resultant nutrient reduction and risks posed to human health.

Keywords

botanicals, proximate and phytochemical composition, Walnut seeds

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INTRODUCTION

African walnut (Tetracarpidiumconophorum Mull. Arg) is aneconomic tropical climbertree widelygrown for its edible nut (2;17). The plant is mostly known as African walnutorconophor nut. It belongs to the family Euphorbiaceae. In Nigeria, it is known by different names among the Yoruba tribe, Igbo, Hausa and the Bini tribe of Edo State (7; 13). It is grown mainly in SierreLeone, Angola, Nigeria and different nations in West Africa (7).

African walnut is very rich in nutrients composition, it has high content of essential fatty acids, polyunsaturated fatty acids and proteins (2; 17; 32; 20).

African walnutis used as food (21), as raw material in the cosmetic industry formanufacturing dry skin anti-wrinkle and anti-aging creams (17).It is also useful in medicinedue to itsmale fertility enhancing, antioxidant and immune-stimulatingactivities (2),anti-malarial, anti-diarrheal, and anti-diabetic decoctions for human health; because of theirbiochemical composition of polyunsaturated fatty acids and high protein value; as well as income generation for farmers (2; 32; 19).

Several diseases affect and render the edible nuts unfit for consumption,alter their visual appearance and alter theirnutrient composition.As a result, they becomesour-tasting, foul-smelling and unpleasant for saleand consumption (36). The protein, oils including omega-3 fatty acids, vitamins and minerals can be considerably reduced due to the rotting of the seeds (22).Also, fungi-derived toxins i.e., mycotoxinssuch as aflatoxins and fumonisincan be deposited in infected walnuts which will impact human health negatively wheneaten (30). Hence, there is a need to determine the level of walnut nutrient deterioration and possible ways to reduce the impact of fungi rot on walnut nutrient composition.Therefore, the objectives of this study are to assess the efficacy of extractsof seeds clove (Syzygiumaromaticum)locally known as kanaperi and West African pepper (Piper guineense) locally called uzizato reduce fungi deteriorating effects on the proximate and phytochemical composition of walnut seeds.

MATERIALS AND METHODS

Experimental Location

The experiment was conducted in the laboratory of the Department of Plant Health Management, Michael Okpara University of Agriculture, Umudike and the Bioscience Laboratory of National Root Crop Research Institute, Umudike.

Collection of walnut seeds

Freshly harvested and healthy walnut fruits were collected from walnut growing areas inMbaise, Imo State into a sterile polythene bag andbrought to the laboratory for use in the investigation.Fungi used for this study were isolated and identified from fungi-infected walnut seeds in a previous study. Two Fusariumspecies (F. solani and F. oxysporium), Rhizopusstolonifer, Lasidioplodiatheobromae,and two Aspergillusspecies (A. nigerand A. flavus) were used for this study.

Source and Preparation of Plant Extracts

The dried seeds of clove (SyzygiumaromaticumL.) and West African pepper (Piper guineenseSchumach and Thonn) were bought and taken to the laboratory, washed under running tap water and then with sterile distilled water and weighed (100g) each and grounded to fine powder then stirred vigorously in 200 ml distilled water and then allowed to stand for 6 hours, filtered using 3 folds of cheesecloth and the filtrate used as plant extract (23).

Walnut seed treatment with extracts and Inoculation with fungi

The modification of the methods of (24,25) was used for the study. Healthy walnut seeds were surface sterilized with 1% sodium hypochlorite; rinsed in three changes of sterile water and dried with a sterile paper towel. A 5mm diameter flame-sterilized cork borer was used to remove a cylindrical core deep into the seed, plant extracts were applied into the deep core 20 minutes before being replaced aseptically with 5mm diameter disc from the advancing edge of 7-day old fungal culture of each isolate for each of the walnut seeds;then sealed with the removed disc of walnut seed in the cork borer.Vaseline applied around the cylindrical core to prevent external infection before incubation. The controls were separately inoculated with a 5mm diameter disc of 7-day-old fungal culture alone. Three seeds each were wasinoculated for each fungus, this served as three replicates.The treated walnut seeds were incubated for 7 days and observed only for rot development.The experiment was laid out in a completely randomized design.

Proximate Analysis:

Moisture content was determined by drying fresh samples to constant weight in a hot air circulating oven at 100oC. Proximate compositions which included percentage moisture, fat, crude protein, fibre, and ash were determined according to the standard methods of the AOAC (9;10;11).

The total percentage carbohydrate content was determined by the difference method as reported by (28). This method involved adding the total values of crude protein, crude fat, crude fibre, moisture,and ash constituents of the sample and subtracting it from 100. The value obtained is the percentage carbohydrate constituent of the sample. The total energy value was calculated thus:

Data Analysis

Data from proximate and nutrient composition analysis were analyzedusing Statistical Package and Service Solutions (SPSS) version 23. Means were compared and separated using least significant difference (F-LSD) and at 5 % probability level.

RESULTS AND DISCUSSION

Effect of Crude Extracts of Botanicals on Proximate Composition of African Walnut Seeds Inoculated with Aspergillusflavus and Aspergillusniger

Table 1presents the effect of botanical treatment on fungal activities on the proximate composition of African walnut seeds inoculated with Aspergillusflavus and Aspergillusnigerafter treatment with botanicals.For Aspergillusflavus, moisture level (3.06%), fibre(3.40 %), protein (2.51 %), fat (1.78 %) and ash content (2.07 %)were higher in the treated samples than control; however, there isa decrease in carbohydrate (87.20 %) for clove treated walnut samples. A similar trend was observed inClove+West African pepper and West African pepper treatments.

For Aspergillusnigermoisture content (3.45 %), protein (6.73 %), content,fibre(2.94 %), fat content (3.16 %), ash (2.99 %) was higher, however,with slight decrease in carbohydrate 9(80.73 %) compared to control (85.40 %).  A similar trend was observed in West African pepper treatments. However, for clove+West African pepper treated samples, moisture (2.73 %), crude protein (2.05 %), CHO (85.40 %) and energy value were lower compared to the respective controls.

Generally, botanical treatments showed increased protein, and fat contents compared to the pathogen-inoculated control samples. Combined treatment withclove and West African pepper treatment had positive effects, on the impact of the pathogens on the proximate composition of walnuts although they were all significantly lower than the uninoculated control (Table1).

Effect of Crude Extracts of Botanicals on Proximate Composition of African Walnut Seeds Inoculated with Lasidioplodiatheobromaeand Rhizopussp

The proximate composition of African walnut seeds treated with crude extracts of clove (S.aromaticum) and West African pepper (P.guineense) before inoculation of Lasidioplodiasp and Rhizopussp is shown in Table 2. Walnut seeds treated with botanicals prior to inoculation with Lasidioplodiasp had higher moisture, protein, crude fiber, fat and ash content compared to the control samples except forclove+West African pepper ((3.25%) treatment where moisture content was observed to be slightly lower than the control (3.44 %). 

Also, the CHO and energy valueswere slightly higher in the fungi inoculated control than the treatments; although they were not significantly different but differed from the uninoculated control which had higher percentage of the different compositions analyzed. For walnut seeds treated and inoculated withRhizopussp higher moisture, protein, crude fibre, fat and ash content were recorded compared to the control samples except for treated samples, where the CHO (87.20 %) and energy value (376.28 KCAL), were slightly lower than the inoculated control (CHO = 88.81%), (KCAL EV = 378.97); although they were not significantly(p≤0.05) different but they were all lower than that recorded in the uninoculated control.

On the average, the nutritional composition of the African walnut seeds treated with clove extract prior to inoculationwithRhizopushad higher moisture, protein, crude fibre, fat and ash contents compared to those treated withclove+West African pepper and West African pepper (Table 2).

3.1.3Effect of crude extracts of botanicals on proximate composition of African walnut seeds inoculated with Fusariumsolaniand Fusarium oxysporium

Table 3shows the proximate composition of African walnut seeds treated with crude extracts of clove and West African pepper before inoculation of Fusarium oxysporium and Fusariumsolani. Clove 2.57 % and clove+West African pepper (2.44 %) recorded significantly(p≤0.05)lower moisture content than the control (2.62 %).  However, crude protein and fibre contents recorded were lower than that of the control as well as fat, ash carbohydrate contentand energy value compared to the control but were not significantly different. For Fusariumsolani, the control recorded the highest moisture content (3.53 %) while clove treated walnut seeds (3.05 %) had the least. Crude protein in the treated samples were higher than the control; Clove (3.95 %), clove + West African pepper (3.69 %) had significantly higher crude protein content than the control (3.55 %). A similartrend was observed for crude fibre, fat, ash, carbohydrateand energy value (Table 3). On the average,treatment with botanicals had a considerable effect in moisture content reduction and improved nutrient content compared to the pathogen-inoculated samples; but were all significantly lower than that recorded in the uninoculated control for all the walnut components analysed.

3.1.4: Effect of crude extracts of botanicals on alkaloid and trypsin inhibitor (mg/100g) content of African walnut seeds inoculated with different fungi

Table 4showsthe botanical treatments’ different fungi effects on alkaloid and trypsin content of African walnut seeds. Alkaloid content was mostly reduced by thefungi, compared to the controls except for Fusariumsolani, where the alkaloid level in clove+West African pepper treated samples was significantly higher than the control (0.61mg/100g); although it was significantly lower than that recorded in the uninoculated control (2.26 mg/100g).

For trypsin content, a similar trend was observed, clove+West African pepper and West African pepper treatmentswere also generally higher than the controls. However, combining clove andWest African pepper extracts generally led to higher alkaloid (0.27 mg/100g) and trypsin (2.12 mg/100g) content compared to their respective inoculatedcontrol (0.22, 1.77 %); but lower than the uninoculated control (2.26mg/100g) (Table 4).

DISCUSSION

Walnut seed plays vital role in human health and nutrition; being an excellent food material with potential for use in combating food insecurity. The results of this study revealed that clove, West African pepper and clove+ West African pepper influenced the activities of Fusariumsolani, Fusarium oxysporium, Rhizopusstolonifer, Lasidioplodiatheobromae, AspergillusnigerandAspergillusflavusin walnut seeds.The phytochemical and proximate composition of walnut seeds revealed a high concentration of alkaloids (14). However, fungi rot can lead to deterioration in the nutrients and the major bioactive compounds found in T. conophorum.  The proximate composition of African walnut seeds showed that the fungi majorly had negative effects on the proximate components of walnut seeds by a slight increase in moisture content compared to the control. This suggests that the fungi contributed to higher moisture levels in the walnut seeds, potentially due to decay or fungal growth activities, as well as metabolic activities of the infecting organisms(1).The results from this study also showed that the crude protein and crude fibre contents of the seed were significantly higher than the control.A decrease in the nutrients and the major bioactive compounds indicated that these fungi could have utilized or broken-down these constituents in the walnut seeds. The ash, crude fiber, ash and carbohydrate and anti-nutritional factors revealed trypsin and alkaloid recorded in botanical treated walnut seeds to be comparable to those reported by (27),  (37), (13) and (3). However, in some cases, no decrease in walnut nutrient and phytochemical components was recorded; for example, A. flavusinfection has been reported to not always affect the nutrient content of most food crops as observed in this study but deposits toxic metabolites in them. This is supported by the study of (29) who reported a negative correlation between aflatoxin and protein content of grains. The fungi affected the carbohydrate content of some samples. This was however not the case with total energy, where in most of the treatments, there was no significant difference between treatments and the control.

The general decrease in carbohydrate content indicated the potential breakdown or utilization of carbohydrates by the fungi. These observations indicate that the fungi infection influenced the nutritional composition of African walnut seedswhich could have implications for their overall nutritional value and potential health benefits.

The botanical treatments particularly clove andclove+West African pepper showed lower moisture content compared to the pathogen-inoculated samples, indicating a potential preservative or antifungal effect. Application of clove and West African pepper combined, showed potential for mitigating the impact of the pathogens, with improvements observed in moisture content reduction, and increased protein and fat content.Thetreatment with crude extracts of S.aromaticumand P.guineenseimpactedfungi activities and alkaloid and trypsin inhibitor contents of African walnut seeds. Treatment with clove resulted in higher phytochemical content, while the West African pepper treatment led to lower content in some cases. The combined treatment of clove and West African pepper increased alkaloid content compared to individual treatments and the control for all the fungistudied.For trypsin content, the combined treatment with clove and West African pepper generally recorded higher trypsin content compared to individual treatments and was significantly different from the controls.Clove+West African pepper appears to have a synergistic effect in enhancing both Alkaloid and Trypsin content in walnuts across different fungi compared to individual treatments and the control. Several authors (15), (33), (18) reported the presence of antifungal compounds in plants could be responsible for the control of fungal pathogens by the plants. Greater efficiency of these plants extract was shown to be due to the phenolic substances they contain(8). The effect of P.  guineense and S. aromaticum on fungi causing rots of African walnut is also consistent with the findings of (26) and(31) who reported that phenolic constituents have adverse effect on the protein component of fungal cells which act by disrupting their growth. The botanical extracts were more effective in combined application than in a single application; this suggests that they had synergistic suppressive effect on the fungi activities.

CONCLUSION

The results from the study showed that treatment with crude extracts of clove, West African pepper and clove+African pepper influenced the effect of fungi on the proximate composition, alkaloid, and trypsin content ofAfrican walnut seeds.There isan increase in moisture content and a reduction in crude protein and fibercontent by fungi. Considering the numerous benefits that can be derived from the consumption of walnuts, it is very crucial to protect the nuts from post-harvest losses due to fungi attacks. In this study, the botanical treatments considerably reduced the effects of the fungi on the composition of walnut seeds.

RECOMMENDATION

Therefore, these botanicals can also be usefully exploited in the protection of walnuts from fungi deterioration and nutrient reduction, which will minimize pesticide hazards to the environment and dangers that consumption of infected walnut seeds may pose to human health.

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