Utilization of a novel feedstuff: effects of additives on the fermentation characteristics, chemical composition and in vitro digestibility of tetraploid black locust (Robinia pseudoacacia) silage / Utilização de um novo alimento: efeitos de aditivos nas características de fermentação, composição química e digestibilidade in vitro da silagem de gafanhoto negro tetraplóide (Robinia pseudoacacia)

ABSTRACT: This study was conducted to evaluate the effects of additives on the fermentation characteristics, chemical composition and in vitro digestibility of tetraploid black locust (TBL). The TBL leaves silage was either untreated (control) or treated with 1 × 106 cfu/g FM Lactobacillus plantarum (L), 1% glucose (G), 3% molasses (M), a combination of 1% glucose and Lactobacillus plantarum (L+G), or a combination of 3% molasses and Lactobacillus plantarum (L+M). Fermentation quality, chemical composition and nutrient digestibility were then analyzed. Ethanol and acetic acid concentrations were the dominant fermentation products in all silages except L+M silage. The L, G and L+G treatments failed to influence the fermentation. The M treatment increased (P<0.05) the lactic acid concentration and lowered (P<0.05) the pH when compared with control silage. The best fermentation properties were observed in L+M silage, as indicated by the dominance of lactic acid over ethanol in fermentation products. The M and L+M silages exhibited higher (P<0.05) dry matter, and M silage showed higher residual water-soluble carbohydrates than the control. Ensiling increased (P<0.05) the in vitro dry matter, neutral detergent fiber and acid detergent fiber degradability of TBL. Among the silages, M silage had the highest levels of dry matter, neutral detergent fiber and acid detergent fiber degradability. The obtained results suggested that application of lactic acid bacteria together with 3% molasses could be an effective strategy to prevent the occurrence of ethanol fermentation and improve fermentation quality of TBL silage; addition of fermentable sugars to TBL improves nutrient availability to ruminants.

RESUMO: Este estudo foi conduzido para avaliar os efeitos de aditivos nas características de fermentação, composição química e digestibilidade in vitro do gafanhoto preto tetraplóide (TBL). A silagem de folhas TBL não foi tratada (controle) ou foi tratada com 1 × 106 ufc / g FM Lactobacillus plantarum (L), 1% glicose (G), 3% melaço (M), uma combinação de 1% glicose e Lactobacillus plantarum ( L + G), ou uma combinação de 3% de melaço e Lactobacillus plantarum (L + M). A qualidade da fermentação, a composição química e a digestibilidade dos nutrientes foram analisadas. As concentrações de etanol e ácido acético foram os produtos de fermentação dominantes em todas as silagens, com exceção da silagem L + M. Os tratamentos L, G e L + G não influenciaram na fermentação. O tratamento com M aumentou (P<0,05) a concentração de ácido láctico e diminuiu (P<0,05) o pH, quando comparado com a silagem controle. As melhores propriedades de fermentação foram observadas na silagem L + M, como indicado pela dominância do ácido lático sobre o etanol nos produtos de fermentação. As silagens M e L + M apresentaram maior teor de matéria seca (P<0,05), e a silagem M apresentou maior carboidrato solúvel em água residual que o controle. A ensilagem aumentou (P<0,05) a matéria seca in vitro, a fibra em detergente neutro e a degradabilidade da fibra em detergente ácido de TBL. Entre as silagens, a silagem M apresentou os maiores teores de matéria seca, fibra em detergente neutro e degradabilidade da fibra em detergente ácido. Os resultados obtidos sugerem que a aplicação de bactérias lácticas em conjunto com 3% de melaço pode ser uma estratégia eficaz para evitar a ocorrência de fermentação alcoólica e melhorar a qualidade da fermentação da silagem TBL. A adição de açúcares fermentáveis à TBL aumenta a disponibilidade de nutrientes para ruminantes.

Efficient expression and characterization of a cold-active endo-1, 4-β -glucanase from Citrobacter farmeri by co-expression of Myxococcus xanthus protein S

Background: Cold-active endo-1, 4-β-glucanase (EglC) can decrease energy costs and prevent product denaturation in biotechnological processes. However, the nature EglC from C. farmeri A1 showed very low activity (800 U/L). In an attempt to increase its expression level, C. farmeri EglC was expressed in Escherichia coli as an N-terminal fusion to protein S (ProS) from Myxococcus xanthus. Results: A novel expression vector, pET(ProS-EglC), was successfully constructed for the expression of C. farmeri EglC in E. coli. SDS-PAGE showed that the recombinant protein (ProS-EglC) was approximately 60 kDa. The activity of ProS-EglC was 12,400 U/L, which was considerably higher than that of the nature EglC (800 U/L). ProS-EglC was active at pH 6.5-pH 8.0, with optimum activity at pH 7.0. The recombinant protein was stable at pH 3.5-pH 6.5 for 30 min. The optimal temperature for activity of ProS-EglC was 30°C-40°C. It showed greater than 50% of maximum activity even at 5°C, indicating that the ProS-EglC is a cold-active enzyme. Its activity was increased by Co2+ and Fe2+, but decreased by Cd2+, Zn2+, Li+, methanol, Triton-X-100, acetonitrile, Tween 80, and SDS. Conclusions: The ProS-EglC is promising in application of various biotechnological processes because of its cold-active characterizations. This study also suggests a useful strategy for the expression of foreign proteins in E. coli using a ProS tag.