2021 SCIENCELINE

O nline J ournal of A nimal and F eed R esearch

Volume 11, Issue 1: 01-07; January 25, 2021

ISSN 2228-7701

DOI: https://dx.doi.org/10.51227/ojafr.2021.1

TOXICITY OF AUTO-DETOXIFIED Jatropha curcas Linnaeus, 1753

KERNEL CAKE MIXTURES WITH BOVINE BLOOD (ADMJKC/BB) USING

BRINE SHRIMP Artemia salina Linnaeus, 1758



Divine EWANE¹, Benedicta O. OBEN², Kenneth Jacob Ngoh NDAMUKONG¹, Kingsley A. ETCHU³, Eugene

E. EHABE^3,Jane M. CHAH⁴, Kennedy F. CHAH⁵and Pius MBU OBEN²

¹Department of Animal Science, Faculty of Agriculture and Veterinary Medicine, University of Buea, PO Box 63, Buea, Cameroon

²Department of Fisheries and Aquatic Resources Management, Faculty of Agriculture and Veterinary Medicine, University of Buea, PO Box 63,

Buea, Cameroon

³Directorate of Scientific Research, Institute of Agricultural Research for Development, P.O. Box 2123, Yaounde, Cameroon

⁴Department of Agricultural Extension, Faculty of Agriculture, University of Nigeria, Nsukka, Enugu State, Nigeria

⁵Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Enugu State, Nigeria



Email: ewane.divine@ubuea.cm ;

: 0000-0002-7807-4056

^Supporting Information

ABSTRACT: In present study, brine shrimp (Artemia salina L.) was used to determine the toxicity of auto-

detoxified Jatropha kernel cake (JKC) mixed with bovine blood (ADMJKC/bb). The powdered-JKC was mixed

with bovine blood (bb) at three ratios (1:1=X, 2:1=Y and 3:1=Z of JKC: bb) and the resultant mixtures processed

using four protocols: Heated, Spread Dry = 1, Unheated Spread Dry = 2, Heated Spread Remoisten to 66% dry

matter (DM) = 3 and Unheated Spread Remoistened to 66% DM = 4). The resultant 12 treatment combinations

(X1, X2, X3, X4, Y1, Y2, Y3, Y4, Z1, Z2, Z3 and Z4) were placed in a Solar J. curcas auto-detoxification

apparatus from where samples were retrieved periodically and evaluated for detoxification using the brine

shrimp lethality test. There were no significant differences within the same ratio of mixes among the four

protocols. However, there was a tendency for mean LC₅₀values to increase between the ratios. Specifically,

Protocol 2 recorded a significant difference between X2 and Z2 treatments, having 1:1 and 3:1 JKC: bb mixes

respectively. Upon ranking the level of auto-detoxification, the most detoxified treatments (Z2 with LC₅₀=4674

and Z4 with LC₅₀=3692) differed significantly from the least two (X1 with LC₅₀=1383 and X2 with LC₅₀=1459).

Addition of bovine blood to JKC increased the dynamics of JKC auto-detoxification, probably due to the

presence of some innate auto-detoxifying microbial inoculum and bovine blood which boost the rapid growth,

development and succession of these microbes. Thus combining JKC with bovine blood is complementary for

JKC auto-detoxification, with the most detoxified ingredients (Z2, Z4 and Y3) appearing most suitable for

further development and testing as feed ingredient for farm animals.

Keywords: Auto-detoxification, Bovine blood, Feedstuff, Jatropha, Shrimp.

Abbreviations: ADJKC; auto-detoxified Jatropha kernel cake; ADMJKC/bb: auto-detoxified mixtures Jatropha kernel cake and bovine blood;

ANOVA: analysis of variance; BSLT: Brine Shrimp Lethality Test; bb: bovine blood; CMJKC/bbE: crude methanol auto-detoxified mixtures of

Jatropha kernel cake with bovine blood extracts; DJADA: Diffuse daylight Jatropha curcas auto-detoxification apparatus; DMSO: dimethyl

sulfoxide; DMRT: Duncan's Multiple Range Test; FAO: Food and Agriculture Organization of the United Nations; IBM: International Business

Machines; JKC: Jatropha kernel cake; LC_50:Lethal concentration killing 50% of test organisms; NRC: National Research Council; SJADA: Solar

Jatropha curcas auto-detoxification apparatus; SPSS: Statistical package for social sciences; UDC: un-moistened diffuse daylight spread; USS:

un-moistened solar spread; X1: Jatropha kernel cake and bovine blood, mixed at a ratio of 1:1. Heated, spread dried without remoistening; X2:

Jatropha kernel cake and bovine blood, mixed at a ratio of 1:1. Unheated, spread dried without remoistening; X3: Jatropha kernel cake and

bovine blood, mixed at a ratio of 1:1. Heated, spread dried, remoisten to 66% dry matter; X4: Jatropha kernel cake and bovine blood, mixed at

a ratio of 1:1. Unheated, spread dried, Remoistened to 66% dry matter; Y1: Jatropha kernel cake and bovine blood, mixed at a ratio of 2:1.

Heated, spread dried without remoistening; Y2: Jatropha kernel cake and bovine blood, mixed at a ratio of 2:1. Unheated, spread dried without

remoistening; Y3: Jatropha kernel cake and bovine blood, mixed at a ratio of 2:1. Heated, spread dried, remoisten to 66% dry matter; Y4:

Jatropha kernel cake and bovine blood, mixed at a ratio of 2:1. Unheated, spread dried, remoistened to 66% dry matter; Z1: Jatropha kernel

cake and bovine blood, mixed at a ratio of 3:1. Heated, spread dried without remoistening; Z2: Jatropha kernel cake and bovine blood, mixed

at a ratio of 3:1. Unheated, spread dried without remoistening; Z3: Jatropha kernel cake and bovine blood, mixed at a ratio of 3:1. Heated,

spread dried, remoisten to 66% dry matter; Z4: Jatropha kernel cake and bovine blood, mixed at a ratio of 3:1. Unheated, spread dried,

Remoistened to 66% dry matter.

INTRODUCTION

Jatropha curcas has shown some promise as a crop from which biofuels can be produced without compromising human

food needs (Nithiyanantham et al., 2012; Maghuly and Laimer, 2013). However, despite global excitement on its

potentials, at current levels of technology, value-addition from its by-products and co-products is necessary in order for

viability of the sector to be ensured (Makkar et al., 2012). One of the by-products being evaluated in this regard is

Jatropha kernel cake (JKC), the remnants obtained after extraction of oil from Jatropha kernels that have a potential for

use as animal feed (Nithiyanantham et al., 2012; Che Hamzah et al., 2020). Except for lysine, its nutrient profile

surpasses the Food and Agriculture Organization of the United Nations (FAO)’s reference protein (Makkar et al., 1998).

Citation: Ewane D, Oben BO, Ndamukong KJN, Etchu KA, Ehabe EE, Chah JM, Chah KF and Mbu Oben P (2021). Toxicity of auto-detoxified Jatropha curcas Linnaeus,

1753 kernel cake mixtures with bovine blood (ADMJKC/bb) using brine shrimp Artemia salina Linnaeus, 1758. Online J. Anim. Feed Res., 11(1): 01-07. DOI:

https://dx.doi.org/10.51227/ojafr.2021.1

However, it is toxic to humans and animals because of its phorbol esters and other anti- nutrients (trypsin inhibitors,

lectins and phytate) and therefore requires detoxification prior to use (Sharath et al., 2014; Rodríguez-González et al.,

2018).

Several methods targeting the larger industries and Jatropha farm holdings have been employed and categorised

with varying degrees of success to detoxify JKC (Ewane et al., 2017). However, the need for simple cottage techniques for

small and large- scale operations persists. Auto-detoxification of JKC has therefore been developed as a pro-poor and pro-

rural detoxification method for JKC (Ewane et al., 2017).

Present study aimed to determine the toxicity of auto-detoxified mixtures JKC and bovine blood (ADMJKC/bb) on

brine shrimp (Artemia salina L.). The strategy was to manipulate endogenous and environmental factors that can enhance

self-detoxification in mixtures of JKC and bovine blood and identify the most promising treatment combinations. Bovine

blood, often used to produce blood meal, highly supports microbial growth, and also represents one of the richest sources

of lysine (NRC, 1994). Hence, the (ADMJKC/bb) mix shall enhance the nutrient profile and detoxification rate of JKC.

Furthermore, the powdery ground JKC shall easily trap the liquid bovine blood and reduce the need for further processing

of blood meal by small-scale farmers (Nithiyanantham and Francis, 2012).

MATERIALS AND METHODS

Study Location

The study used the facilities of the Teaching and Research

Farm, University of Buea, Cameroon (located at 4.1667⁰N, 9.2333⁰

E, 578 m asl). J. curcas seeds were harvested from farms, live fences

and plantations in four of Cameroon’s five agro-ecological zones

(Figure 1): Maroua in the Sudano-Sahel zone, Ngaoundere in the High

Guinean Savana, Bamenda in the Western Highlands and Mamfe in

the Humid Forest zone with a mono-modal rainfall pattern. Bovine

blood was collected from the main abattoir in the town of Buea.

Preparation and pre-treatment of J. curcas kernel cake and

bovine blood mix

J. curcas seeds were cracked open (using two hard boards) and

the extracted kernels de-oiled using a hydraulic press. The J. curcas

kernel cake (JKC) obtained was then finely ground using a plate mill

and the powder homogenized by hand mixing before further mixing

with bovine blood in three different proportions (1/1, 2/1 and 3/1).

Twelve combinations were developed for production and

evaluation based on four processing protocols of JKC mixtures with

bovine blood (bb) at three mix ratios (Table 1). The mixes were

chosen by serially increasing the quantity of JKC in a fixed amount

Figure 1 - Location map of Cameroon showing the

of bovine blood. This gave combination protocols from three ratio

mixes and four auto-detoxification treatments. These protocols

were inspired by previous studies on the effects of moisture (Abou-

agro-ecological zones where J. curcas seeds were

collected.

Arab and Abu-Salem, 2010), spreading (Schmidt and Hecker, 1975) and heat (Aregheore et al., 2003; Martinez-Herrera et

al., 2006) on degradation of phobol esters and/or associated anti- nutrients of JKC.

Each combination was replicated 4 times and placed in steel plates arranged on two tables within the performant

SJADA (Ewane et al., 2017), operated at full detoxification mode with the air access inlet closed to the minimum level of

50 cm². Six of the 12 treatment combinations (Protocol code 3 and code 4: X3, Y3, Z3, X4, Y4 and Z4) were remoistened

to 66 % dry matter daily, while the other six (Protocol code 1 and code2: X1, Y1, Z1, X2, Y2 and Z2) were not remoistened.

About 10 g of sample were collected weekly from each replicate to evaluate the level of auto-detoxification

Table 1 - Treatment codes of JKC/bb mixing ratios, auto-detoxification and moistening cycles.

JKC: bb mixing ratio

Protocol

Code

Auto-detoxification treatment of JKC: bb combination Protocol

X (1:1)

Y (2:1)

Z (3:1)

Heat, spread and dried - No remoistening

Heat, spread and dried - Remoistened to 66% dry matter

No heat, spread and dried - No remoistening

No heat, spread and dried - Remoistened to 66% dry matter

Citation: Ewane D, Oben BO, Ndamukong KJN, Etchu KA, Ehabe EE, Chah JM, Chah KF and Mbu Oben P (2021). Toxicity of auto-detoxified Jatropha curcas Linnaeus,

1753 kernel cake mixtures with bovine blood (ADMJKC/bb) using brine shrimp Artemia salina Linnaeus, 1758. Online J. Anim. Feed Res., 11(1): 01-07. DOI:

https://dx.doi.org/10.51227/ojafr.2021.1

Preparation of crude methanol auto-detoxified mixtures of JKC/bovine blood extracts (CMJKC/bbE)

A sample of about 40 g (10 g per replicate) was collected weekly from each treatment and analysed to determine

the progress of auto-detoxification. The replicates of each treatment were pooled and further dried within the apparatus

for another week, homogenised and powdered. From the powdered mass, 20 g were placed in an extraction bottle

containing 200 mL methanol and the methanolic extract obtained by regularly stirring the whole for 72 h using a rotatory

stirrer. The extract was then filtered through Whatman No. 541 filter papers and the crude methanol auto-detoxified

mixtures of JKC/bovine blood extracts (CMJKC/bbE) obtained by evaporation of the solvent using a rotary evaporator.

Brine shrimp bioassay

The brine shrimp lethality test (Meyer et al., 1982) was used, with some modifications, to test the toxicity of

CMJKC/bbE. Brine shrimps (Artemia salina Leach) were hatched from eggs in rectangular dishes containing sea water.

The dishes were sub-divided by a perforated barrier into two unequal chambers and kept under constant aeration. The

bigger chamber receiving the eggs was in the dark while the smaller chamber receiving the hatched napuli in anticipation

was under constant light. Forty-eight hours was allowed for the eggs to hatch and the phototropic napuli to mature. At

each turn, 10 napuli were drawn using a pipette and placed in a marked vial containing 4 ml of natural seawater. Prior to

this, a CMJKC/bbE stock solution of 100,000 ppm was prepared by dissolving 200 mg of CMJKC/bbE in 2 ml of dimethyl

sulfoxide (DMSO). The stock solution was then diluted serially with natural seawater to give a series of concentrations for

testing (10 000, 1000, 100, 10 and 1 ppm). Three replicates of each concentration were prepared. From each pre-

dilution, 1 ml containing CMJKC/bbE, seawater and DMSO was added to the pre-marked vials containing 4 ml of natural

sea water and 10 napuli to make a total volume of 5 ml. Negative controls were just dilutions of DMSO in seawater

without CMJKC/bbE while un-detoxified whole Jatropha kernel cake served as the positive control.

The vials were incubated under light for 24 h, after which manual counting of dead and immobile napuli at bottom

of vial commenced, against a lit background with the aid of a 3X magnifying hand lens. The mortality was calculated as

the percentage ratio of the number of dead napuli to the total number of napuli tested after corrections to account for

mortalities recorded in the control (Abbott, 1925) as shown in equation 1. Subsequently corrections were made for 0%

and 100% as proposed by Ghosh (1984) and presented in equation 2 and equation 3, respectively.

(

)

{(

)⁄(

)}

퐶표푟푟푒푐푡푒푑 푚표푟푡푎푙푖푡푦 % = 푀_ꢀ푏푠− 푀_ꢁꢀ푛100 − 푀_ꢁꢀ푛푥100

… …

Eqn. 1

Where,

M_obsand M_conwere the respective observed and control mortalities.

(

)

(

)

0% 퐶표푟푟푒푐푡푒푑 푚표푟푡푎푙푖푡푦 % = 100 푥 0.25 푥 ꢂ

… …

Eqn. 2

Eqn. 3

)

⁄

100 % 퐶표푟푟푒푐푡푒푑 푚표푟푡푎푙푖푡푦 % = 100 푥 (ꢂ − 0.25 ꢂ)

Where,

n is the number of test animals in each group.

Determination of lethal concentration

The lethal concentration of CMJKC/bbE resulting in 50 % mortality of brine shrimp (LC₅₀) was determined from the

24 h counts by a plot of percentage of the shrimps killed against the logarithm of the CMJKC/bbE concentration and the

best-fit line was obtained from the curve data by means of regression analysis (MS Excel version 7). The LC₅₀was derived

from the slope of the best-fit line obtained.

Statistical analysis

Levene's Test for Equality of Variances was performed on LC₅₀. Also LC₅₀values were subjected to a one-way

analysis of variance (ANOVA) and the significance of the differences between means tested using Duncan's Multiple

Range Test (DMRT) (P<0.05). The software used was the IBM SPSS Statistics version 22 (IBM Corp. Released 2013). The

most promising ADMJKC/bb treatments were selected after ranking the LC₅₀from the largest to the smallest values, with

the largest values indicating the least toxicity. ANOVA and DMRT were used to detect significant difference of the LC₅₀

values of ADMJKC/bb treatments

RESULTS

The evolution of LC₅₀of crude methanol auto-detoxified mixtures of JKC/bovine blood extracts (CMJKC/bbE) with duration

of incubation have been presented in Figure 2, while: comparison of 3 week means of LC₅₀of crude methanol auto-

detoxified mixtures of JKC/bovine blood extracts (CMJKC/bbE) is in Table 2 . By the third week of the trial, all the 12

treatments had LC₅₀values above 1000. This indicates they were all substantially detoxified. There were no significant

differences (P>0.05) among the four Auto-detoxification treatment of JKC: bb combination protocols when compared

within rations for each ratio of mixes.

However, there was a general tendency for mean LC₅₀values to increase across ratios as quantity of JKC increased

in the mixture. The only exception was Protocol code3 (the Heat, spread and dried - Remoistened to 66% dry matter

Citation: Ewane D, Oben BO, Ndamukong KJN, Etchu KA, Ehabe EE, Chah JM, Chah KF and Mbu Oben P (2021). Toxicity of auto-detoxified Jatropha curcas Linnaeus,

1753 kernel cake mixtures with bovine blood (ADMJKC/bb) using brine shrimp Artemia salina Linnaeus, 1758. Online J. Anim. Feed Res., 11(1): 01-07. DOI:

https://dx.doi.org/10.51227/ojafr.2021.1

protocol). In Protocol code 3, the 2:1 ratio (Y3) is higher than both the 1:1ratio (X3) and 1:3 ratios (Z3). In addition,

Protocol code2 (the no heat, spread and dried - no remoistening protocol) recorded a significant difference (P<0.05)

between X2 and Z2 treatments which are compared across ratios.

The ranking of mean LC₅₀(Table 3) indicates that the most detoxified treatments were Z2=3,047.22, Z4=2,

0130.67 and Y3=1,997.64, while the least detoxified were X2=653.65 X1=736.96 and Y1= 1,092.93. The top two most

detoxified treatments (Z2 and Z4) differed significantly (P<0.05) from the least two (X1 and X2).

Of all the treatments evaluated, the extent of lethality was found to be proportional to the concentration of the

CMJKCE/bbE. High mortalities were recorded at 1000 ppm and 10,000 ppm, while lower mortalities were recorded at 1

ppm and 10 ppm. The graphical representations of the corrected brine shrimp mortalities in week 3 for the most and

least detoxified treatments as well as the un treated whole Jatropha kernel cake control are shown in Figure 3.

Figure 2 - Evolution of LC₅₀of crude methanolic auto-detoxified mixtures of JKC/bovine blood extracts (CMJKC/bbE) with

duration of incubation

Table 2 - Comparison, LC₅₀of crude methanol auto-detoxified mixtures of JKC/bovine blood extracts (CMJKC/bbE) mean

of 3 weeks

JKC: bb mixing ratio

Protocol

Code

Auto-detoxification treatment of

JKC: bb combination Protocol

X (1:1)

Y (2:1)

Z (3:1)

Heat, spread an d dried - No

remoistening

737.0 ± 844.7 ^a

1092.93 ± 783.83 ^ab

1110.46 ± 652.12 ^ab

No heat, spread and dried - No

remoistening

653.7 ± 604.5 ^a

1700.64 ± 771.72 ^ab

3047.22 ± 553.13 ^b

Heat, spread and dried -

Remoistened to 66% dry matter

1248.5 ± 815.2 ^ab

1997.64 ± 643.05 ^ab

1498.18 ± 406.18 ^ab

No heat, spread and dried -

Remoistened to 66% dry matter

1796.5 ± 323.7 ^ab

1794.53 ± 470.58 ^ab

2130.67 ± 563.01 ^b

^a,b,c:Mean LC₅₀values with different superscript differ significantly (P<0.05).

Table 3 - Ranking of LC₅₀of crude methanol auto-detoxified mixtures of JKC/bovine blood extracts (CMJKC/bbE)

Treatment

Mean (± sem) LC₅₀

Group

RANK

Best fit equation

R²

3047.22 ± 553.13

2130.67 ± 563.01

1997.64 ± 643.05

1796.54 ± 323.73

1794.53 ± 470.58

1700.64 ± 771.72

1498.18 ± 406.18

1248.46 ± 815.19

1110.46 ± 652.12

1092.93 ± 783.83

736.96 ± 844.73

653.65 ± 604.48

0.05

y = 22.568x - 32.795

0.8502

y = 23.337x - 32.795

0.8941

y = 21.415x - 21.645

y = 21.799x - 18.954

y = 8.7287x + 61.229

0.8254

0.9534

0.6719

JS Control

Values are means ± standard deviations of results obtained after 3 weeks. ^a,b,c:Mean LC₅₀values with different superscript differ significantly

(P<0.05).

Citation: Ewane D, Oben BO, Ndamukong KJN, Etchu KA, Ehabe EE, Chah JM, Chah KF and Mbu Oben P (2021). Toxicity of auto-detoxified Jatropha curcas Linnaeus,

1753 kernel cake mixtures with bovine blood (ADMJKC/bb) using brine shrimp Artemia salina Linnaeus, 1758. Online J. Anim. Feed Res., 11(1): 01-07. DOI:

https://dx.doi.org/10.51227/ojafr.2021.1

Figure 3 - Corrected brine shrimp mortality rates for different protocols and mixtures of Jatropha kernel subjected to

different levels of auto-detoxification

DISCUSSION

According to Meyer et al. (1982), several extracts derived from natural products which had LC₅₀≤ 1000 μg/ml using Brine

Shrimp Lethality Test (BSLT) were known to contain physiologically active principles while those with LC₅₀values > 1000

ppm were considered inactive. This indicates that all the 12 ADMJKC/bb ingredients were substantially detoxified after 3

weeks of exposure in the SJADA. The evolution of the LC₅₀values indicates that detoxification was more rapid with

increasing content of JKC in the ADMJKC/bb ingredients. Two ingredients containing a ratio of 3:1 JKC: bb (Z2 and Z4) and

two containing JKC: bb ratio of 2:1 (Y3 and Y4) all had LC₅₀values above 1000 within the first week of auto detoxification

compared to just one ingredient containing a ratio of 1:1 JKC: bb (X4). Nonetheless, this level of auto-detoxification for

even the least ratio mix of JKC with bovine blood is remarkably rapid, when compared to LC₅₀values of auto-detoxified

JKC (ADJKC) ingredients produced solo without addition of bovine blood as reported in Ewane et al. (2017). In that study

which was performed under similar conditions as the current study, it took 4 weeks for just 3 out of 14 ADJKC test

ingredients on trial, to attain LC₅₀values above 1000.

Therefore, the addition of bovine blood to JKC probably changed the dynamics of auto- detoxification. According to

Thomas (1988), in the live animal blood is generally a sterile medium, possessing innate bacteriostatic and bactericidal

abilities. Such antibacterial effects are clearer with gram negative bacteria such as E-coli, and non-virulent strains of Vibro

cholerae, Haemophilus influenae, salmonellae and shigellae. Out of the animal, however, bovine blood is a rich nutrient

medium encouraging the growth of several microbes. Adding bovine blood to JKC may therefore increase the range of

microbes in the mixture which would rapidly increase the level of auto-detoxification. Kasuya et al. (2013) increased the

level of bio- detoxification of JSC by adding 10% eucalyptus bark. From the data obtained, they deduced that the

importance of adding eucalyptus bark served to balance carbon and nitrogen and decrease the fat content, thus resulting

in improved fungal growth. They concluded that their results support the hypothesis that phorbol ester degradation occurs

because of co-metabolism by the enzymes responsible for lignin de-polymerization.

Even though the differences were not significant (P>0.05), ADMJKC/bb produced by subsequent remoistening

irrespective of whether they were preheated or not (Protocol code 3 and code 4: Z3, Y3, X3, Z4, Y4, X4) as a group were

among the top most detoxified, while the ADMJKC/bb preheated without subsequent remoistening (Protocol code 1: X1,

Y1 and Z1) were the least detoxified. Interestingly, the actual most detoxified, median most detoxified and actual least

detoxified ADMJKC/bb ingredients were respectively Z2, Y2 and X2, from protocol code 2. These ingredients were

unheated and un-remoistened; the difference between the three ingredients was the ratio of mixing JKC with bovine

blood. They were respectively mixed at 3:1, 2:1 and 1:1 for Z2, Y2 and X2. The protocol code 1 (Z1, Y1, and X1)

ADMJKC/bb ingredients which were the least detoxified as a group followed a similar detoxification ranking with Z1 as the

most detoxified and X1 as the least detoxified for that group. What was common among the Protocol code 1 and code 2:

Z1, Y1, X1 Z2, Y2, X2 groups was that they were all undergoing a form of unperturbed solid state auto-detoxification

compared to their daily remoistened counterparts of Protocol code 3 and code 4 (Z3, Y3, X3 & Z4, Y4, X4). The only

Citation: Ewane D, Oben BO, Ndamukong KJN, Etchu KA, Ehabe EE, Chah JM, Chah KF and Mbu Oben P (2021). Toxicity of auto-detoxified Jatropha curcas Linnaeus,

1753 kernel cake mixtures with bovine blood (ADMJKC/bb) using brine shrimp Artemia salina Linnaeus, 1758. Online J. Anim. Feed Res., 11(1): 01-07. DOI:

https://dx.doi.org/10.51227/ojafr.2021.1

difference is that while the Protocol Code 1 (Z1, Y1, X1) group was heated, the Protocol code 2 (Z2, Y2, X2) group was

unheated. Similarly, while the Protocol code 3 (Z3, Y3, X3) group was preheated, the protocol code 4 (Z4, Y4, X4) group

was unheated.

The differences observed among the various ratio mixes of JKC with blood therefore, probably indicate that the

higher the ratio of JKC in the mixture, the higher the level of innate auto-detoxifying microbial inoculum. The hypothesis

that needs to be verified is that “Microbes supporting auto-detoxification are present on the JKC and either act or wait for

their opportunity to act in an ecological succession. Bovine blood provides a trigger (probably nutrients in simple

molecules plus additional microbes) which boosts the rapid growth, development and succession of these microbes”.

Evidence from an earlier study by Ewane et al. (2017) in support of this hypothesis is that powdered JKC treatments with

neither addition of water nor lye (un-moistened solar spread-USS- and un-moistened diffuse daylight spread-UDC-)

treatments were substantially detoxified, having LC₅₀of 63.89 and 5.22 respectively after four weeks of exposure in the

Solar J. curcas Auto-detoxification Apparatus (SJADA) and Diffuse Daylight J. curcas Auto-detoxification Apparatus

(DJADA) respectively. The observation that the ADMJKC/bb preheated without subsequent remoistening (Protocol code 1:

X1, Y1 and Z1) as a group were the least detoxified is a pointer that the initial inoculum was probably destroyed by heat

and a succession was slower to take off in the absence of added moisture. The abnormal higher ranking of Y3 over Z3 in

Protcol code 3 is possibly an indication that release of nutrients and other factors from bovine blood as a proportion to

available microbial inoculum to auto-detoxifying microbes in heat treated and subsequently remoistened ADMJKC/bb

could also be a determinant to the rate of auto- detoxification.

Furthermore, the results of Chikpah and Demuyakor (2012), who observed that approximately 60% reduction in

crude phorbol ester levels can be achieved within 21 days of spontaneous fermentation of J. curcas kernel meal, supports

the hypothesis that JKC contains some innate auto-detoxifying microbial inoculum. As defined in Ewane et al. (2017),

auto-detoxification is a self-detoxification process induced by endogenous and environmental factors including enzymes,

microbes, sunlight, temperature, humidity and wind. It is the natural way to transform the toxic J. curcas seeds into an

innocuous material. These processes take considerable time under natural conditions but their duration can be shortened

by human manipulation. The results of this present study therefore highlight the possible role of microbes in the auto-

detoxification process. Working with pure microbial cultures, some authors including Belewu and Akande (2010), Belewu

et al. (2010), Bose and Keharia (2013), Kasuya et al. (2013) and Azhar et al. (2014) have confirmed the role of fungi in

the detoxification of JKC, while others including El-Zelaky et al. (2011), Phengnuam and Suntornsuk (2013), Widiyastuti et

al. (2013) and Chang et al. (2014) have confirmed the role of bacteria in the detoxification of JKC.

All the 12 ADMJKC/bb ingredients were substantially detoxified; however, the top three most detoxified ingredients

(Z2, Z4 and Y3) have been selected for further development. Any of the bottom nine (X4, Y4,Y2, Z3,X3,Z1,Y1,X1, and X2)

may still be tested with animal models for further differentiation.

CONCLUSION

The addition of bovine blood to Jatropha Kernel Cake (JKC) changed the dynamics of auto-detoxification. Microbes

supporting auto- detoxification are present on the JKC and either act or wait for their opportunity to act in an ecological

succession. Bovine blood provides a trigger (probably nutrients in simple molecules plus additional microbes) which

boosts the rapid growth development and succession of these microbes. Also, blood, which is the richest natural source of

lysine, could supplement the reported lysine deficiency in JKC and the combined auto-detoxified mixtures Jatropha kernel

cake and bovine blood; (ADMJKC/bb), would potentially become a better feed ingredient than either blood meal alone or

JKC alone. Consequently, the top three most detoxified ingredients (Z2, Z4 and Y3) have been selected for further

development and testing as feed ingredient for farm animals. However any of the bottom nine (X4, Y4,Y2,

Z3,X3,Z1,Y1,X1, and X2) may still be tested with animal models for further differentiation.

DECLARARATIONS

Corresponding author’s Email: ewane.divine@ubuea.cm ;

Authors’ contribution:

: 0000-0002-7807-4056-

D.Ewane conceived the study, designed the study, collected data, contributed in data analysis, and writing the

manuscript, coordinated the inputs of all the other authors; B.O.Oben and K.J.N.Ndamukong performed critical reviewing

of the manuscript and supervision of the study; K.A.Etchu performed critical reviewing of the manuscript; E.E.Ehabe

contributed in design of study, data analysis and writing the manuscript; J.M.Chah contributed in data collection and write

up of the manuscript; K.F.Chah contributed in conception of the study, design of study, data analysis and critical reviewing

of the manuscript; P.M.Oben performed critical reviewing of manuscript and coordinated the study.

Competing interest

The authors have not declared any conflict of interest

Acknowledgements

Thanks to the University of Buea for financial support to purchase the Jatropha seeds.

Citation: Ewane D, Oben BO, Ndamukong KJN, Etchu KA, Ehabe EE, Chah JM, Chah KF and Mbu Oben P (2021). Toxicity of auto-detoxified Jatropha curcas Linnaeus,

1753 kernel cake mixtures with bovine blood (ADMJKC/bb) using brine shrimp Artemia salina Linnaeus, 1758. Online J. Anim. Feed Res., 11(1): 01-07. DOI:

https://dx.doi.org/10.51227/ojafr.2021.1

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Citation: Ewane D, Oben BO, Ndamukong KJN, Etchu KA, Ehabe EE, Chah JM, Chah KF and Mbu Oben P (2021). Toxicity of auto-detoxified Jatropha curcas Linnaeus,

1753 kernel cake mixtures with bovine blood (ADMJKC/bb) using brine shrimp Artemia salina Linnaeus, 1758. Online J. Anim. Feed Res., 11(1): 01-07. DOI:

https://dx.doi.org/10.51227/ojafr.2021.1