Characterization of Condensed Tannins Purified From Legume Forages: Chromophore Production,Protein Precipitation,and Inhibitory Effects on Cellulose Digestion

To identify simple screening tools for selecting condensed tannin (CT)-containing forages as candidate sources for further study, CT were isolated from nine legumes, and their molecular weights (MW), chromophore production, capacity to precipitate bovine serum albumin (BSA) and Fraction 1 protein (Rubisco) isolated from alfalfa, and inhibition of filter paper digestion were compared. Sources were as follows: leaves of sericea lespedeza (Lespedeza cuneata Dum.-Cours.), crown vetch (Coronilla varia L.), and sainfoin (Onobrychis viciifolia Scop.); stems of hedysarum (Hedysarum alpinum L.); seeds of alfalfa (Medicago sativa L.); and whole plants of birdsfoot trefoil (Lotus corniculatus var. corniculatus L.) and three varieties of big trefoil (Lotus pedunculatus Cav.), viz., Lotus uliginosus Schkuhr, L. uliginosus var. glabriusculus, and L. uliginosus var. villosus. Molecular weights and sizes (degrees of polymerization) of the CT varied considerably within and among plant species. Average MW ranged from 3036 Da (crown vetch) to 7143 Da (lespedeza). All CT exhibited greater capacity (w/w basis) to bind alfalfa Rubisco than BSA. Relative astringencies (μg CT required to precipitate 1 mg protein) against BSA ranged from 262.5 for CT from lespedeza to 435.5 for CT from L. corniculatus, and against Rubisco, from 49.6 (sainfoin) to 108.2 (alfalfa seed). Including CT at 300 μg/ml in cultures of Fibrobacter succinogenes reduced digestion of cellulose filter paper by 19.8% (sainfoin) to 92.4% (crown vetch) and increased the specific activity of cell-associated endoglucanase. There were no correlations between inhibitory effects of CT on filter paper digestion and (1) chromophore formation during CT assay by butanol–HCl, vanillin–HCl, or H₂SO₄; (2) precipitation of BSA or alfalfa Rubisco; and (3) MW of CT. The most inhibitory CT for cellulose digestion included those with broad and with narrow MW distributions. Sainfoin was the most desirable source of CT, as it had the highest capacity to bind alfalfa protein and was least inhibitory to cellulose digestion by F. succinogenes. This study suggests that these properties are not easily defined via chemical means, and that biological assays using rumen bacteria may help identify those CT with properties of nutritional interest.     

Actinobacillus succinogenes NJ113厌氧发酵产丁二酸培养条件的优化

采用Plackett-Burman设计法(Plackett-Burman,PB),对影响Actinobacillus succinogenes NJ113厌氧发酵生产丁二酸的11个因子进行了筛选。结果表明,影响该菌厌氧发酵产丁二酸的主要因子为葡萄糖、酵母膏、玉米浆。在此基础上,采用响应曲面法(Response Surface Methodology,RSM)对这3个因子的影响进行了研究,得出丁二酸产量的数学模型,通过对二次多项回归方程求解,得到3因子的最优用量:葡萄糖107g/L,酵母膏16 g/L,玉米浆12 g/L,在优化条件下培养48 h,丁二酸的产量由原始培养条件下的62g/L增到84 g/L,收率从62%提高到78.5%,生产强度达1.75 g/(L·h)。     

Simultaneous refolding,purification, and immobilization of recombinant Fibrobacter succinogenes 1,3‐1,4‐β‐D‐glucanase on artificial oil bodies

BACKGROUND: 1,3‐1,4‐β‐D‐glucanase (1,3‐1,4‐β‐D‐glucan 4‐glucanohydrolase; EC 3.2.1.73) has been used in a range of industrial processes. As a biocatalyst, it is better to use immobilized enzymes than free enzymes, therefore, the immobilization of 1,3‐1,4‐β‐D‐glucanase was investigated. RESULTS: A 1,3‐1,4‐β‐D‐glucanase gene from Fibrobacter succinogenes was overexpressed in Escherichia coli as a recombinant protein fused to the N terminus of oleosin, a unique structural protein of seed oil bodies. With the reconstitution of the artificial oil bodies (AOBs), refolding, purification, and immobilization of active 1,3‐1,4‐β‐D‐glucanase was accomplished simultaneously. Response surface modeling (RSM), with central composite design (CCD), and regression analysis were successfully applied to determine the optimal temperature and pH conditions of the AOB‐immobilized 1,3‐1,4‐β‐D‐glucanase. The optimal conditions for the highest immobilized 1,3‐1,4‐β‐D‐glucanase activity (7.1 IU mg^?1 of total protein) were observed at 39 °C and pH 8.8. Furthermore, AOB‐immobilized 1,3‐1,4‐β‐D‐glucanase retained more than 70% of its initial activity after 120 min at 39 °C, and it was easily and simply recovered from the surface of the solution by brief centrifugation; it could be reused eight times while retaining more than 80% of its activity. CONCLUSIONS: These results indicate that the AOB‐based system is a comparatively simple and effective method for simultaneous refolding, purification, and immobilization of 1,3‐1,4‐β‐D‐glucanase. Copyright © 2009 Society of Chemical Industry