2009年--外文翻譯--含副干酪乳桿菌nfbc338噴霧干燥的益生菌酸奶的研制

1、<p>　　中文6600字，4200單詞，21700英文字符</p><p>　　出處：Kearney N, Meng X C, Stanton C, et al. Development of a spray dried probiotic yoghurt containing Lactobacillus paracasei NFBC 338[J]. International Dairy Journ

2、al, 2009, 19(11): 684-689.</p><p>　　本科畢業(yè)設(shè)計(論文)</p><p>　　外文參考文獻譯文及原文</p><p>　　學 院 輕工化工學院 </p><p>　　專 業(yè) 食品科學與工程 </p><p>　　2010 年 6月 </

3、p><p>　　含副干酪乳桿菌NFBC338噴霧干燥的益生菌酸奶的研制</p><p><b>　　摘要</b></p><p>　　本研究的目的是研究副干酪乳桿菌NFBC338這益生菌的可行性，通過：（a）在嗜熱鏈球菌和德氏乳桿菌保加利亞亞種發(fā)酵劑下的兩個酸奶發(fā)酵階段，（b）經(jīng)過噴霧干燥，以及（c）42天的儲藏。在初始發(fā)酵階段（10小時），益生菌

4、乳桿菌的數(shù)目增加了7倍達到3.9 × 109 cfu g?1，且在進一步發(fā)酵3小時后，酸奶發(fā)酵劑中仍保持著這個數(shù)目。經(jīng)過噴霧干燥，益生菌培養(yǎng)基存活性最好，其次是嗜熱鏈球桿菌和德氏乳桿菌保加利亞亞種（產(chǎn)量分別是3.4 × 108, 1.2 × 108 和 4.0 × 105 cfu 

5、;g?1 ）。副干酪乳桿菌NFBC338和嗜熱鏈球桿菌在儲藏溫度為4 °C 和 15 °C穩(wěn)定，活菌的數(shù)目超過107 cfu g?1,而在儲存期間德氏乳桿菌保加利亞亞種的存活性大大的降低。</p><p><b>　　目 錄</b></p><p><b>　　1.導言4</b>

6、</p><p><b>　　2.方法和材料6</b></p><p>　　2.1微生物發(fā)酵劑6</p><p>　　2.2CH-1酸奶發(fā)酵劑在牛奶中的發(fā)酵6</p><p>　　2.3在益生菌乳桿菌338和 CH-1酸奶發(fā)酵劑中牛奶的發(fā)酵6</p><p>　　2.4酸奶的噴霧干燥、儲藏

7、測試以及發(fā)酵奶和干燥酸奶粉中微生物的計算7</p><p>　　2.5 通過RAPD-PCR確認噴霧干燥酸奶中Lb.338（副干酪乳桿菌338）的</p><p><b>　　存在8</b></p><p>　　2.6在儲藏中對干燥粉末的水分活力(aw)和pH的測定8</p><p><b>　　2.7數(shù)

8、據(jù)分析8</b></p><p><b>　　3.結(jié)果和討論9</b></p><p>　　3.1在酸奶生產(chǎn)中嗜熱鏈球菌、德氏乳桿菌保加利亞亞種和Lb.338的生長9</p><p>　　3.2 噴霧發(fā)酵對酸奶發(fā)酵劑的影響10</p><p>　　3.3 酸奶粉中嗜熱鏈球桿菌、德氏乳桿菌保加利亞亞種和

9、Lb.338在儲藏過</p><p><b>　　程中的存活性11</b></p><p><b>　　4.總結(jié)14</b></p><p><b>　　1.導言</b></p><p>　　酸奶是眾多食品中的一種益生菌微生物的載體(Adolfsson, Meydani,

10、Russell,2004),其中含有至少1 × 106個雙歧桿菌活菌(FAO/WHO, 2003)。對于食物成功的遞送，益生菌必須存活于產(chǎn)品成熟期（例如，發(fā)酵產(chǎn)品）和保質(zhì)期間的食品加工和儲藏過程。此外，食物產(chǎn)品必須定期的消耗相關(guān)“劑量”的活菌進行遞送，而在胃腸道遇到不利的條件，會引起細胞活力的降低 (Ross et al., 2005 R.P. Ross, C. Desmond, G.F. Fit

11、zgerald and C. Stanton, Overcoming the technological hurdles in the development of probiotic foods, Journal of Applied Microbiology 98 (2005), pp. 1410–1417. Full Text via CrossRef | View Record in Scopus | Cited By in S

12、copus (34)Ross, Desmond, Fitzgerald, & </p><p>　　有關(guān)于噴霧干燥酸奶中的培養(yǎng)物的存活和隨后在儲藏中的存活的刊物資料是有限的。在出風溫度為60°C簡單噴霧干燥的酸奶的pH為4.2，導致嗜熱鏈球菌和德氏乳桿菌保加利亞亞種的存活率分別為3%和0.7%(Kim & Bhowmik, 1990)。在出風溫度為70–75 °C，可

13、以獲得符合要求的德氏乳桿菌保加利亞亞種和嗜熱鏈球桿菌的存活率，分別為13.7-15.8%和51.6-54.7%；以及干燥酸奶產(chǎn)品可以維持好的感官特性和5.1-6.3%的水分含量。然而，在80 ℃出風溫度干燥發(fā)酵的階段，會大大減少微生物的存活率(Bielecka和 Majkowska, 2000)。據(jù)Kumar and Mishra (2004)所說，經(jīng)過噴霧干燥的酸奶，與德氏乳桿菌保加利亞亞種相比，嗜熱鏈球菌表現(xiàn)出更低的敏感性

14、。酸奶發(fā)酵劑的存活性受儲藏條件的影響，特別是溫度。酸奶微生物在儲藏溫度為5-10°C比在室溫下能存活更長的時間。另外，在儲藏期間，酸奶粉末的最適宜水分活度應(yīng)該是0-0.2%(Kumar 和 Mishra, 2004)。干燥酸奶的應(yīng)用有很多方面，比如糖果膜，湯料的制造，調(diào)味羹和油膠狀產(chǎn)品，家禽飼料，速</p><p><b>　　2.方法和材料</b></p><

15、p><b>　　2.1微生物發(fā)酵劑</b></p><p>　　益生菌菌種接種物副干酪乳桿菌NFBC338（此后稱為Lb.338）在液氮下被做成粒狀的，其中的數(shù)量有cfu·g-1。這些制成粒狀的發(fā)酵劑在使用之前被儲藏在-80°C的溫度里。這些用于研究的喜溫酸奶發(fā)酵劑是由嗜熱鏈球桿菌和德氏乳桿菌保加利亞亞種混合組成制成冷凍干燥的粒狀，其中含有每一種類（FD-DVS C

16、H-1 – Yo-Flex, Chr. Hansen, Hørsholm, Denmark）中的一種菌種。這些冷凍干燥的發(fā)酵劑一直保存在-20°C直到用為止。冷凍益生菌發(fā)酵劑和冷凍干燥酸奶發(fā)酵劑都是可直接使用的發(fā)酵劑，因此在使用之前要立即從冷藏室中拿出并且直接拖入到已經(jīng)巴氏消毒的牛奶發(fā)酵媒介物中。</p><p>　　2.2CH-1酸奶發(fā)酵劑在牛奶中的發(fā)酵</p><p&g

17、t;　　將奶粉（SMP:16%,w/v）溶解在50°C去離子水中保持1個小時以制得發(fā)酵培養(yǎng)基。往其中添加1%（w/v）蔗糖可增加其風味。采用一批高剪切攪拌機取得其再結(jié)合(Silverson Mixer Model AXR; Silverson Machines Ltd., Chesham, UK)。培養(yǎng)基在APV巴氏消毒設(shè)備溫度為95°C維持5分鐘進行巴氏消毒(APV Danmark A/S, Silkeborg,

18、Denmark; 流量為150 L h?1)，接著冷卻至42°C 。用10升的發(fā)酵罐收集20公升的培養(yǎng)基，然后放到水浴鍋中在42°C中進行恒溫培養(yǎng)。已經(jīng)121°C、15分鐘消毒的酵母濃縮物(YE; 0.05%, w/v) (Merck Chemicals Ltd., Nottingham, UK)和DVS CH-1 發(fā)酵劑(0.0052%, w/v)添加到其中，在繁殖期都需要監(jiān)測凝結(jié)奶直

19、至pH約為4.7 。酸奶（無益生菌酸奶）利用高速混合器進行混合(Silverson Machines Ltd.)，而后用干燥機設(shè)備進行噴霧干燥(F1 Lab dryer, Anhydro, Cope</p><p>　　2.3在益生菌乳桿菌338和 CH-1酸奶發(fā)酵劑中牛奶的發(fā)酵</p><p>　　在復原乳中添加1%(w/v)的蔗糖，如上條件進行巴氏消毒。已消毒的YE（促進Lb.338的

20、生長）加入到其中，1%(w/v)Lb.338接種到酸奶中的基礎(chǔ)物中。在10升的發(fā)酵罐處于37°C的恒溫槽中接種，以獲得在酸奶發(fā)酵物加入前高產(chǎn)量的益生菌物質(zhì)。在37°C中進行10小時的發(fā)酵，當溫度上升至42°C（酸奶發(fā)酵的最佳生長和酸度的溫度）加入0.0052% (w/v)的CH-1發(fā)酵物。監(jiān)控pH直至其達到終點得到pH為4.7，然后將酸奶（益生菌酸奶）進行混合和噴霧干燥。所有實驗數(shù)據(jù)都由重復的三次實驗所得。

21、</p><p>　　2.4酸奶的噴霧干燥、儲藏測試以及發(fā)酵奶和干燥酸奶粉中微生物的計算</p><p>　　酸奶產(chǎn)品（無論是參照實驗還是含有益生菌）在進風溫度170 °C ± 2 °C反復的進行噴霧干燥。當出風口溫度變化范圍為80-85°C時，獲得的產(chǎn)品水分含量不超過4%,水分活度值低于0.6([Alzamo

22、ra et al., 2003] and [Masters, 1985]) 。酸奶粉末經(jīng)分離后，密封包裝在聚乙烯袋里，分別儲藏在4 °C, 15 °C 和 37 °C中42天。所有噴霧干燥所得的實驗數(shù)據(jù)來源于相同的三次實驗。存活的百分率計算如下：</p><p>　　其中，No干燥前酸奶中微生物的數(shù)目，N為噴霧干燥粉末中微生物的數(shù)目，No 、N

23、表示的是每克干燥物料中的含量。</p><p>　　對于活微生物的計算，1 mL的牛奶或是酸奶應(yīng)加入9 mL的最大恢復稀釋液(MRD; Oxoid c/o Fannin Healthcare, Dublin, Ireland)，而后準備相對應(yīng)的一系列稀釋液在選擇性培養(yǎng)基未凝固時倒入其中，覆蓋在培養(yǎng)基上面用以培養(yǎng)每一種菌種。對于噴霧干燥酸奶粉末中活菌的恢復，在覆滿精心挑選的瓊脂之前用1g的制造好的粉末與9mL的MR

24、D在室溫下1小時進行水合。嗜熱鏈球菌在M17的培養(yǎng)基中顯示出其好氧性(Difco Laboratories, Detroit, MI, USA)，添加已透過膜過濾的無菌10%乳糖溶液(Oxoid; 1% 終濃度)在45°C中培養(yǎng)2-3天。使用5M HCL(AGB Scientific Ltd., Dublin, Ireland)調(diào)節(jié)MRS媒介物(Difco Laboratories)的pH至5.2，用此在45°C厭氧

25、的條件下培養(yǎng)2-3天挑選出德氏乳桿菌保加利亞亞種。使用5M HCL將乳桿菌發(fā)酵液（LC）媒介物的pH調(diào)至5.1，添加經(jīng)膜過濾的無菌10% (w/v) D-核糖溶液(Sigma Aldrich; 1% 終濃度)和0.2</p><p>　　2.5 通過RAPD-PCR確認噴霧干燥酸奶中Lb.338（副干酪乳桿菌338）的存在</p><p>　　1g儲藏在4°C的益生菌酸奶粉末，加

26、入9 mL的MRD在37°C下1小時能逐漸形成懸浮狀，在LC瓊脂培養(yǎng)基中能挑選出來（粉末樣本一式三份）。在同批中抽取出來接種到10 mL的MRS肉湯中在厭氧、37°C的條件下培養(yǎng)生長一夜。利用Hoffman and Winston (1987) 所概述的方法在培養(yǎng)一夜的培養(yǎng)基中萃取基因組DNA。接著將5 μL的DNA萃取液放入到含有1.25 U BIOTAQ DNA聚合酶、0.2

27、0;mm dNTP、50 mm MgCl2 和無MgCl2 緩沖液的10 × NH4 (Bioline c/o Labplan Ltd., Naas, Ireland)的50 μL的容器中進行聚合酶鏈式反應(yīng)擴大。濃度為1 μm的引物P2 (5′ ATGTAACGCC 3′； Simpson, Stanton, Fitzgerald, 和 Ross, 2002)用于隨機擴增多態(tài)D

28、NA的反應(yīng)。擴增的步驟</p><p>　　2.6在儲藏中對干燥粉末的水分活力(aw)和pH的測定</p><p>　　使用便攜式水分活度儀CX-2 (Decagon Devices, Pullman, WA, USA)對噴霧干燥酸奶粉末進行水分活力的測定。噴霧干燥粉末的pH的測定是經(jīng)過粉末(10%, w/v)在無菌的HPLC水中，保持室溫1個小時進行水合，再用帶有電極表刻度(Mettle

29、r-Toledo InLab® 413, Mettler-Toledo)的pH計(model MP220, Mettler-Toledo, Greifensee, Switzerland)進行測量。</p><p><b>　　2.7數(shù)據(jù)分析</b></p><p>　　所有實驗結(jié)果是通過學生對經(jīng)過噴霧干燥和儲藏后在p < 0.05

30、這一個可能的水平的存活率的顯著測量的t-實驗（雙尾配對）所得，在選擇性培養(yǎng)基中活菌的數(shù)目可變換為記錄為cfu g?1。誤差棒在圖表中表示的是數(shù)據(jù)點的標準差。</p><p><b>　　3.結(jié)果和討論</b></p><p>　　3.1在酸奶生產(chǎn)中嗜熱鏈球菌、德氏乳桿菌保加利亞亞種和Lb.338的生長</p><p>　　酸奶發(fā)酵微生

31、物嗜熱鏈球桿菌和德氏乳桿菌保加利亞亞種在發(fā)酵過程中會產(chǎn)生抑制物質(zhì)如酸和過氧化氫，可能會降低益生菌微生物的存活性(Shah, 2000)?；谶@個原因（同時也為了盡量減少所需益生菌的數(shù)量），我們使用兩步發(fā)酵的方法。.在初始發(fā)酵階段，Lb.338的數(shù)目增加了6.8倍從5.7 × 108 cfu g?1 增加至3.9 × 109 cfu g?1

32、，嗜熱鏈球桿菌和德氏乳桿菌保加利亞亞種加入時的數(shù)量分別為1.5 × 106 cfu g?1 and 5.9 × 105 cfu g?1。終發(fā)酵階段中，嗜熱鏈球桿菌和德氏乳桿菌保加利亞亞種的數(shù)目分別增加到1.6 × 109 cfu g?1 and 2.1 × 1

33、08 cfu g?1，而酸奶中的益生菌Lb.338的數(shù)量稍微減少至3.0 × 109 cfu g?1，但是這種數(shù)量上的減少并非是不可忽略的（圖1）。結(jié)果是，在發(fā)酵的全過程中，Lb.338的數(shù)量增加了5.3倍。據(jù)Shah (2000)報導，在兩步發(fā)酵過程里，初計數(shù)酸奶中益生菌微生物增加了4-5倍；后計數(shù)經(jīng)過42天的儲藏后產(chǎn)品中的數(shù)目 >107 cfu&#

34、160;g?1。嗜熱鏈球桿菌和德氏乳桿菌保加</p><p>　　圖1是兩步發(fā)酵的結(jié)果展示。在牛奶中培養(yǎng)生長10小時，至發(fā)酵終點，經(jīng)過噴霧干燥，Lb.338的初計數(shù)的存活用(■)在途中左邊表示出來，接著的是嗜熱鏈球桿菌( )和德氏乳桿菌保加利亞亞種(□)。全發(fā)酵過程中pH ( )也在圖中表示出來。誤差棒表示的是根據(jù)重復三次實驗數(shù)據(jù)的標準誤。星號代表經(jīng)噴霧干燥與后發(fā)酵形成菌落形成單位數(shù)量的不同(p <

35、; 0.05)。RAPD（隨機擴增多態(tài)DNA）-PCR（聚合酶鏈式反應(yīng)）圖作為從噴霧干燥益生菌酸奶粉中分離的乳桿菌的數(shù)量的典型的圖表在上圖表中表示出來。映象1和19表示的是標準分子量，映象2-16表示的是從酸奶粉中分離15乳桿菌的RAPD圖，映象17和18分別表示的是反面對照和正面對照。</p><p>　　圖2，嗜熱鏈球桿菌和德氏乳桿菌保加利亞亞種在牛奶中接種CH-1發(fā)酵劑的初計數(shù)和后發(fā)酵的存活率以及

36、經(jīng)噴霧干燥后的全部pH圖( )。誤差棒表示的是根據(jù)重復三次實驗數(shù)據(jù)的標準誤。星號代表經(jīng)噴霧干燥與后發(fā)酵形成菌落形成單位數(shù)量的不同(p < 0.05)。</p><p>　　3.2 噴霧發(fā)酵對酸奶發(fā)酵劑的影響</p><p>　　選擇Lb.338用于此研究是因為此菌種已經(jīng)過臨床實驗。在健康成人中進行雙盲法和安慰劑對照試驗，發(fā)現(xiàn)菌種的吸收可從糞便中分離得到的乳桿菌的明

37、顯增加得出奇之間有很大的關(guān)聯(lián)（未發(fā)表資料）。此外，菌種在噴霧干燥中的堅固性已得到證明([Desmond et al., 2002], [Desmond et al., 2001] 和 [Gardiner et al., 1998])。噴霧干燥后，在對照試驗中嗜熱鏈球桿菌和德氏乳桿菌保加利亞亞種的存活數(shù)分別是6.5 × 107 cfu g?1 and 3.0&

38、#160;× 105 cfu g?1。在噴霧干燥時的出風溫度為80–85 °C，德氏乳桿菌保加利亞亞種表現(xiàn)出較低的存活率。相同條件下，與嗜熱鏈球桿菌相比，這看起來似乎是比較易受到噴霧干燥此過程的影響([Bielecka 和 Majkowska, 2000]和 [Kim 和 Bhowmik, 1990])。另外，酸奶中的德氏乳桿菌保加利亞亞種在干燥前存活數(shù)目比起嗜熱鏈球桿菌要低，

39、這也是另外一個影響它的存活率的因素。在益生菌酸奶粉末中，與耐熱性較低的德氏乳桿菌保加利亞亞種存活數(shù)目為4.0 × 105 cfu g</p><p>　　3.3 酸奶粉中嗜熱鏈球桿菌、德氏乳桿菌保加利亞亞種和Lb.338在儲藏過程中的存活性</p><p>　　噴霧干燥被用于益生菌發(fā)酵劑的貯藏，在室溫保藏幾周它們的存活率通常都會有所降低。這

40、與壓力有關(guān)，此種壓力通過溫度的改變，減數(shù)分裂的改變，干燥，結(jié)合物對細胞質(zhì)膜進行破壞，且與蛋白質(zhì)有關(guān)(Anal & Singh, 2007)。正如所料，發(fā)現(xiàn)在噴霧干燥酸奶粉中發(fā)酵劑(Fig. 3A,B)的存活性與儲藏溫度成相反關(guān)系([Ananta et al., 2005] 和[Desmond et al., 2002])。儲藏溫度為4 °C時，發(fā)現(xiàn)酸奶粉中Lb.338的存活數(shù)目沒

41、有出現(xiàn)明顯的減少。相比之下，在15 °C保藏14天，發(fā)現(xiàn)其存活性沒有很明顯的減少(p < 0.05)，其在儲藏了42天后還能在酸奶粉中存在這活菌的數(shù)目為2.27 × 107 cfu g?1，這符合益生菌產(chǎn)品中每g至少含有106個活菌細胞的要求。這樣，在生產(chǎn)產(chǎn)品的全過程中，從開始的接種到后來的貨架期，益生菌Lb.338在4 °

42、;C 時存活率降低了2.5倍，在15 °C時降低了22.9倍。益生菌種經(jīng)歷了高損耗的存活率，然而，在37°C儲藏7天，其存活的數(shù)目大大的減少(p < 0.05)。在酸奶粉的嗜熱鏈球桿菌在在4 °C 和 15 °C保</p><p>　　圖3 A、B圖分別表示益生菌酸奶粉中Lb.338、嗜熱鏈球桿菌、德氏乳桿菌保加利亞亞

43、種和空白對照試驗組酸奶粉中嗜熱鏈球桿菌、德氏乳桿菌保加利亞亞種在儲藏長達42天的存活率。柱狀圖( ), ( ), ( ),分別表示 Lb. 338在 4 °C, 15 °C 和37 °C儲藏溫度下的存活率； ( ), ( ), ( ), 分別表示 嗜熱鏈球桿菌在 4 °C, 15 °C 和37 °C儲藏溫度下的存活率；

44、 ( ), ( ), ( ) 分別表示德氏乳桿菌保加利亞亞種在 4 °C, 15 °C 和37 °C儲藏溫度下的存活率；此結(jié)果是從重復三次實驗的數(shù)據(jù)而得，誤差棒表示標準誤。</p><p>　　空白試驗組的酸奶粉和益生菌酸奶粉在各儲藏溫度下保存長達42天，其pH值都很穩(wěn)定，分別是4.48 ± 0.01 和 4.82 

45、;± 0.01，由此得出pH并不影響菌的存活率。益生菌酸奶粉和空白試驗組的酸奶粉中的aw也沒有很明顯的區(qū)別，aw值的范圍都在0.21–0.32之間。脫水食物的aw值控制在0～0.3，這不僅因為微生物的生長，而且還有些對食物顏色、組織、風味和營養(yǎng)價值產(chǎn)生有害的物化和生化反應(yīng)。</p><p><b>　　4.總結(jié)</b></p><p>　　本研究表

46、明，生產(chǎn)具有高益生菌數(shù)目干燥酸奶粉可適當?shù)膽?yīng)用于功能食物中。數(shù)據(jù)表明，有一種技術(shù)的獲得就是在酸奶發(fā)酵的過程中使用兩步發(fā)酵方法，不影響嗜熱鏈球桿菌和保加利亞亞種的發(fā)酵效力的同時提高了Lb.338的活菌數(shù)目。噴霧干燥粉活菌細胞的總數(shù)一方面取決于其對噴霧干燥溫度的耐受性，另一方面取決于其在干燥前的酸奶中的數(shù)量。與在酸奶粉儲藏在4 °C 、15 °C時德氏乳桿菌保加利亞亞種、Lb.338、嗜熱鏈球桿菌能保

47、持高存活率（這些微生物存活率的減少是有限的），但儲藏37 °C時卻快速的降低相比，Lb.338和嗜熱鏈球桿菌表現(xiàn)出較高的存活率。比起新鮮食品，干燥益生菌酸奶粉有著很大的發(fā)展優(yōu)勢：較低的水分活度，因此有著較長的保存期，運輸費用也減少了。最后，值得強調(diào)的是，本文的干燥粉末生產(chǎn)可歸類于酸奶的組成部分的應(yīng)用。</p><p>　　Development of a spray dried probiot

48、ic yoghurt containing Lactobacillus paracasei NFBC 338</p><p>　　N. Kearneya, X.C. Mengb, C. Stantona, d, J. Kellya, G.F. Fitzgeraldc, d and R.P. Rossa, d, , </p><p>　　aTeagasc, Moorepark Food Re

49、search Centre, Fermoy, County Cork, Ireland</p><p>　　bKey Laboratory of Dairy Science (Ministry of Education) and Food Science & Technology College, Northeast Agricultural University, Harbin, China</p

50、><p>　　cMicrobiology Department, University College Cork, Ireland</p><p>　　dAlimentary Pharmabiotic Centre, University College Cork, Ireland</p><p>　　Received 20 January 2009;  &l

51、t;/p><p>　　revised 11 May 2009;  </p><p>　　accepted 12 May 2009.  </p><p>　　Available online 27 May 2009. </p><p><b>　　Abstract</b></p><p&

52、gt;　　The aim of this study was to monitor viability of probiotic Lactobacillus paracasei NFBC 338 during: (a) two-stage yoghurt fermentation with starter cultures Streptococcus thermophilus and Lactobacillus delbrueckii

53、subsp. bulgaricus, (b) following spray drying, and (c) during storage for 42 days. During the initial fermentation phase (10 h), probiotic Lactobacillus numbers increased 7-fold to 3.9 × 109 cfu&

54、#160;g?1 and these numbers were maintained during fermentation for a further 3 h in the presenc</p><p>　　Article Outline</p><p>　　1. Introduction </p><p>　　2. Materials and met

55、hods </p><p>　　2.1. Starter microorganisms </p><p>　　2.2. Fermentation of milk with CH-1 yoghurt culture </p><p>　　2.3. Fermentation of milk with probiotic Lb. 338 and CH-1 yoghurt

56、culture </p><p>　　2.4. Spray drying yoghurt, storage tests and enumeration of microorganisms from fermented milk and dried yoghurt powder </p><p>　　2.5. Confirmation of the identification of Lb.

57、 338 in spray dried yoghurt powder by RAPD–PCR </p><p>　　2.6. Determination of water activity (aw) measurements and pH of spray-dried powders during storage </p><p>　　2.7. Statistical analysis&l

58、t;/p><p>　　3. Results and discussion </p><p>　　3.1. Growth of S. thermophilus, L. delbrueckii subsp. bulgaricus and Lb. 338 during yoghurt production </p><p>　　3.2. Effect of spray-dry

59、ing on yoghurt cultures </p><p>　　3.3. Viability of S. thermophilus, L. delbrueckii subsp. bulgaricus and Lb. 338 in yoghurt powders during storage</p><p>　　4. Conclusions </p><p>　

60、　Acknowledgements </p><p>　　References</p><p>　　1. Introduction</p><p>　　Yoghurt is among the most popular food vehicles for the carriage of probiotic microorganisms (Adolfsson, Mey

61、dani, & Russell, 2004), and should contain at least 1 × 106 viable cells of Lactobacillus and/or Bifidobacterium g?1 (FAO/WHO, 2003). For successful delivery in foods, probiotics must survive food proc

62、essing and storage during product maturation (e.g., fermented products) and shelf-life. Moreover, the food product must be consumed regularly to deliver the relevant “dose” of live bacteria t</p><p>　　Publis

63、hed data relating to survival of cultures spray dried in yoghurt, and subsequent survival during storage are limited. Spray drying plain yoghurt with a pH of 4.2 at an outlet air temperature of 60 °C resulted i

64、n survival rates of 3% and 0.7% for Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively (Kim & Bhowmik, 1990). At outlet temperatures of 70–75 °C, satisfactory survival

65、of L. delbrueckii subsp. bulgaricus (13.7–15.8%) and S. thermophilus (51.6–54.</p><p>　　This study involved the optimisation of fermentation to achieve high viable counts of L. paracasei NFBC 338 in yoghurt

66、along with the commercial starter cultures S. thermophilus and L. delbrueckii subsp. bulgaricus. The effect of spray drying conditions on the survival of L. paracasei NFBC 338, S. thermophilus and L. delbrueckii subsp. b

67、ulgaricus in yoghurt and the storage stability of the cultures at 4 °C, 15 °C and 37 °C over 42 days were investigated. The primary objective was to maintain hig</p><p>　　2. Mat

68、erials and methods</p><p>　　2.1. Starter microorganisms</p><p>　　The probiotic strain L. paracasei NFBC 338 (hereafter referred to as Lb. 338) inoculum was pelletized under liquid nitrogen and c

69、ontained 2 × 1010 cfu g?1. The pelletized cultures were stored at ?80 °C until use. The thermophilic yoghurt culture used in this study consisted of a defined mixed strain culture of S.

70、 thermophilus and L. delbrueckii subsp. bulgaricus in a freeze-dried pellet form containing only one strain of each species (FD-DVS CH-1 – Yo-Flex®, Chr. Hansen, Hørsholm, Denmark). The </p><p>　　2

71、.2. Fermentation of milk with CH-1 yoghurt culture</p><p>　　The fermentation substrate was prepared by dissolving skim milk powder (SMP; 16%, w/v) in deionised water for 1 h at 50 °C. It was s

72、upplemented with 1% (w/v) sucrose (Sigma Aldrich, Wicklow, Ireland) which was added to enhance flavour. Recombination was achieved using a high shear batch mixer (Silverson Mixer Model AXR; Silverson Machines Ltd., Chesh

73、am, UK). The substrate was pasteurised at 95 °C for 5 min in an APV pilot plant pasteurizer (APV Danmark A/S, Silkeborg, Denmark; 150 L h?1 flow rat</p><p>　　2.3. Fermentation of mil

74、k with probiotic Lb. 338 and CH-1 yoghurt culture</p><p>　　Reconstituted skim milk (RSM), supplemented with 1% sucrose (w/v), was pasteurised using the same conditions as above. The sterilised YE was added (

75、promoting the growth of Lb. 338) and the yoghurt base was then inoculated at 0.1% (w/v) with Lb. 338 frozen pellets. The 10 L fermentation vessels were incubated in a waterbath at 37 °C to acquire high num

76、bers of the probiotic culture before addition of the yoghurt cultures. Following 10 h of fermentation at 37 °C, the temperature was increased to 42</p><p>　　2.4. Spray drying yoghurt, storage

77、tests and enumeration of microorganisms from fermented milk and dried yoghurt powder</p><p>　　The yoghurt products (both control and probiotic) were spray-dried at a constant air inlet temperature of 170

78、0;°C ± 2 °C. The outlet temperature ranged from 80 to 85 °C, to obtain product with moisture content not exceeding 4% and with water activity (aw) values below 0.6 ([Alzamora et

79、60;al., 2003] and [Masters, 1985]). The yoghurt powders were segregated, sealed in polyethylene bags, and stored at 4 °C, 15 °C and 37 °C for 42 days. All spray drying data are based on thre

80、e separate trials. The perc</p><p><b>　　(1)</b></p><p>　　where No represents the number of bacteria in the yoghurt before drying, and N is the number of bacteria in the spray-dried y

81、oghurt powder. Both N and No were expressed per gram of dry matter. </p><p>　　For the enumeration of viable microorganisms, 1 mL aliquots of either milk or yoghurt was added to 9 mL of maximum reco

82、very diluent (MRD; Oxoid c/o Fannin Healthcare, Dublin, Ireland) and the appropriate serial dilutions were prepared before pour plating onto the appropriate selective medium for each strain. For the recovery of viable ce

83、lls from the spray-dried yoghurt powders, 1 g of the manufactured powder was rehydrated in 9 mL of MRD for 1 h at room temperature prior to pour plating onto the</p><p>　　2.5. Confirmation of

84、the identification of Lb. 338 in spray dried yoghurt powder by RAPD–PCR</p><p>　　One gram of the probiotic yoghurt powder stored at 4 °C was resuspended in 9 mL of MRD, incubated for 1 h

85、at 37 °C and selected on LC agar (powder samples were taken in triplicate). Random colonies were inoculated into 10 mL of fresh MRS broth and were grown overnight at 37 °C under anaerobic conditi

86、ons. Genomic DNA was extracted from overnight cultures by the method outlined in Hoffman and Winston (1987). Aliquots of 5 μL of extracted DNA were subsequently used per polymerase chain reaction (PC</p><

87、p>　　2.6. Determination of water activity (aw) measurements and pH of spray-dried powders during storage</p><p>　　The water activity (aw) in the spray-dried yoghurt powders was measured using the Aqualab C

88、X-2 (Decagon Devices, Pullman, WA, USA) according to the manufacturer's instructions. The pH of the spray-dried powders was measured following rehydration of the powder (10%, w/v) in sterile HPLC grade water for 1

89、60;h at room temperature using a pH meter (model MP220, Mettler-Toledo, Greifensee, Switzerland), with calibrated electrode (Mettler-Toledo InLab® 413, Mettler-Toledo).</p><p>　　2.7. Statistical analysi

90、s</p><p>　　All results were compared by the Student's t-test (two-tail paired) with significance measured at a probability level of p < 0.05 for survival following spray drying and storage.

91、Viable counts on selective media were converted to log cfu g?1. The error bars on the figures indicate the standard error of the mean (SEM) for the data points.</p><p>　　3. Results and discuss

92、ion</p><p>　　3.1. Growth of S. thermophilus, L. delbrueckii subsp. bulgaricus and Lb. 338 during yoghurt production</p><p>　　Yoghurt starter bacteria S. thermophilus and L. delbrueckii subsp. bu

93、lgaricus produce inhibitory substances such as acid and hydrogen peroxide during fermentation which could potentially reduce the viability of probiotic bacteria (Shah, 2000). For this reason (and also in an effort to red

94、uce costs by minimising the amount of necessary probiotic), we used the two-step fermentation method. During the initial phase of fermentation, the numbers of Lb. 338 increased 6.8-fold from 5.7 × 108 

95、;cfu g?1 to</p><p>　　Fig. 1. Progress of the two-step fermentation. Initial counts of Lb. 338 (■) are shown on the far left, then growth in milk for 10 h, followed by inoculation with S.

96、thermophilus ( ) and L. delbrueckii subsp. bulgaricus (□), then viability at the end of fermentation and after spray drying. The pH profile ( ) of the entire process is also shown. The error bars represent the standard e

97、rror of the mean (SEM) based on data from triplicate trials. An asterisk denotes significant difference (p < 0.05)</p><p>　　View Within Article</p><p>　　Fig. 2. Initial co

98、unts of S. thermophilus ( ) and L. delbrueckii subsp. bulgaricus (□) in milk inoculated with CH-1 and viability at the end of fermentation and after spray drying, with the pH profile ( ) of the entire process. The error

99、bars represent the SEM based on data from triplicate trials. An asterisk denotes significant difference (p < 0.05) in cfu counts after spray drying compared with end of fermentation. </p><p>　　V

100、iew Within Article</p><p>　　3.2. Effect of spray-drying on yoghurt cultures</p><p>　　Lb. 338 was chosen for this study because the strain already has been clinically tested. A double-blinded pla

101、cebo-controlled feeding trial in healthy adults showed that ingestion of the strain was associated with significant increases in total Lactobacillus isolated from faeces (unpublished data). Futhermore, the robustness of

102、this strain in terms of spray-drying has already been demonstrated ([Desmond et al., 2002], [Desmond et al., 2001] and [Gardiner et al., 1998]). After spray drying, the li</p><p>　　3.3. Viabil

103、ity of S. thermophilus, L. delbrueckii subsp. bulgaricus and Lb. 338 in yoghurt powders during storage</p><p>　　When spray-drying is used for the preservation of potential probiotic cultures, much of their v

104、iability is typically lost after a few weeks of storage at room temperature. This is associated with stress that is induced by temperature change, phase changes and drying, a combination of which tend to damage cell memb

105、ranes and associated proteins (Anal & Singh, 2007). As expected, it was found that culture viability in the spray dried yoghurt powders (Fig. 3A,B) was inversely related to storage temp</p><p>　　Fig

106、. 3. Viability of (A) Lb. 338, S. thermophilus, and L. delbrueckii subsp. bulgaricus in probiotic yoghurt powders and (B) S. thermophilus and L. delbrueckii subsp. bulgaricus in control yoghurt powders stored f

107、or up to 42 days. Histograms are ( ), ( ), ( ), Lb. 338 stored at 4 °C, 15 °C and 37 °C, respectively; ( ), ( ), ( ), S. thermophilus stored at 4 °C ，15 °C and 37 &#

108、176;C, respectively; ( ), ( ), ( ) Lactobacillus delbrueckii subsp. bulgaricus stored at 4 °C, 15 °C and 37 °C, respectively. T</p><p>　　View Within Article</p><p>

109、　　The pH values of the control and probiotic yoghurt powders were stable during storage for up to 42 days at each of the storage temperatures, with values of 4.48 ± 0.01 and 4.82 ± 0.01, res

110、pectively, therefore not affecting viability. The aw values of the probiotic and the control powders did not differ significantly. Both powders had aw values within the range 0.21–0.32. Fully dehydrated foods have aw val