Comparing the efficiency of rumen bacteria isolates from lambs, cows, and buffalo in producing the enzyme esterase

Introduction

Nutritionists animal feeding facing challenges to reduce the cost of ruminants products, to reach this point they using roughages due to its low cost, low-quality roughages like crops and cereal straws remains a essential strategy in ruminant nutrition in the world (Sultan et al., 2023) [32] . At any rate, the intake and nutritional value of these fibrous feeds were severely constrained due to the difficult structural of the plant cell wall (Beauchemin et al., 2020). As well as, cell wall composite of cellulose and hemicellulose, and lignin, to break this structural strong, research's biotechnological were focused on enzyme supplementation to destroy these cross-links. Different enzymes using and among these enzymes were esterases particularly ferulic acid esterase (FAE, EC 3.1.1.73) (Ribeiro et al., 2022) [29] . Breaking these bridges disrupts the lignocellulosic complex then release cell wall-bound phenolic compounds which help to attack carbohydrates by microbial colonization rapidly and fermentation in the rumen (Azzaz et al., 2021; Wang et al., 2023) [35] . The aim of this study is to compare different isolation and characterization of biosafety-compliant bacterial strains from rumen of ruminants, and evaluation of their efficiency in producing esterase enzyme to enhance fiber digestion and improve feed efficiency.

Material and methods

Sample collection and bacterial isolation

Fresh rumen samples of lambs, cows and buffalo were collected from slaughter house, Iraq. The nature of nutrition's were roughages, concentrate, hard plants and grass. Rumens of slaughtered animals opened in the field and samples were collected by straining through three -layer of cheesecloth in a small container 1000 ml, then covered with 5 ml of oil paraffin to provide anaerobic condition through minimizing the exposure to oxygen then Samples were kept in ice and transferred to the laboratory within 48 h to isolate bacteria (Fruntina 2022). Serial dilution of each ruminant sample were made, in which one milliter of sample was diluted on nine milliter of phosphate normal saline to obtain 10 -1 , then dilutions were made to 10 -8 (Collin. 1989; Marutin 2001) [11] . For facultative anaerobes and obligate aerobes, selective media were applied to propagate bacterial isolate selectively. Each medium was prepared as their manufacture's instruction. Nutrient agar for total bacterial count, MacCkongy agar for gram negative bacteria, Cetrimide for Pseudomonas spp, Pfizer agar for fecal Streptococcus faecalis and urea based agar augmented with urea for urease positive bacteria. Bacterial enumeration was done by streaking 0.1 ml of each diluent on the top of agar plates and incubated for 24-72 h at 37-39 °C. Meanwhile, counting bacterial colonies on the surface of each medium type and bacterial count should between 30-300 colonies on the top of agar. The estimation of bacterial count would obey the equation: colony forming unit/ ml= colonies number×dilution factor×10 (Cappuccino and Welsh 2020) [8] . Isolated colony was picked from each medium and propagated on the top of nutrient agar to screen for other characters. Bacterial isolates were studied and characterized for their appearance on the top of their corresponding medium and their microscopically cell shape after staining using Gram stain. Subsequently, isolated colonies from each sample were put into groups to study their characters, group gram negative, group Pseudomonas, group Streptococcus and group Lactic acid bacteria (Cheesbrough, 2006) [9] .

Methylene blue test

Methylene blue test serves as a redox indicator to evaluate the metabolic efficiency and enzymatic activity of each bacterial isolate under study. Inoculate bacterial isolates on nutrient broth medium for 24 hours at 37°C in a shaking incubator to ensure adequate aeration and continuous mixing of the culture components. Meanwhile, 0.1 ml of methylene blue (0.03% ) was added to 0.9 ml of the bacterial culture and mix thoroughly by stirring several times and allow to stand, then note and record the time required for the blue color to disappear from middle to bottom of samples. Positive result indicated by time required for the enzymatic reduction of methylene blue to leuco-methylene blue and the scores of activities were listed in table 1 following Cockcroft& Jackson (2002) [10] . Qualitative screening was done using nutrient agar amended with 0.5% of 2-naphthyl acetate. Each bacterial isolate was streaked over agar and incubated at 37-39°C for 72h. A clear zone around growth was indicated as a Positive result. For Quantitative determination, nutrient broth inoculated with each isolate and incubated at 37-39°C for 24 h. Meanwhile, using cooled centrifuge, samples were centrifuged at 10000rpm for 10 min to extract crude enzyme from bacterial isolates. The substrate 1-naphthyl acetate was applied for enzymatic activity assay. Briefly, substrate was prepared as 0.5% in phosphate buffer pH 7.5 and used through the assay. To 0.9 ml of substrate only 0.1 ml of crude extract was used from each isolate. Mixtures were incubated for 30 min in dark then absorbance was measured using UV-VIS spectronic at a wavelength 320 nm (He 2003).

Total volume=1ml, time =30min, enzyme volume 0.1ml, €=2.19×10 3

Screening of ferulic acid

Following Sharma et al., (2020) [31] , Successive steps were carried out for determination of ferulic acid released from wheat bran by bacterial fermentation. Initially, wheat bran was washed using distelled water several time then, was spread in a thin layer and left it for air-drying. Dried wheat bran was ground to fine particles and used as a precursor and a medium for bacterial isolates. Medium preparation was done at a ratio 2:10 using distilled water in which 4 gram was weighted and mixed with 20 ml disilled water, then, sterilized using autoclave for 15 min. After cooling each medium was inoculated with 1ml of bacterial culture equivalent to 1× 10 8 and incubated at 37-39°C for 72 h with moderate agitation 50 rpm. Cultures were centrifuged at 10000rpm for 10 min, supernatant was applied as a source to estimate ferulic acid produced by bacterial esterase. Each extract was measured at a wavelength 320 nm to determine ferulic acid.

Using the equation for quantifying ferulic acid in gram of wheat bran:

A= absorbance, V total volume, D= dilution factor, W= weight of wheat bran For ferulic concentration mg/L using equation:

Concentration mg/L= A×12.14 Complete evaluation of degradation and hydrolysis of roughages (wheat bran) by measuring the reducing sugar was applied by dinitrosalycilic acid method (DNS method). Briefly, 0.2 of cell free extract of fermented wheat bran was mixed with 0.3ml of 3, 5 dinitrosalicylic acid reagent (DNS reagent), boiling for 10 min in a water bath, then reducing sugar was estimated by spectrophotometer at 540 nm against reagent blank (Miller, 1972) [24] . Standard curve of Glucose was used to determine the concentration of released reducing sugars. Positive result indicates by increasing the concentration of reducing sugar through hydrolysis of fiber by bacterial enzyme beside ferulic acid.

Statistical Analysis:

The statistical analysis system genestat program was used.

Duncan range tests were used to significant compare between means in this study.

Statistical model: Yijk = μ + Ai+ eijk.

Results and Discussion:

Results indicated differences in the facultative anaerobes among samples of cows, buffalos and lamb. Table (2) presented the total count of each group of bacteria in corresponding to their selective agar. Mean value of total count was approximately equal among the samples. Gram negative bacteria grew on MacConkey were nearly identical in cows and lamb and they were non-lactose ferment while buffalo samples showed more gram-negative bacteria with lactose ferment and non-ferment. Pseudomonales bacteria not exceeded 100 CFU/ml in sample of buffalo and cattle and around 1000 CFU/ml in lamb and were non-pigment producers. Agreement with other studies, variations in bacterial counts among these three ruminants can be attributed to differences in anatomical physiology, dietary fiber content, and other management conditions. However, diet composition is the most influential factor in altering the ruminal microbial population (Al-Dahami, 2019; Al-Rubaiee, & Al-Zubaidy.2021) [2,3] . [30,27] . Almost, Pseudomonas species are either obligating aerobes or microaerophiles that using oxygen as a final electron accepter (Arai, 2011; Deusch et al., 2017) [4,14] .

On the recent study, isolated colonies of Streptococcus picked from Pfizer agar and streaked over MacConkey agar, all bacterial isolate from the cows, lambs and buffaloes exhibited characteristic growth, indicating their ability to grow in the presence of bile salts. A highly desirable characteristic in ruminant feed manufacturing application. Iraqi investigations highlighted the functional importance of bile salt resistance for lactic acid bacteria in their fermentation process through degradation of bile salt and diminished its bactericidal activity (Abdulhussien & Isa, 2025; Othman & Karim, 2023) [1,26] .

Recent results indicated the differences among isolates in their fermentation efficiency. Blue color of methylene blue changed to luco-color at different time interval and the scores listed in table (3). The score 4 recognized for growth culture of Streptococcus spp isolated from Lamb and cattle followed by score 3 by Pseudomonas from lamb. Score 2 by gram negative bacteria isolated from cattle sample. We chose seven isolated bacterial species due to their metabolic activity for further analysis. 4). This result of the analysis of variance (ANOVA) noticed highly significant differences (p < 0.005) in esterase enzyme activity between the different bacterial isolates that isolated from different animal rumen.

The bacterial isolate (lamb Streptococcus) isolated from rumen of lamb was the highest average enzyme activity with level 117.67 U/ml (2.52 U/ml) comparing to all other isolates.

While the buffalo isolate (Gram-Negative) came next with an activity level 100.57 U/ml (1.08 U/ml). Table showed there is no significant differences were found between the cattle isolates (Cattle Gram-Negative) and gram positive Streptococcus spp it was recorded similar activity levels (72.83 U/ml and 72.63 U/ml, respectively). Furthermore, the (Buffalo-Gram-Positive) isolate was the lowest esterase enzyme activity level among all measured isolates with rate reaching 45.70U/ml and 0.36units/ml. In the present study, quantitative assessment of esterase activity showed that the isolate Streptococcus sp from lamb rumen exhibited a significantly higher esterase productivity compared to other bacterial isolates under identical cultural conditions. This could attribute to the rapid growth kinetic, highly protein secretion and metabolic adaptability characteristic of Streptococcus species found in ruminal environment (Wong, 2006; Dilokpimol et al., 2016) [38,15] . On the other hand, Streptococcus belonging to the ruminal lactic acid bacterial consortium that possess gene clusters responsible for carbohydrate esterase enzyme, facilitating lignocellulose degradation (Crepin et al., 2004) [12] . These finding strongly accepted by Zhang et al 2025, when screened whole genome sequence and catalytic profiling that lamb derived streptococcus isolates express noticeably elevated esterase enzymes relative to other microflora. Figure (4) demonstrated significant variability's among the bacterial isolates from cows, buffaloes and lamb regarding their capability to degrade lignocellulosic material. Ferulic acid and increased in the concentration of reducing sugar reflected the degradation of wheat bran by esterase enzyme Morgavi et al., 2013 [25] . Ferulic acid resemble the primary chemical bridge cross linking lignin to hemicellulose via ester bonds thus presenting a structural barrier that restrict the activity of cellulolytic enzyme (Morgavi et al., 2013) [25] . Results under study showed that gram positive; Streptococcus isolated from cattle rumen exhibited the highest production of ferulic acid followed by gram positive2 from buffalo with activity reached to 138 µg/ml after 72h and 100.5 µg/ml at 144 h. The significant increase in ferulic acid indicated a high production of feruloyl esterace by gram positive isolates. The enzymatic activity to cleave ester bond weakened the structural integrity of cell wall and enhanced synergistic attack of cellulose and xylanase to digest lignocellulosic matrix coupled with high sugar released exceeding 1200 µg/ml finding agree with Paul et al., 2004 and Dilokpimol et al., 2016 [28,15] . The analysis of enzymatic kinetics in fermentation process between 72h and 144h revealed a progressive synergistic activity between ferulyte esterase in response to ferulic acid concentration and the increased in reducing sugar quantity among all isolates. Linear regression analysis revealed a positive correlation between ferulic acid concentration and sugar released among the evaluated bacterial isolate after 144 h of incubation (figure 5). As reflected by upward of the regression line, increased ferulic acid levels generally corresponded to higher sugar accumulation in the medium. However, metabolic efficiency varied significantly among isolates. Gram negative isolate from buffalo exhibited low sugar concentration less than 200 µg/ml with lower ferulic acid 64 µg/ml. Other isolates, Pseudomonas and Streptococcus demonstrated high metabolic activity, achieving peak sugar concentration between 1200 and 1500 µg/ml. on the other hand, the width of 95% confidence interval at elevated ferulic acid concentration more than 100 µg/ml indicated greater variability in metabolic pathways under higher substrate availability. Our results suggested that hydrolysis of lignocelluosic fiber generated by ferulyte esterase was bacterial isolate dependent and accordance with Varel & Jung, 1986. [12] .

Conclusion:

In conclusion, this study demonstrated that indigenous ruminal facultative eanaerobic microflora specifically Streptococcus and Pseudomonas isolates grew from cattle and lamb possessed high fibrolytic and esterase producing capacities. The functional activity of these isolates successfully disrupted the lignocellulosic matrix of wheat bran, facilitating release of ferulic acid and significantly increased hydrolysis of complex carbohydrates into reducing sugars.

Remarkably, Streptococcus sp isolated from lamb exhibited the highest enzymatic efficiency in both total esterase activity and reducing sugar liberation. These finding highlighted the potential of Streptococcus isolate as bio-safe bio-inoculant and feed additives to overcome low quality of roughages and enhancing rumin performance and livestock productivity.

Recommendations:

 Genotypic identification and safety assessment should performed using 16rRNA and blood hemolysis test for bio-safety status.  Studying enzyme kinetic as well as specific activity towards various lignocellulosic substrates.  Optimize the condition of solid state fermentation using statistical experimental design and laboratory experiments to scale up enzymes production using low cost agricultural residues.  Investigate the synergistic effects between different isolates to establish multi enzyme complexes.  Perform in vitro gas production assay and digestion trial in lamb and cattle to assess the actual nutrient digestibility, volatile fatty acids profile and animal performance.

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