TW202300161A - 植物乳桿菌hg-23菌株及其應用 - Google Patents
植物乳桿菌hg-23菌株及其應用 Download PDFInfo
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Abstract
本發明涉及一種植物乳桿菌HG-23菌株及其應用,所述植物乳桿菌HG-23已於2021年4月6日保藏於中國典型培養物保藏中心,其保藏號為CCTCC No: M2021330。本發明的植物乳桿菌HG-23可用於製備降低血脂的食品或降低血脂的藥物。本發明篩選所得植物乳桿菌HG-23安全性較好,可以耐受胃酸和膽鹽,存活和定殖於腸道內,調解改善宿主的腸道微生態平衡,發揮其有益作用,產生確切的健康功效;體外及動物試驗表明其具有較強降低血液中甘油三酯和膽固醇的功能。
Description
本發明屬於微生物技術領域,具體涉及一種植物乳桿菌HG-23菌株及其在調節血脂方面的應用。
高膽固醇血症也稱高低密度脂蛋白膽固醇血症,是導致動脈粥樣硬化性心血管病的首要危險因素。最新研究顯示,各種心血管病風險人群低密度脂蛋白每降低1mmol/L,主要血管事件可降低21%。據2013年的統計顯示,我國約有2億臨界高膽固醇血症人群以及8800萬高膽固醇血症患者。
目前主要採用藥物治療和飲食干預的方法來控制和降低血脂水平。臨床上使用較多的是他汀類和貝特類藥物,藥物初期使用療效較為顯著,但隨著長期服用會產生不同程度的副作用,加重患者的健康問題,如長期服用他汀類藥物容易誘發肌病、橫紋肌溶解、血糖異常、肝炎及腎功能障礙等,長期服用貝特類藥物會導致肝損傷嚴重,增加患膽結石的風險。低脂或低飽和脂肪食物的膳食能有效減少血清中膽固醇的含量,但低脂食物的口感和風味比較簡單,這個膳食建議仍未被大眾接受或長期堅持,稍不注意就容易反彈,因此,食用降血脂功能性食品是長期控制血脂更理想的方法。
目前,對於益生菌降血脂的研究已經持續將近半個世紀,國內針對降血脂功能研究比較深入的兩個菌株同屬於植物乳桿菌,分別為L.plantarum P8和植物乳桿菌ST-III。L.plantarum P8來自內蒙古農業大學的張和平領導的團隊,動物試驗表明,L.plantarum P8具有顯著的降血脂作用。曾任東北農業大學食品學院副院長,現任光明乳業股份有限公司總經理郭本恒領導的團隊和江南大學合作研發的植物乳桿菌ST-III,成功通過動物實驗和人群臨床試驗的證實其降膽固醇的效果,並已產業化,成為光明公司的一個具有自主知識產權的益生菌產品。
因此,開發出安全、有效,具有調節血脂作用的乳酸菌在發酵食品和醫藥領域具有廣泛的應用前景。
鑑於此,申請此專利。
有鑑於此,吾等發明人乃潛心進一步研究,並著手進行研發及改良,期以一較佳發明以解決上述問題,且在經過不斷試驗及修改後而有本發明之問世。
為了解決現有技術存在的問題,本發明提供了一種植物乳桿菌HG-23菌株及其應用,菌株可以耐受胃酸和膽鹽,存活和定殖於腸道內,調解改善宿主的腸道微生態平衡,調節血脂,具體表現為降低膽固醇和甘油三酯。
本發明的目的是提供一種植物乳桿菌HG-23。
本發明的另一目的是提供上述植物乳桿菌HG-23的應用。
根據本發明的具體實施方式的植物乳桿菌HG-23,所述植物乳桿菌HG-23的保藏號為CCTCC No: M2021330。
根據本發明的具體實施方式的植物乳桿菌HG-23在製備食品或藥物中的應用,所述食品或藥物為降低血脂的食品或降低血脂的藥物。
優選的,所述食品或藥物為降低膽固醇和甘油三酯的食品或藥物。
根據本發明的具體實施方式的植物乳桿菌HG-23在製備藥物中的應用,進一步的,所述藥物包含所述的植物乳桿菌HG-23;以及醫藥用無毒載體。
根據本發明的具體實施方式的植物乳桿菌HG-23在製備藥物中的應用,進一步的,所述藥物的劑型選自片劑、顆粒劑、散劑、膠囊劑、懸浮劑、乳劑、凍幹製劑的至少一種;優選的,所述藥物為複合益生菌片。
本發明所述的藥物中還包含藥學上可接受的輔料。所述藥學上可接受的輔料包括但不限於:藥物可接受的載體、稀釋劑、填充劑、結合劑及其它賦形劑。所述載體包括但不限於澱粉或其衍生物、乳糖、聚乙二醇;所述稀釋劑包括但不限於澱粉或其衍生物、乳糖、蔗糖、植物油、蠟、脂肪酸;填充劑包括但不限於滑石粉;所述輔料還可以包括如防腐劑、潤滑劑、分散劑、矯味劑、保濕劑、抗氧化劑、著色劑、穩定劑、緩衝劑、pH值調節劑。上述物質根據需要幫助配方的穩定性或有助於提高活性或生物有效性或在口服的情況下產生可接受的口感或氣味。
根據本發明的具體實施方式的植物乳桿菌HG-23在製備食品中的應用,進一步的,所述食品包含所述的植物乳桿菌HG-23,以及食品學上可接受的添加劑。
本發明中所使用的術語“食品”是指廣義理解,其可以是任何可被使用的形式,即除了常規的食品形式外,本發明的食品還可以為保健品、飲品、發酵食品等。
優選地,所述食品為益生菌固體粉劑。將在實施例中詳細闡明一種益生菌固體粉劑的配方。
根據本發明的具體實施方式的植物乳桿菌HG-23在製備食品中的應用,更進一步的,所述益生菌固體粉劑由低聚木糖、菊粉、低聚果糖、檸檬水果粉和植物乳桿菌HG-23製成。
根據本發明的具體實施方式的植物乳桿菌HG-23在製備食品或藥物中的應用,更進一步的,所述植物乳桿菌HG-23為凍乾粉;所述凍乾粉採用凍幹機將植物乳桿菌HG-23的菌懸液在無菌環境下冷凍真空乾燥而成。
與現有技術相比,本發明具有如下有益效果:
(1)本發明篩選所得植物乳桿菌HG-23可以耐受胃酸和膽鹽,存活和定殖於腸道內,調解改善宿主的腸道微生態平衡,發揮其有益作用,產生確切的健康功效;具體表現降低血液中甘油三酯和膽固醇。
(2)本發明篩選所得植物乳桿菌HG-23可用於製備降膽固醇及甘油三酯的食品及藥品,試驗表明,得到的食品也同樣具有降低膽固醇及甘油三酯功效。
關於吾等發明人之技術手段,茲舉數種較佳實施例配合圖式於下文進行詳細說明,俾供 鈞上深入瞭解並認同本發明。
為使本發明的目的、技術方案和優點更加清楚,下面將對本發明的技術方案進行詳細的描述。顯然,所描述的實施例僅僅是本發明一部分實施例,而不是全部的實施例。基於本發明中的實施例,本領域普通技術人員在沒有做出創造性勞動的前提下所得到的所有其它實施方式,都屬於本發明所保護的範圍。
本發明的植物乳桿菌HG-23是一種乳酸菌,能夠有效的降低膽固醇和甘油三酯, 可作為食品及藥物的成分,改善人體高血脂症狀。
本發明的新菌株植物乳桿菌HG-23(分類名稱:植物乳桿菌;拉丁名稱:Lactobacillus plantarum)已經於2021年4月6日在中國典型培養物保藏中心進行了保藏,保藏單位的地址為:中國.武漢.武漢大學,保藏在中國典型培養物保藏中心,保藏號為CCTCC No: M2021330;存活性由該保藏中心於2021年4月6日進行檢測,結果為:存活。
在一些較為具體的實施方案中,植物乳桿菌HG-23在製備食品或藥物中的應用,所述食品或藥物為降低膽固醇的食品或降低膽固醇的藥物。
進一步的,所述藥物包含所述的植物乳桿菌HG-23;以及醫藥用無毒載體。
優選的,所述藥物為複合益生菌片。
所述食品包含所述的植物乳桿菌HG-23,以及食品學上可接受的添加劑。
優選地,所述食品為益生菌固體粉劑。
更進一步的,所述益生菌固體粉劑由低聚木糖、菊粉、低聚果糖、檸檬水果粉和植物乳桿菌HG-23製成;所述植物乳桿菌HG-23為凍乾粉;所述凍乾粉採用凍幹機將植物乳桿菌HG-23的菌懸液在無菌環境下冷凍真空乾燥而成。
以下通過實施例並結合附圖進一步詳細說明本發明的技術方案。然而,所選的實施例僅用於說明本發明,而不限制本發明的範圍。
實施例
1
植物乳桿菌
HG-23
的分離鑑定
1.培養基配方優化
分組進行優化培養基實驗,最後得到乳酸菌培養基最佳配方:
MRS固體培養基1L配方:酪蛋白腖10.0g/L,牛肉浸膏10.0g/L,酵母浸膏5.0g/L,葡萄糖20.0g/L,磷酸氫二鉀2.0g/L,吐溫801.0g/L,檸檬酸三銨2.0g/L,醋酸鈉5.0g/L,硫酸鎂0.1g/L,硫酸錳0.05g/L,瓊脂17.5g;pH6.5,121℃滅菌20min。
MRS肉湯培養基配方:酪蛋白腖10.0g/L,牛肉浸膏10.0g/L,酵母浸膏5.0g/L,葡萄糖20.0g/L,磷酸氫二鉀2.0g/L,吐溫801.0g/L,檸檬酸三銨2.0g/L,醋酸鈉5.0g/L,硫酸鎂0.1g/L,硫酸錳0.05g/L,pH6.5,121℃滅菌20min。
2.菌株篩選
植物乳桿菌HG-23菌種來源於健康人腸道,從健康的成年人糞便中分離得到。取健康的成年人糞便,用無菌生理鹽水,10倍梯度稀釋樣品後至10-3倍,將其塗布於MRS固體培養基,置於37℃培養箱中培養24-48h,觀察菌落形態特徵,並挑取MRS固體培養基上乳酸菌的疑似菌落,分別轉接到MRS液體培養基中進行純培養,共分離出5株菌,對每株菌進行編號。
對分離純化得到的這若干株菌進行酸及膽鹽耐受力及降膽固醇能力的測定,詳細實驗步驟見實施例2和實施例3,從中篩選得到酸及膽鹽耐受力較強,降膽固醇能力較強的植物乳桿菌HG-23。
菌種鑑定
對篩選到的植物乳桿菌HG-23抽提細菌總DNA,進行16s rDNA擴增,引物為:
sgF:5'-AGAGTTTGATCATGGCTCAG-3'(SEQ ID NO:1)
sgR:5'-TAGGGTTACCTTGTTACGACTT-3'(SEQ ID NO:2)
利用上述引物進行PCR擴增和瓊脂糖凝膠電泳,然後切膠回收、測序。
分離獲得的菌株16s DNA測序結果如SEQ ID NO:3所示。
實施例
2
植物乳桿菌
HG-23
的體外膽固醇去除率試驗
按照以下步驟檢測菌株體外膽固醇去除率,以商業菌株植物乳桿菌299V作為實驗對照組,具體如下:
將供試菌株轉接於MRS液體培養基中,於37℃培養24h,以1%的接種量傳代2次後,於37℃培養15h,待用。配製液體MRS培養基,滅菌待用;
配製含有膽固醇的MRS培養基(培養液MRSO-CHOL):由MRS液體培養基、巰基乙酸鈉、膽鹽和膽固醇組成,其中,巰基乙酸鈉在培養液MRSO-CHOL中的濃度為2g/L,膽鹽在培養液MRSO-CHOL中的濃度為0.3%,膽固醇的濃度為120ug/ml。
實驗組:活化本發明的植物乳桿菌HG-23菌液按1%接種量接種於10ml培養液MRSO-CHOL中。
陽性對照組:活化商業菌株的菌液按1%接種量接種於10ml培養液MRSO-CHOL中。
空白對照組:10ml沒有接種菌株的培養液MRSO-CHOL。
將這兩組樣品放置於37℃恒溫培養箱培養24h後,取出搖勻,4000r/m離心15min,取上清液,用鄰苯二甲醛顯色劑測定上清中膽固醇含量,並計算膽固醇的去除率。
其中,膽固醇去除率=對照組膽固醇與實驗組膽固醇的差值/對照組膽固醇含量。
實驗結果見圖1,植物乳桿菌HG-23降膽固醇降低率為70.94%,表明植物乳桿菌HG-23有較強體外降低膽固醇能力。
實施例
3
植物乳桿菌
HG-23
的細菌安全性試驗
(1)抗生素敏感性測試
採用微量稀釋法檢測HG-23抗生素抗性。將測試抗菌藥物(Gentamicin ,Kanamycin, Tetracycline, Erythromycin, Clindamycin, Chloramphenicol, Amplicilin,起始濃度分別為1024 ug/ml,256 ug/ml, 64 ug/ml,8 ug/ml,16 ug/ml,64 ug/ml,16 ug/ml) 做一系列2倍比稀釋共12次並分別加入96孔板對應孔,然後對應孔加入2ml稀釋的試驗菌液,經48h培養後 觀察,以抗菌藥物最小抑菌濃度(MIC)孔,即為試驗菌株的敏感度。實驗結果如見[表1]所示:
[表1] 植物乳桿菌HG-23對抗生素的抗性測試結果
抗生素(mg/mL) | HG-23 | EFSA規定安全值(植物乳桿菌屬) |
Gentamicin | 8 | 16 |
Kanamycin | 32 | 64 |
Tetracycline | 16 | 32 |
Erythromycin | 0.5 | 1 |
Clindamycin | 0.5 | 2 |
Chloramphenicol | 2 | 8 |
Amplicilin | 1 | 2 |
從[表1]中可以看出,植物乳桿菌HG-23對抗生素的抗性測試結果表明抗生素MIC都在安全範圍內。
(2)代謝物毒性檢測
乳酸旋光性檢測:使用愛爾蘭Megazyme公司的D-/L-乳酸檢測試劑盒進行檢測。實驗結果不產生D-乳酸。
硝酸鹽還原酶活性檢測:在無菌條件下,將活化好的菌株按3%的接菌量接入到硝酸鹽培養基中37℃培養5d後,滴加碘化鉀溶液和澱粉溶液各10滴,觀察實驗結果。同時做空白實驗。實驗結果顯示所有菌液均未變藍,為陰性反應。
吲哚實驗:無菌條件下,將活化好的菌株按3%的接菌量接入到蛋白腖水養基中。37℃培養72h,加入吲哚試劑8~10滴,觀察實驗結果。同時做空白實驗。實驗結果未出現紅色圓環,表明其代謝不產生吲哚。
氨基脫羧酶活性檢測:具有氨基酸脫羧酶的細菌,能分解氨基酸使其脫羧生成胺(賴氨酸→屍胺,鳥氨酸→腐胺,精氨酸→精胺)和二氧化碳,使培養基變鹼性,滴加指示劑(溴甲酚紫),呈黃色為陰性,呈紫色為陽性。實驗結果顯示測定管呈黃色,為陰性。
(3)細胞黏附能力的測定
6孔板中的細胞單層培養物用不完全DMEM(不添加血清和雙抗)液37℃恒溫培養1h;將培養液吸出,用無菌PBS洗滌3次,將洗滌液吸幹;取500μL 10
8cfu/mL的實驗菌株與500μL不完全DMEM混勻後,加入到6孔培養板中,同時設無細胞僅有菌液的空白對照,37℃,恒溫培養1.5h;取出培養板,將培養液吸幹,用無菌PBS洗滌5次,吸幹洗滌液;每孔加入10%甲醛,室溫固定2h;吸幹固定液,用無菌PBS洗滌3遍,吸幹洗滌液,室溫乾燥;革蘭氏染色後鏡檢,隨機選取20個視野進行計數,並計算黏附指數:
黏附指數=細菌/細胞數(即平均每個細胞黏附的細菌個數)
菌株隨機選取20個視野,對視野中的細胞上黏附的細菌的總數和細胞數進行計數並測定黏附指數,實驗結果HG-23對人結腸癌細胞系HT-29黏附指數為5.6,具有較好的細胞黏附能力。
實施例
4
植物乳桿菌
HG-23
的體外酸及膽鹽耐受力試驗
配製模擬胃液SGF(2.0g NaCl+3.2g胃蛋白酶(pepsin)+適量濃鹽酸調PH值3.0),用0.2μm微孔濾膜過濾後待用。挑取斜面菌種於MRS液體培養基中,37℃培養16~18h。將菌懸液4000r/min離心15min,去上清,稱量菌體濕重, 按照0.1g/ml的比列重新懸浮菌體於生理鹽水中,在按照1:10的比列加入到SGF液中,充分混勻後置於37℃恒溫培養箱中培養2h,進行細胞計數。實驗結果見圖2,活菌數保持在一個數量級,表明植物乳桿菌HG-23體外有較強的耐酸能力。
將活化兩次的菌液按1%量接入滅菌的含有0.3%膽鹽的10mL MRS液體培養基中培養並進行生長曲線測定,實驗結果見圖3,表明HG-23體外有較強的抗膽汁能力。
實施例5 植物乳桿菌HG-23的體內有效性研究(動物實驗)
(1)實驗菌株的製備:
將活化兩次的植物乳桿菌HG-23接種於MRS液體培養基中,於37℃培養18h,6000r/m離心10min,用滅菌生理鹽水洗滌後收集菌體。然後,加入0.85%生理鹽水,調整菌數約1.0×10
9CFU/ml,然後將活菌按每日使用量分裝於15ml離心管,服用劑量為每天2ml/只,每組10只,共4組,需灌胃28天。
(2)實驗動物分組及飼養方式:
Sprague-Dawley系5周齡雄性大鼠,共40只,自由飼喂至第28天,平均分為4組,每組10只:
HG-23組:灌胃活菌懸液,飼喂高脂飼料;
模型組:灌胃0.85%生理鹽水,飼喂高脂飼料;
正常組:灌胃0.85%生理鹽水,飼喂標準飼料;
血脂康組:灌胃標準劑量血脂康,飼喂高脂飼料。
每天每只2ml灌胃,持續28天,然後停止灌胃,各組飼料、水不變持續28天。
(3)樣品採集與分析測試:
正式試驗前、第28天以及時間段進行采血。采血方法為絕食一夜後,於大鼠股靜脈采血,凝血後4000r/m離心10min分離血清,利用試劑盒(試劑盒名稱:總膽固醇測定試劑盒,公司:浙江東甌診斷產品有限公司)和)和變色酸顯色法,酶標儀(公司:Molecular Devices,型號:Spectra MAX190)分別檢測膽固醇及甘油三酯。
(4)實驗結果
實驗結果見圖4和圖5。用含HG-23菌株菌懸液餵養高脂大鼠28天后股靜脈抽血檢測,發現與對照相比大鼠血液中甘油三酯(TG)及膽固醇(TC)分別下降25%和27.6%,表明植物乳桿菌HG-23在大鼠體內確有降低膽固醇及甘油三酯功效。
實施例6
一種調節血脂的複合益生菌固體粉劑的製備。其由以下重量百分比的組分組成:檸檬水果粉30%、低聚果糖22%、低聚木糖20%、菊粉20%、植物乳桿菌HG-23菌粉8%。
動物實驗證明植物乳桿菌HG-23有效降低血清中膽固醇和甘油三酯水平。低聚果糖、低聚木糖和菊粉為益生元,可以促進益生菌的增殖。此外,國內外多項研究表明低聚果糖、低聚木糖和菊粉能夠降低血清中甘油三酯和膽固醇的濃度, 進而改善脂類代謝。該配方還具有組分天然,配方科學,營養均衡,服用方便,溶解性好,不黏牙的優點。
複合益生菌固體粉劑按以下工藝步驟製備而成:
(1)凍乾粉製備:採用凍幹機將植物乳桿菌HG-23的菌懸液在無菌環境下冷凍真空乾燥製成植物乳桿菌HG-23凍乾粉,真空度6Pa,冷凍溫度-40℃,凍幹時間為7〜10小時。其中每克植物乳桿菌HG-23凍乾粉中活菌數為5×10
11cfu,水分含量小於5%;
(2)配料:將植物乳桿菌HG-23、檸檬水果粉、低聚果糖、低聚木糖、菊粉、原料轉入10萬級潔淨區域,取樣進行檢驗;
(3)過篩:檢驗合格後,分別過80目篩,按照配比稱取原料;
(4)混合:將稱量的原料邊混合邊攪拌,混合成色澤一致,無色差,得到總混合粉;
(5)包裝:將混合粉按計量重量進行包裝,貼簽,裝箱,得到成品。
將實施例6得到的調節血脂的複合益生菌固體粉劑進行藥效試驗。
試驗方案:實驗方案同實施例5,其中灌胃的樣品改為上述益生菌固體粉劑加無菌水製成的懸液,調整菌數約1×10
9CFU/mL,服用劑量為每天2mL/只。
用上述益生菌固體粉劑的懸液餵養高脂大鼠28天后股靜脈抽血檢測,結果見圖6和圖7,發現與對照相比大鼠血液中甘油三酯及膽固醇都有明顯下降的情況,植物乳桿菌HG-23在大鼠體內定植並發揮了作用,表明灌胃上述益生菌固體粉劑在大鼠體內確有降低甘油三酯及膽固醇功效。
以上所述,僅為本發明的具體實施方式,但本發明的保護範圍並不局限於此,任何熟悉本技術領域的技術人員在本發明揭露的技術範圍內,可輕易想到變化或替換,都應涵蓋在本發明的保護範圍之內。因此,本發明的保護範圍應以所述權利要求的保護範圍為准。
綜上所述,本發明所揭露之技術手段確能有效解決習知等問題,並達致預期之目的與功效,且申請前未見諸於刊物、未曾公開使用且具長遠進步性,誠屬專利法所稱之發明無誤,爰依法提出申請,懇祈 鈞上惠予詳審並賜准發明專利,至感德馨。
惟以上所述者,僅為本發明之數種較佳實施例,當不能以此限定本發明實施之範圍,即大凡依本發明申請專利範圍及發明說明書內容所作之等效變化與修飾,皆應仍屬本發明專利涵蓋之範圍內。
〔本發明〕
無
[圖1]顯示根據本發明的具體實施例2植物乳桿菌HG-23對膽固醇的降解率,橫坐標為分組(空白對照、商業菌株、HG-23),縱坐標為體外對膽固醇的降解率;
[圖2]為根據本發明的具體實施例4植物乳桿菌HG-23的耐胃酸能力,橫坐標為時間,縱坐標為HG-23菌株在pH為2的模擬胃液中的存活菌數;
[圖3]為根據本發明的具體實施例4植物乳桿菌HG-23耐膽鹽能力,橫坐標為時間,縱坐標為HG-23菌株在MRS培養基和含0.3%膽鹽的MRS培養基中的OD值;
[圖4]為根據本發明的具體實施例5植物乳桿菌HG-23降低體內TG含量的能力,橫坐標代表組別,縱坐標代表甘油三酯的濃度;
[圖5]為根據本發明的具體實施例5植物乳桿菌HG-23降低體內TC含量的能力,橫坐標代表組別,縱坐標代表膽固醇的濃度;
[圖6]為根據本發明的具體實施例6益生菌固體粉劑降解甘油三酯(TG)動物實驗的結果,橫坐標代表組別,縱坐標代表甘油三酯的濃度;
[圖7]為根據本發明的具體實施例6益生菌固體粉劑降解膽固醇(TC)動物實驗的結果,橫坐標代表組別,縱坐標代表膽固醇的濃度。
CN中國大陸 中國典型培養物保藏中心 2021/4/6 CCTCC No: M2021330
Claims (6)
- 一種植物乳桿菌HG-23,其特徵在於,所述植物乳桿菌HG-23的保藏號為CCTCC No: M2021330。
- 如請求項1所述之植物乳桿菌HG-23在製備食品或藥物中的應用。
- 如請求項2所述之應用,其中,所述食品或藥物為降低血脂的食品或降低血脂的藥物;優選的,所述食品或藥物作用於降低膽固醇或降低甘油三酯。
- 如請求項3所述之應用,其中,所述藥物包含如請求項1所述之植物乳桿菌HG-23;以及醫藥用無毒載體。
- 如請求項4所述之應用,其中,所述藥物的劑型選自片劑、顆粒劑、散劑、膠囊劑、懸浮劑、乳劑、凍幹製劑的至少一種;優選的,所述藥物為複合益生菌片。
- 如請求項3所述之應用,其中,所述食品包含如請求項1所述之植物乳桿菌HG-23,以及食品學上可接受的添加劑;優選地,所述食品為益生菌固體粉劑。
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