自食品中分離鑑定可產生脂肪酶之微生物應用於製造人造奶油

Abstract

人造奶油 (margarine) 是常見的奶油替代品，為世界上許多地區的日常食物，可用於烹調、烘焙與調味等。與奶油不同，人造奶油需要添加食品添加劑將油相及水相混合均勻，單酸甘油酯 (monoglyceride) 及雙酸甘油酯 (diglyceride) 為廣泛使用之食品乳化劑。近年食安意識抬頭，各種食安事件促使消費者關注飲食安全，並逐漸傾向選擇配方簡單及添加物少之食品，這種新的食品趨勢被歸納為潔淨標示 (clean label) 。目前市售人造奶油包含許多化學合成之食品添加劑，因此希望利用來自微生物之生物性脂肪酶 (lipase, triacylglycerol acylhydrolases) 切解棕櫚油中的三酸甘油酯 (triglyceride) 生產脂肪酸甘油酯做為乳化基質。然而，大部分脂肪酶生產之微生物不屬於公認安全 (generally recognized as safe, GRAS) 的分類，且不允許用於食品中。本研究之目的為篩選出具分解棕櫚油產生乳化劑之脂肪酶的GRAS微生物，以減少食品添加劑的使用。 自市售奶油分離之46株分離菌株及自克弗爾發酵乳及康門貝爾乾酪分離之124株分離菌株中，分別以革蘭氏染色 (Gram staining) 及油滴乳化試驗 (oil spreading assay) 篩選生產脂肪酶且具乳化能力之革蘭氏陽性菌 (Gram-positive bacteria) 及酵母菌 (yeast) ，初篩選25株及58株具乳化能力的細菌及酵母菌後，藉由腸桿菌基因間重複序列聚合酶連鎖反應 (Enterobacterial repetitive intergenic consensus polymerase chain reaction, ERIC-PCR) 進行分類 (typing) ，排除重複菌株。以滴定法測定18株細菌及1%商業用脂肪酶的脂肪酶活性，細菌處理組以經破菌處理之菌液上清液作為粗脂肪酶，最終選擇了平均活性較佳的B043 (40.67±2.40 U/mL) 及B013 (32.67±13.91 U/mL) 作為候選菌株。此外，17株酵母菌則以API ZYM商業套組篩選，留下具脂肪酶相關酵素活性的10株酵母菌，以脂肪酶活性分析商業套組測定脂肪酶活性，選擇與實際製程應用之0.58%商業脂肪酶 (61.80±21.24 U/L) 無顯著差異的CC11 (82.13±7.84 U/L) 及CC05 (39.93±9.05 U/L) 兩株酵母菌作為候選菌株。 經16S rRNA基因及持家基因 (housekeeping gene) 序列分析，與National Center for Biotechnology Information (NCBI) 資料庫進行BLAST分析，並根據neighbor-joining法繪製系統分類樹，B043均具有100%的支持度 (bootstrap value) 被認為屬於Leuconostoc pseudomesenteroides，B013之pheS及rpoA系統分類樹分別具有100%及93%的支持度被認為屬於Lactobacillus plantarum subsp. plantarum。酵母菌則以內轉錄間隔區1 (internal transcribed spacers 1) 、5.8S rRNA及內轉錄間隔區2區域定序結果繪製系統分類樹，CC05具有100%的支持度被認為屬於Yarrowia lipolytica，CC11具有100%的支持度被認為屬於Candida cylindracea。經菌種鑑定後確定四株候選株均為GRAS菌株。 將Y. lipolytica CC05、C. cylindracea CC11、Lb. plantarum subsp. plantarum B013和Leu. pseudomesenteroides B043之粗脂肪酶與0.58%商業酵素同時進行水解反應測試，測定酸價的改變，以薄層液相分析 (thin-layer chromatography, TLC) 進行定性，再以膠體管柱層析 (column chromatography) 進行定量，商業酵素具有顯著較高的水解能力 (53.77±4.44 mg KOH/g) ，而微生物粗脂肪酶處理組之間，Y. lipolytica CC05之酸價 (5.58±0.77 mg KOH/g) 顯著高於其他菌株粗脂肪酶處理組 (P<0.05) ，經過定性及定量後，切解液之組成為三酸甘油酯、雙酸甘油酯及單酸甘油酯分別為85.21%、10.66%及1.31%，水解程度無商業酵素強效，然而確實產生出單酸甘油酯，證實Y. lipolytica CC05具有水解棕櫚油生產乳化劑之能力。 本研究從食品分離純化之微生物，經過篩選之GRAS菌株，Y. lipolytica CC05，具有水解棕櫚油產生脂肪酸甘油酯之能力，希望未來將Y. lipolytica CC05經適當培養提升脂肪酶產量及活性，分解棕櫚油中的三酸甘油酯產生的脂肪酸甘油酯做為乳化基質，應用於人造奶油製程，減少化學食品添加劑的使用，創造符合市場需求之無添加產品，以迎合目前食安意識抬頭、潔競標示觀念盛行的社會現況。

Margarine is a common replacement for butter. As a water-in-oil emulsion, margarine needs emulsifiers to homogenize oil with its water-soluble components. However, to keep up with the worldwide trend of clean label, usage of food additives should be reduced. Using lipases (triacylglycerol acylhydrolases) to produce monoglyceride and diglyceride to replace chemical emulsifiers may be a novel solution. Nevertheless, most lipase-producing microorganisms are not generally recognized as safe (GRAS) and are not allowed in food products. Thus, in the present study, we aim to screen lipase-producing GRAS microorganisms to reduce the usage of food additives in margarine processing. First, 46 isolates from butter and 124 isolates from kefir and Camembert cheese were pre-selected for their emulsion activity by oil spreading assay. We identified 25 Gram-positive bacteria and 58 yeasts with emulsifying activities. We further typed these isolates using Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) and kept 18 Gram-positive bacteria and 17 yeasts as representative strains. After typing, we screened the crude lipases of the Gram-positive bacteria by their lipase activities with titration method. As a result, bacteria strains B043 and B013 showed the highest activities, 40.67±2.40 U/mL and 32.67±13.91 U/mL, respectively. We also tested the enzymatic activities of the yeasts with the API ZYM kit and quantified the lipase activities of 10 positive strains with lipase activity kit. Lipase activities of yeast strains CC11 (82.13±7.84 U/L) and CC05 (39.93±9.05 U/L) showed no significant difference when compared to the 0.58% commercial lipase (61.80±21.24 U/L) used for commercial margarine production (P>0.05). In summary, two bacteria and two yeasts were chosen for further investigation by their lipase activities. We identified the 2 bacteria using 16S rRNA gene and housekeeping gene analysis. The sequence analysis-based phylogenetic tree constructed by the neighbor-joining method confirmed isolate B043 as Leuconostoc pseudomesenteroides with 100% pheS and rpoA gene sequence similarity and confirmed isolate B013 as Lactobacillus plantarum subsp. plantarum with 100% and 93% pheS gene and rpoA gene sequence similarity, respectively. We also identified the 2 yeasts using internal transcribed spacers 1, 5.8S rRNA, and internal transcribed spacers 2 (ITS1-5.8S-ITS2) region sequence analysis, which confirmed isolate CC05 as Yarrowia lipolytica with 100% sequence similarity and confirmed isolate CC11 as Candida cylindracea with 100% sequence similarity. These strains are all generally recognized as safe (GRAS). We further compared the crude lipases from the four selected strains with 0.58% commercial lipase on their hydrolyzing abilities with acid value analysis in palm oil to ensure that they could be applied in margarine production. While 0.58% commercial lipase had the highest acid value (53.77±4.44 mg KOH/g), Y. lipolytica CC05 had the highest acid value (5.58±0.77 mg KOH/g) among the four microorganisms (P<0.05). We also assessed their monoglyceride and diglyceride producing abilities with thin-layer chromatography (TLC) and column chromatography. Components of hydrolyzed product by crude lipase from Y. lipolytica CC05 were 85.21% triglyceride, 10.66% diglyceride, and 1.31% monoglyceride. These results showed the potential of Y. lipolytica CC05 as an emulsifier producer. In the future, we will further enhance the production and activity of the lipase from Y. lipolytica CC05 by optimizing culture condition and purifying the enzyme. The final aim of this study is to develop a novel lipase that could be applied in margarine production to reduce its usage of food additives, keeping pace with the clean label world trend.