Bacillus licheniformis CK1降低玉米烯酮毒素之評估

Abstract

玉米烯酮為黴菌所產生之次級代謝物，為鐮菌屬真菌所產生之黴菌毒素，常見於玉米、小麥、大麥等穀物中。玉米烯酮可能對家畜造成不孕、流產等繁殖性能上的影響，亦具有致癌性、基因毒性、血液毒性或免疫毒性等，對家畜或人體健康造成威脅。研究顯示Bacillus licheniformis CK1在in vitro試驗中可降低培養液中玉米烯酮含量，其機制可能為降解作用及吸附作用。本研究探討B. licheniformis CK1與玉米烯酮吸附作用之成分與效果，及是否可於體內降低玉米烯酮之毒性。 本研究首先分析活菌、熱致死及酸致死形式之B. licheniformis CK1吸附玉米烯酮之能力，結果顯示，熱致死及酸致死菌之吸附效果較活菌好，而活菌及熱致死菌在高濃度玉米烯酮（50 ppm）下之吸附作用趨於飽和。為了解菌體成分與玉米烯酮吸附之相關性，B. licheniformis CK1以不同化學試劑或酵素進行前處理，再分析其吸附效果變化。結果顯示，以pronase E處理後，酸致死之B. licheniformis CK1吸附玉米烯酮的濃度顯著降低，以8 M尿素處理後，活菌及熱致菌體吸附之濃度亦顯著降低，推測B. licheniformis CK1與玉米烯酮吸附之成分主要為細胞壁表面之蛋白質。此外，不同金屬離子強度（125-1000 mM的Na+或Ca2+）或不同pH（pH 1.5-8.5）之環境，皆不影響其吸附效果，推測其吸附機制與靜電作用或氫鍵的形成無關，並且於胃腸道中各階段皆可進行吸附作用。 進一步以六週齡雌性BALB/c小鼠進行試驗，結果顯示，以口服方式連續28天每天給予小鼠玉米烯酮40 mg/kg BW，造成小鼠肝臟相對重量有下降的趨勢，並使血清丙胺酸轉胺酶（ALT）含量上升及血液尿素氮（BUN）含量下降，肝臟抗氧化酵素過氧化氫酶（CAT）活性亦有下降之趨勢，B. licheniformis CK1的餵予則可降低這些毒性影響；然而，licheniformis CK1的餵予無法改善玉米烯酮造成的血清總免疫球蛋白G（IgG）及細胞激素-1β（IL-1β）含量之變化。 綜上所述，B. licheniformis CK1具有良好的吸附玉米烯酮能力，於不同環境下其吸附效果仍相當穩定，推測其細胞壁表面與玉米烯酮吸附之成分主要為蛋白質，其詳細作用機制仍需進一步研究。而以口服方式餵予B. licheniformis CK1可部分降低玉米烯酮對小鼠之毒性影響，待更多研究以確認其於體內去除玉米烯酮毒性之效果。

Zearalenone (ZEN), one kind of mycotoxins, is a phenolic resorcylic acid lactone produced by certain species of Fusarium that infects cereals and grains, such as corn, oats, wheat and hey. ZEN can induce infertility, abortion or other breeding problems in livestock. Furthermore, it is carcinogenic, genotoxic, hepatotoxic and immunotoxic in both livestock and humans. Bacillus licheniformis CK1 was found to be capable to decrease ZEN residue in vitro, and the mechanisms might be both adsorption and degradation. This study is aimed at investigation of the binding interaction between B. licheniformis CK1 and ZEN and evaluating the efficacy of detoxification of ZEN by B. licheniformis CK1 in BALB/c mice. First, the extent of ZEN binding over the range of 5-50 ppm (37℃, 30 min) was studied for viable, heat- and acid-killed B. licheniformis CK1. Both heat- and acid-killed bacteria showed greater ZEN-binding ability than viable bacteria; the absorption of ZEN by viable and heat-killed bacteria seemed to be saturated at higher initial ZEN concentration. To identify the type of chemical moieties and interactions involved in binding with ZEN, B. licheniformis CK1 was subjected to different chemical and enzymatical treatments prior to the binding experiments. Pre-treating acid-killed B. licheniformis CK1 with pronase E significantly decreased ZEN bound concentration, and pre-treating viable and heat-killed bacteria with 8 M urea significantly decreased ZEN bound concentration, suggesting that ZEN binds predominantly to protein components. Besides, ionic strength (125-1000 mM of Ca2+ or Na+) and pH value (pH 1.5-8.5) did not affect the concentration of ZEN bound with B. licheniformis CK1, suggesting that electrostatic interactions and hydrogen bonding do have minor effects on binding and that bacterial binding of ZEN might occur at any point along the gastrointestinal tract. In animal trial, fifty 7-week old BALB/c mice were divided into five groups (10 mice/group) and orally administered 1) PBS; 2) olive oil; 3) 109 CFU/kg BW of B. licheniformis CK1 in PBS; 4) 40 mg/kg BW of ZEN in olive oil; 5) 40 mg/kg BW of ZEN in olive oil + 109 CFU/kg BW of B. licheniformis CK1 in PBS each day for 28 days. The mice were sacrificed at the end of experiment and the blood and organ samples were collected and analyzed. The results showed that, compared with control groups, liver relative weight tended to decrease in ZEN group but not in ZEN + B. licheniformis CK1 group. ZEN treatment resulted in a significant increase of alanine aminotransferase (ALT) level in plasma and a decrease of blood urea nitrogen (BUN) level. Moreover, ZEN treatment also caused a slight decrease of liver catalase (CAT) activity, indicating that the liver might be injured. Treatment with ZEN and B. licheniformis CK1 simultaneously seemed to restore these changes. However, treatment with ZEN and B. licheniformis CK1 simultaneously did not decrease the elevated level of plasma interleukin-1β (IL-1β) and increase the decreased level of total immunoglobulin G (IgG) induced by ZEN. In conclusion, the ZEN binding ability of B. licheniformis CK1 is stable in various conditions. The exact binding sites on the surface of B. licheniformis CK1 might be protein components. Oral administration of B. licheniformis CK1 could partially ameliorate the harmful effect of ZEN in mice. More studies needs to be investigated to confirm the protective effects of B. licheniformis CK1 on ZEN toxicity in vivo.