The effects of Pinus brutia bark extract on pure and mixed continuous cultures of rumen bacteria and archaea, and fermentation characteristics in vitro

Abstract

The aim of the study was to investigate the effects of Pinus brutia bark extract, which is rich in polyphenolic compounds of tannins, on both pure and mixed continuous cultures of rumen bacteria and archaea, as well as on rumen fermentation characteristics in vitro. Antimicrobial susceptibility assay with pure cultures was carried out in an anaerobic chamber. Pinus brutia bark extract exhibited a potential inhibitor activity (P<0.05) against pure cultures of Ruminococcus flavefaciens, Eubacterium ruminantium, and Methanobacterium formicicum while a growth stimulatory effect (P<0.05) was observed for Ruminoccocus albus, Butyrivibrio fibrisolvens, and Streptococcus bovis. Pinus brutia bark extract only had a potential inhibitor effect (P<0.05) on R. albus at the highest dose (1200 µg/mL). Pinus brutia bark extract also stimulated (P<0.05) the growth of pure cultures of Fibrobacter succinogenes, while it did not affect Megasphaera elsdenii, except at the highest dose. The effects of two doses (75 and 375 mg/L) of P. brutia bark extract on in vitro mixed cultures and rumen fermentation parameters were determined by the rumen simulation technique (Rusitec). Supplementation with P. brutia bark extract led to a quadratic decrease (P<0.05) in the cell numbers of R. flavefaciens. Production of total and individual short chain fatty acids (SCFA), acetate to propionate ratio (C2/C3), total protozoa, ruminal pH, and dry matter digestibility (DMD) did not change in the presence of P. brutia bark extract. Supplementation with both doses of P. brutia bark extract decreased (P<0.05) the ammonia-N concentrations. Ammonia-N concentration was lowest in the high-supplemented group (P<0.05). As a conclusion, inhibitory effects of P. brutia bark extract on some species in the pure cultures were in the same direction as with mixed ruminal cultures, while stimulatory effects disappeared. The lack of inhibitory effects on protozoa and on a large number of Gram-positive rumen bacteria in the mixed cultures suggests that its mechanism of action is not exactly similar to antibiotics. Although P. brutia bark extract did not alter ruminal SCFA, it could have potential to improve ruminal protein utilization without depressing rumen microbial fermentation.

Preliminary study of the effects of <i>Apilactobacillus kunkeei</i> EIR/BG-1 and <i>Enterococcus hirae</i> EIR/CM-2 supplementation on<i> in vitro</i> rumen fermentation and microbial population

“…Five millilitres of samples collected for each analysis were stored at −20 °C until measurements. Fluid samples for DNA extraction were collected daily from the fermenters during the feed bag exchange, thus they contained both planktonic and solid-phase-associated microorganisms (Demirtas et al, 2021). The collected samples were immediately transferred to liquid nitrogen and stored at −20 °C until DNA extraction.…”

“…Subsequently, the feed bags were dried at 65 °C for 48 h. To calculate the dry matter digestibility (DMD) after 48 h, the difference between the weight of the original dry matter (DM) sample and the weight of the DM residue was divided by the original sample weight. Then, this value was multiplied by 100 to obtain the percentage DMD (Demirtas et al, 2021).…”

“…From the pooled sample, 4-ml aliquots were used for DNA extraction. After thawing, the samples were centrifuged at 10000 rpm for 10 min at room temperature, and the resulting pellets were washed twice with phosphatebuffered saline (Demirtas et al, 2021). Total DNA was extracted from the pellets using the E.Z.N.A.™ stool DNA isolation kit (Omega Bio-tek, Norcross, GA, USA).…”

Development of bioactive films loaded with extract and polysaccharide of Pinus brutia bark

“…For example, pine fractions have been reported to be effective in treating gastrointestinal diseases and neuronal problems. At the same time, findings have been found to prevent chronic diseases, such as hypertension, arteriosclerosis, and diabetes (Demirtas et al, 2021). P. brutia bark (PBB) is abundant in bioactive compounds such as phenolic compounds (flavonoids and phenolic acids), mainly tannins, and antimicrobial compounds.…”