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標題: 脈衝照射之舞菇黃豆對骨質疏鬆症之影響及胚芽乳酸桿菌發酵菇類多醣產物之抗發炎活性
Effect of pulsed light irradiated Grifola frondosa fermented-soybean on osteoporosis and anti-inflammatory activity of Lactobacillus plantarum fermented-fungal polysaccharide products
作者: 郭芷瑜
Chih-Yu Kuo
關鍵字: 骨質疏鬆症;維生素 D2;脈衝光照射;舞菇;黃豆;食用菇;多醣;胚芽乳酸桿菌;小鼠巨噬細胞Raw 264.7;發炎;Osteoporosis;Vitamin D2;Grifola fiondosa;Soybeans;Edible mushroom;polysaccharide;Lactobacillus plantarum;macrophage raw 264.7;inflammation
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在全球人口結構逐漸老化的社會,老化所引起之骨質疏鬆症 (osteoporosis) 已經成為日益嚴重的公共衛生議題。骨質疏鬆症是一種因骨密度降低、骨組織破壞與骨脆度增加的疾病,因老化使鈣質過度流失是主要病因,會使骨質健康下降,而具有骨折的危險。而維生素D有助於血鈣與血磷的平衡,且為鈣質吸收的調控因子,因此與骨質疏鬆症關聯性密不可分。

黃豆中富含植物雌激素之一的異黃酮,大豆異黃酮可以與雌激素受體結合,進而達到預防心臟病、癌症與骨質疏鬆症的功能。菇類所含有之麥角固醇為維生素 D2 之前驅物,受到陽光中紫外線照射會轉化為維生素 D2,而經由脈衝光照射可以有效率的提升菇類維生素D2的含量。

以脈衝光照射舞菇黃豆製備出含有高含量維生素D2 (2.74 μg/g )之發酵舞菇黃豆 (陳,2015) ,並且以卵巢切除之C57BL/6J 品系鼠作為體內試驗模式,模擬並評估脈衝光照射舞菇黃豆對停經後骨質疏鬆症之影響。藉由病理切片分析脈衝舞菇黃豆是否對實驗動物具有毒性,並且透過血清中骨質指標、微電腦斷層影像測定股骨之骨質參數進行分析比較。

實驗分為七組,包括:Control (偽手術+未餵食任何樣品) 、Negative Control (卵巢切除術+未餵食任何樣品) 、NGFS 5X (卵巢切除術+五倍劑量之未照射脈衝照射舞菇黃豆) 、IGFS 0.5X (卵巢切除術+0.5倍之照射脈衝舞菇黃豆) 、IGFS 1X (卵巢切除術+1倍之照射脈衝舞菇黃豆) 、IGFS 5X (卵巢切除術+5倍之照射脈衝舞菇黃豆) 與ISO (卵巢切除術+大豆異黃酮標準品) ,投以樣品28周後,犧牲小鼠並分析其血清生化值與股骨參數。

結果顯示,在病理切片中實驗動物顯示出一些老化常見之疾病,發生頻率與實驗組別無關,亦與實驗樣品劑量無關,因此判定病變發生與樣品無關,且對實驗動物不會造成生理毒性。血清中骨質指標顯示在負控制組中成骨指標的procollagen I C-terminal propeptide (PICP)會顯著下降 (16.46 pg/mL) ,且投以IGFS組別會因所投之劑量高低而有顯著性差異,IGFS 5X具有最高之PICP含量 (110.35 pg/mL) ,顯示IGFS具有提升成骨作用的效果。而在股骨參數中,發現負控制組骨小樑會參數較控制組傾向骨質疏鬆的情況,而IGFS 5X在相對骨體積比值 (BV/TV) 、骨骼表面積與骨骼體積比值 (BS/BV) 、骨表面積與組織體積 (BS/TV) 、骨小樑厚度 (Tb.Th) 、骨小樑間分離度 (Tb.Sp) 、骨小樑數量 (Tb.N) 、骨小樑模型因子 (Tb.Pf) 等數值中,都具有傾向對骨質健康的數值。

總結本實驗之結果及先前之研究,顯示脈衝光照射舞菇黃豆是安全無毒性的,可作為良好的維生素 D 補充來源,且經過投食脈衝光照射舞菇黃豆動物模式中發現可以增強成骨細胞活性而增進骨質強度。未來可以進一步研究以顯示對人類預防骨質疏鬆症的功效。

關鍵字: 骨質疏鬆症、維生素 D2、脈衝光照射、舞菇、黃豆

食用菇子實體其色香味美又含有多種生理活性物質,具有食用與營養價值,廣受人們喜愛,富含蛋白質、礦物質與多醣等營養物質,其中多醣有許多生理活性功能,例如免疫調節、降血糖、降血脂、抗氧化、抗腫瘤等功能。益生菌定義為對宿主體內有正面效果之活菌成份,益生質為無法被宿主可消化但可以選擇性促進腸道中微生物生長,而益生菌具有誘導免疫反應、影響腸道菌相、抗菌活性、降血脂等功能。胚芽乳酸桿菌 (Latobacillus plantarum) 為一種益生菌,可以對宿主體內具有正面效果,可以具有調節免疫的功能,而益生質則為無法被宿主可消化但可以選擇性促進腸道中益生菌生長,為一種健康補充品。

因此本實驗利用食用菇類包含日常較易取得的銀耳與金針菇,以及較高單價之巴西蘑菇與猴頭菇四種食用菇類,萃取菇類多醣,並且評估多醣作為益生質的潛力,並且以乳酸菌菇類多醣發酵除菌液以及熱失活發酵破菌液,探討發酵液對於小鼠巨噬細胞Raw 264.7之發炎反應。

首先進行菌種鑑定,確定實驗用之子實體菌種,隨後對於熱水萃取食用菇類多醣進行成分分析,分別測得銀耳多醣(Tremella fuciformi polysaccharides, TFP)、金針菇多醣(Flammulina velutipes polysaccharides, FVP)、巴西蘑菇多醣(Agaricus blazei polysaccharides, ABP)與猴頭菇多醣(Hericium erinaceu polysaccharides, HEP)中的還原糖含量為3.53%、3.65%、4.48%與4.22%;蛋白質含量分別為2.20%、4.98%、15.40%與15.6%1及醣醛酸分別為15.47%、2.51%、4.83%與3.80%。

益生質評估,將Lactobacillus plantarum接種於不含醣類培養基、含0.5% TFP、0.5% FVP、0.5% ABP與0.5% HEP中,計算菌數的結果發現添加0.5% HEP生長情況最佳,能夠使菌數在培養12小時後,得到10 log CFU/mL的菌數。而在耐酸性及耐鹼性試驗中,四種菇類多醣添加並不會提升存活率,但菌數仍然維持8 log CFU/mL以上;耐鹼性試驗中,添加0.5% HEP的組別會在2小時後,有最低菌數,其他組別不具有顯著差異。L. plantarum在培養於含不同菇類多醣培養基中24小時後,發酵液經過處理得到除菌發酵液與破菌熱失活發酵液,目的為探討乳酸菌菌體是否會影響免疫反應。

將發酵液進行發炎評估,在不會對小鼠巨噬細胞Raw 264.7產生細胞毒性的情況下,除菌發酵液與破菌熱失活發酵液皆可以促進巨噬細胞產生NO、IL-1β、IL-6、TNF-α與IL-10,隨著樣品濃度增加,會使細胞激素分泌上升。以 (IL-1β+ IL-6+ TNF-α) / (IL-10) 比值評估整體細胞激素分泌之狀況,破菌熱失活發酵液會較除菌發酵液具有更顯著之促發炎及抗發炎效果,但銀耳多醣破菌熱失活發酵液 (Tremella fuciformi polysaccharides fermentation heat-inactivated culture TFH) 與其他食用菇破菌熱失活發酵液相比不會顯著促發炎或是抗發炎。

綜合以上實驗與前人的研究結果,推論HEP可以對於L. plantarum生長有提升的影響,但其在耐膽鹽試驗中會降低其生存率。四種食用菇類多醣除菌發酵液皆具有調控發炎反應之能力,破菌熱失活發酵液,在金針菇多醣破菌熱失活發酵液、巴西蘑菇熱失活發酵液與猴頭菇熱失活發酵液中有顯著促發炎及抗發炎效果,而TFH在低濃度與其他組別相比有抗發炎效果,高濃度中則有促發炎效果。未來可以深入探討在發酵液中的成分,是否確實調控細胞激素生成路經。

關鍵字:食用菇、多醣、胚芽乳酸桿菌、小鼠巨噬細胞Raw 264.7、發炎
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