Aceh Journal of Animal Science

Sorghum bicolor is known for its multiple uses and is grown for human grain and livestock feed. The study aimed at assessing the effects of incorporating Lactic Acid bacteria (LAB) on the ensiling properties of sorghum bicolor and an antinutritional factor (ANF) called phytate. Dry matter, pH, ash, crude protein, crude fiber, carbohydrates, and phytate were measured as indices of the grain ferment value from January to August. A feed nutrient quality evaluation was conducted with 3 treatments and having 4 replications. The test was conducted using a completely random design. Laboratory analysis of the variables was done on day 6 of the experiment. Results showed that treating sorghum with LAB significantly (P<0.05) affected the nitrogen-free extracts, pH, and phytate but did not however affect the dry matter, crude fiber, crude protein, and ash content (P>0.05). There was a significant (p<0.05) decrease in phytate and nitrogen-free extracts for the LAB-treated sorghum as compared to non-LAB-treated sorghum. Ash content was significantly lower (p<0.05) in LAB-treated sorghum and interestingly higher in non-LAB-treated sorghum. The study showed that treating sorghum with LAB significantly reduces phytate levels in feed and has little effect on the nutrient status of the feed.


Introduction
Sorghum bicolor is the only species of the sorghum family that is known for its multiple uses and is grown for human grain and livestock feed (Etuk et al., 2012).Sorghum crop produces small grains and it is particularly drought tolerant.Due to climate change in Africa, including Zimbabwe, the need for feed enhancement to counter the adverse effects of changes in weather is now a necessity (FAO, 2020).Change in climatic conditions affect feed raw material availability and, as such, the need for more research on how to sustain poultry production.Sorghum production accounts for a greater percentage in rural areas and as such is available both as food and animal feed (Mugoti et al., 2022).The African rural poultry population accounts for almost 80% of the overall national poultry population, of which an estimated asset value of US$5,750 million has been determined (FAO, 2020).Due to scarcity and the high cost of general feed ingredients such as soya, maize, and fishmeal, there has been a renewal of interest in the use of cheaper and more available feed ingredients such as the Poaceae species.Climate change has also contributed to the short supply of feed ingredients as they are preferably provided to humans first rather than animals.
Sorghum bicolor is mostly grown in arid regions due to its heat resistance capabilities, has been identified as an ingredient of choice to replace general feed ingredients (Prasad and Staggenborg, 2009).However, the amount of sunlight and heat exposure of the sorghum species tends to affect the quantities of the antinutritional factors (ANFs) in a more negative way (Maphosa, 2021).ANFs in sorghum increase upon exposure to intense sunlight and heat as is the case nowadays.Therefore, a need arises to eliminate these anti-nutrients which have deleterious effects if left unchecked and offered to animals.Due Alban et al. to limiting resources, the use of natural remedies to counter these chemicals has been identified, with Lactic acid bacteria (LAB) being the counter-measure of choice together with other measures such as crushing to eliminate ANFs.Besides that, LAB can also play an important role in the digestion of feed so that nutrients can be absorbed flawlessly (Nursyirwani et al., 2017), and also produces antimicrobial compounds (Feliatra et al., 2018).Therefore, the objective of the present study was to assess the effects of incorporating Lactic Acid bacteria (LAB) on the ensiling properties of sorghum bicolor and an antinutritional factor (ANF) called phytate.

Project site description
This study was done between the months of January and August, 2019, at the National University of Science and Technology (NUST) in Matabeleland region of Zimbabwe.The site is located in natural farming region 4 which experiences low rainfalls with an average of 500ml annually.The area is elevated between 600m and 1200m above sea level.NUST lies on a latitude of 20.0954ºS and a longitude of 28.3831ºE.The area possesses deep grey-brown sands and a granite sandy loam.Soil pH ranges between 4.4 and 4.8 whilst the soils are of poor waterholding capacity.Excessive dry spells are experienced even during the rainy season which also includes seasonal droughts.Since crop production is not highly recommended in this area, animal production is therefore highly practiced.Extensive livestock and wildlife production are usually practiced.However, drought-tolerant crops are also produced within this farming zone.Drought-resistant crops which include millet and sorghum varieties are grown.This region is in the South-west of the country, Zimbabwe.

Feed samples preparation
A 20kg bag of the red variety of Sorghum bicolor was bought at Bulawayo local market from local grain traders and a 12kg sample was weighed using a digital scale.Contaminants such as small stones and dirt were removed through the process of winnowing.The weighed sorghum was partly crushed using a mortar and pestle.A total of twelve 5-litter plastic containers were allocated to the three treatments.Each treatment was allocated to four 5-litter plastic containers to cater for the four replications.Of the twelve, eight 5-litter plastic containers were loaded with 1kg sorghum grains each and soaked overnight in chemical-free water.The sorghum and chemicalfree water mixture were then stirred thoroughly until they were drenched in water as a mixture.To avoid mold and undesirable bacteria, the sorghum seeds were left submerged the whole time.From these eight plastic containers with the mixture, four were allocated to treatment 1(trt1) and the remaining four to treatment 2 (trt2).Treatments 1 and 2 involved fermentation of sorghum in which trt1 was inoculated with LAB (LAB-treated) whilst trt2 had no inoculation (non-LAB-treated).A spoonful of juice from the homemade Lacto-ferments was added to trt1 as the inoculum.The process took a total of 5 days to achieve successful fermentation.The third treatment (control/trt3) was not subjected to any fermentation or water soaking in which four 5-litter plastic containers were used for the replications.A comparison between the LAB-treated and the non-LAB-treated grain was made by observing the differences in nutritive value of the two with that of the non-fermented sorghum (control).

Chemical-free water
Chemical-free water was obtained through distillation in order to remove all contaminants and eliminate some variants of undesirable bacteria in the water which would affect successful implementation of the experiment.Chemical-free water use is preferred because it does not influence the processes and functionality of lactic acid bacterium.Preparation of lactose ferment 50ml of fresh milk was covered in a tightly closed container and left to turn sour, in which ferment bacteria is most abundant.A spoonful of sour milk was added to the LAB-treated replications to speed up the fermentation process.

Determination of pH
Potential hydrogen was determined using an electronic pH meter.The 12 samples from the three treatments were ground finely and mixed in water thoroughly until a mixture was formed.With the solid particles removed, the supernatant was then used to measure the pH values.

Determination of phytic acid
Phytic acid composition in sorghum was determined according to Wheeler and Ferrel (1971) method.Two grams of the sorghum grain samples from all treatments were separately weighed in a 125 Alban et al.
ml conical flask.50 ml of 3% Trichloroacetic acid (TCA) was extracted from the sample for 3 hours whilst shaking.The suspension was centrifuged for 5 min.at 2500 rpm.Ten milliliters of an aliquot of the supernatant was then transferred into a 40 ml tube; 4 ml Ferric Chloride (FeCl3) (a solution of 2 mg ferric (Fe +3 ) ion/ml and 3% TCA) were added to the aliquot.A boiling water bath was used to heat the tube for 45 min with a few drops (3 or less) of 3% Sodium sulfate (Na2SO4) being added in 3% TCA in order to produce a precipitate.Cooling the tube followed and then centrifugation for 10 minutes at 2500 rpm was done.The clear fluid was then decanted.Flushing of the precipitate was done twice by diffusing in 25 ml 3% TCA, heating in a boiling water bath for 10 minutes, and finally cooling and centrifugation.Flushing of the precipitate was done for two more times with water and diffused in a few ml of water.3ml of 1.5N Sodium hydroxide (NaOH) was then added to reach a total of 30ml volume together with distilled water.A boiling water bath was again used to heat the tube for 30 min., and hot filtered using Whatman filter paper No. 1.
The precipitate was flushed with 60ml of hot water and the washings decanted.The precipitate was then carefully extracted from the filter paper by dissolving with 40 ml hot 3.2N Nitric acid (HNO3) into a 100 ml volumetric flask and flushing of the paper was repeated with water into the same flask.0.5 ml of the suspension was then moved into a 10 ml volumetric flask.Two milliliters of 1.5N Potassium thiocyanate (KSCN) were added and filled with water to volume, then instantly, the absorbance reading was obtained using a spectrometer (JENWAY 6305 UV) at 48 nm.Ferric ion concentration was used to calculate phytate phosphorus with an estimation of 4:6 iron: phosphorus molar ratio.

Statistical analysis
The three treatments (trt1, trt2, and trt3) were replicated four times, and all measurements were done in duplicate.Data for all the treatments were subjected to analysis of variance (ANOVA) using Graphpad Prism TM software (version 8.3 of 2019).AP value of ≤0.05 was considered statistically significant.Separation of treatment means was done using Duncan's multiple range test (Duncan, 1955).

Ash content
No significant difference (p>0.05) in the ash content of LAB-treated, non-LAB-treated sorghum, and control (sorghum) was obtained which had 2.50%, 2.70%, and 2.90% respectively.

Dry matter (DM)
The dry matter content of the treatments showed that there was a significant difference (P<0.05) with LAB-treated sorghum, non-LAB-treated, and control (sorghum) having 79.59% 82.57%, and 87.17% respectively.LAB-treated sorghum had the lowest DM value of the three treatments.

Crude protein (CP)
The crude protein of LAB-treated sorghum was 13.9% and that of non-LAB-treated sorghum was 13.63% which meant that no significant difference (p>0.05) was observed between the two.The control (sorghum) had 14.33% showing no significant difference (P>0.05)among the three treatments.

Fat content
The fat content of LAB-treated sorghum was 3.5% and that of non-LAB-treated sorghum was 3.73%, whilst that of the control (sorghum) had a lower value of 3.43%.This shows that there is no significant difference (p>0.05)among the three treatments.

Nitrogen Free Extract (NFE)
There was a significant difference (p<0.05) in the nitrogen-free extract (NFE) content of the treatments, with LAB-treated sorghum having 70.4%, while non-LAB-treated was 78.5%.The control (sorghum) had 69.5% which was statistically the lowest of the three (Table 1).pH The pH content for the Lab-treated sorghum fell on the pH value of 4.67 whilst non-Lab-treated had a value of 5.97 (table 1).The control (sorghum) had the highest pH value of 6.03.This shows that there was a significant difference (p<0.05) in the pH values of the treatments.

Phytate
The phytate contents of LAB and non-LAB treated sorghum were presented as mg/100g in table 1.There was a significant difference (P<0.05) in the phytate content of the treatments.The phytate content of LAB-treated sorghum was found to be 252 ±15.1mg/100g that of non-LAB-treated sorghum and control (sorghum) were 314 ±7.51mg/100g and 322±3.61mg/100g(mean ± SEM) respectively.

Discussion
The difference between the treatments, after ensiling, shows that there was no significant difference in the dry matter (DM) content of the three treatments.However, the control had a higher DM content than all the other treatments.The high DM content in treatment 2 (non-LAB treated) and treatment 3(control) as compared to the lower DM of treatment 1(LAB-treated sorghum) could be a result of the differences in the dry matter content of the grains due to the degradation effect of LAB on the fiber (Mtengeti et al., 2013).Lactic acid bacteria by nature can digest fiber into utilizable carbohydrates (Cook et al., 2005).This is supported by Cook et al. (2005) who noted that fermented grains provided less dry matter than non-fermented grains.
Crude protein results after ensiling indicated that no significant differences among the three treatments was obtained.Values from the results of the three treatments, however, show that the two treatments (control and non-LAB treated) have lower crude protein composition than the LAB-treated sorghum.This can also be explained by the fact that the use of microbes as a feed enhancement mechanism is associated with beneficial effects such as microbial protein synthesis, which adds value to the feed protein content (Davis et al., 2012).These results were in agreement with work done by Mtengeti et al. (2013) in which crude protein content of different grain silage feeds had no significant difference, with fermented sorghum having a slightly higher CP value than non-fermented sorghum.

Ash content
Fermenting grains with LAB also had a better result on the ash content than the use of untreated grains although, statistically, there was no significant difference.Results from Table 1 show that the sorghum (control) had more ash than the other two treatments.Non-LAB-treated grains tend to have more ash than LAB-treated sorghum grains.This signifies that Non-LAB treated grain is slightly rich in minerals than its counterpart and as such this can be explained by Mtengeti et al. (2013) who mentioned that microbes tend to utilize minerals in their growth process, therefore, little minerals will be left available in the process.
After ensiling, the pH of the three treatments had a significant difference, having the lowest pH from the LAB-treated grain and the highest from sorghum (control).Lower pH is an indication of good quality silage as it is a representative of lactic acid, the requirement in silage to assure its preservation (Moran, 2005).The low pH status could be a result of the readily available LAB incorporated into the grain, and the high energy status of the grain silage which is readily available to facilitate the formation of lactic acid responsible for lowering the pH (Sarwatt et al., 1992).Non-LAB treated grain had a slightly higher pH value which is characterized by a period of inoculation of the bacteria which affects the amount of lactic acid produced since no LAB was inoculated at the start of the experiment.However, higher values observed can be a result of human error when preparing the samples for analysis.Also, the error can be due to delays in pH sampling in which there is lactic acid reduction once the grain silage is opened hence the high pH values.This was previously reported by Woolford (1990) that opening the buckets causes a rise in pH due to the onset of deterioration because of degradation of the organic acids by aerobes and also, the rise in environmental temperatures.
The phytic content of LAB-treated sorghum was significantly lower than that of both non-LAB and general sorghum.This variation in the percentage reduction of phytate is due to variation in the variety of microbes available in the feed investigated.An extensive number of microflora is well-known to hold and influence phytase activity (Lopez et al., 1983;Yadav and Khetarpaul, 1994).In general, fermentation is known to cause a high reduction in sorghum phytate when compared to other antinutritional factors, and this may be caused by the low pH levels of the fermented samples which are considered excellent for the phytase activity.This is in unison with Valencia et al. (1999) who stated that the fermentation process provides optimal (low) pH conditions for phytase activity in cereals (pH 4.5 -5.0).

Conclusion
From all these observations, it is very much important to implore the new practice of inoculating grains with LAB as it seemed a little more beneficial than the old-fashioned way of using self-inoculation in silage making, although, statistically there were no differences in most outcomes.This however gives evidence that there will be beneficial effects on implementing this new initiative and will prove advantageous to the animals as they benefit from the positive effects of incorporating LAB on the elimination of ANFs on a nutritional basis.

Table 1 .
Effects of treatments on ensiling variables.