摘要:庫德里阿茲威畢赤酵母(Pichia kudriavzevii)是一種常見的東北酸菜腐敗菌,導致東北酸菜成膜、變軟和產(chǎn)生異味等質量缺陷。從自然發(fā)酵的東北酸菜中分離出7株乳酸菌,對Pichia kudriavzevii HSDF-pks-2022(pks)進行抑制和發(fā)酵影響的研究。通過對比pks的抑菌圈大小,從7株乳酸菌中篩選出5株抑制腐敗菌pks明顯的乳酸菌,分別為Levilactobacillus brevis HSDF-lbn-2022(lbn)、Lactobacillus sakei HSDF-lso-2022(qj)、Leuconostoc mesenteroides HSDF-lmo-2022(cm)、Loigolactobacillus coryniformis HSDF-lco-2022(rs)、Lactiplantibacillus xiangfangensis HSDF-lxo-2022(xf);通過乳酸菌和腐敗菌在酸菜中共發(fā)酵,根據(jù)發(fā)酵液產(chǎn)pH、有機酸、腐敗菌活菌數(shù)、生物膜形成量和生物胺情況,從5株乳酸菌中篩選出qj,其抑制pks的效果最好、pH最低(3.34)、有機酸含量最高(62.10 mg/mL)、腐敗菌活菌數(shù)最少(5.00×106 CFU/mL)、腐敗菌生物膜形成量最少(0.021 g)、生物胺種類和含量也較少。不同的乳酸菌抑制pks的能力不同,qj有顯著的抑制效果,該研究為發(fā)酵食品生物法抑制東北酸菜腐敗提供了借鑒。
關鍵詞:清酒乳桿菌;腐敗菌;庫德里阿茲威畢赤酵母;酸菜
中圖分類號:TS201.3文獻標志碼:A文章編號:1000-9973(2025)03-0090-08
Study on Inhibition of Different Lactic Acid Bacteria on Sauerkraut
Spoilage Fungus—Pichia kudriavzevii
TUO Xiao-li, MA Ye, DAI Zhi-peng, ZHAO Kai, FAN Hong-chen*
(School of Food Engineering, Harbin University of Commerce, Harbin 150076, China)
Abstract: Pichia kudriavzevii is a common spoilage fungus in northeast sauerkraut, it causes quality defects of northeast sauerkraut such as film formation, softening and odor production. Seven strains of lactic acid bacteria are isolated from naturally fermented northeast sauerkraut, their inhibition and fermentation effects on Pichia kudriavzevii HSDF-pks-2022 (pks) are studied." By comparing the size of the inhibition zone of pks, five strains with significant inhibition of spoilage fungus pks are screened from the seven strains of lactic acid bacteria. These strains are Levilactobacillus brevis HSDF-lbn-2022(lbn), Lactobacillus sakei HSDF-lso-2022(qj), Leuconostoc mesenteroides HSDF-lmo-2022(cm), Loigolactobacillus coryniformis HSDF-lco-2022(rs) and Lactiplantibacillus xiangfangensis HSDF-lxo-2022(xf) respectively; by co-fermentation of lactic acid bacteria and spoilage fungus in sauerkraut, according to the pH, oganic acid, the number of variable spoilage fungus, the formation amount of biofilm and biogenic amine of fermentation broth, qj is screened out from the five strains of lactic acid bacteria, its inhibition effect on pks is the best, pH is the lowest of 3.34, organic acid content is the highest of 62.10 mg/mL, the number of viable spoilage fungus is "the lowest of 5.00×106 CFU/mL, the formation amount of spoilage fungus biofilm is the lowest of 0.021 g, and the types and content of biogenic amines are also relatively small. Different lactic acid bacteria have varying abilities to inhibit pks, and qj has a significant inhibitory effect. This study has provided references for fermented food biological method to inhibit the spoilage of northeast sauerkraut.
Key words: Lactobacillus sakei; spoilage fungus; Pichia kudriavzevii; sauerkraut
收稿日期:2024-11-21
作者簡介:陀小莉(1997—),女,碩士,研究方向:傳統(tǒng)食品發(fā)酵。
*通信作者:范洪臣(1978—),男,講師,博士,研究方向:傳統(tǒng)食品發(fā)酵。
酸菜是利用鹽漬白菜的汁液自然發(fā)酵而成的一種傳統(tǒng)食品[1]。由于制作過程中蔬菜原料未經(jīng)滅菌處理,原料中存在的多種微生物使酸菜發(fā)酵過程不可控,導致發(fā)酵時間延長和產(chǎn)品質量不穩(wěn)定,甚至發(fā)生腐敗變質,進而成為工業(yè)發(fā)酵的瓶頸[2—3]。腐敗菌一般在發(fā)酵后期利用酸菜鹽鹵中殘留的糖或有機酸作為碳源或能源繁殖代謝,使酸菜體系的pH升高,形成腐敗膜醭,產(chǎn)生令人不悅的氣味;代謝產(chǎn)生的植物細胞壁降解酶使酸菜組織軟化,導致酸菜腐敗變質[4]。酸菜中常見的腐敗菌有畢赤酵母、白地霉和芽孢桿菌等,特別是庫德里阿茲威畢赤酵母(Pichia kudriavzevii)會導致酸菜成膜、變軟和產(chǎn)生異味等,從而導致酸菜發(fā)生腐敗變質現(xiàn)象[5]。Moon等[6]研究發(fā)現(xiàn)從過熟泡菜中分離出的庫德里阿茲威畢赤酵母(Pichia kudriavzevii)導致成膜和產(chǎn)生異味等質量缺陷。饒瑜等[7]研究發(fā)現(xiàn)腐敗泡菜中畢赤酵母屬是產(chǎn)生膜醭的主要真菌,導致泡菜質地軟化和產(chǎn)生異味。腐敗菌存在于酸菜中會造成潛在的食品安全問題,并導致重大的經(jīng)濟損失[8]。
目前控制酸菜腐敗菌的方法主要有化學法、物理法和生物法,化學法和物理法因操作簡便成為主要方法,生物法因其安全性成為近些年主要的研究方向?;瘜W法中,采用高鹽度酸洗能夠抑制腐敗菌,但此方法也有明顯的缺點,如限制了某些正常發(fā)酵菌的生長,從而抑制獨特風味的形成;物理法中,熱處理是最常用的方法,但該方法也會導致酸菜的感官特性和營養(yǎng)特性損失。近年來,研究人員一直在尋找天然防腐劑,以取代目前控制酸菜腐敗菌的化學法或物理法。乳酸菌是發(fā)酵食品主要的生產(chǎn)菌種并且在食品領域作為天然生物防腐劑顯示出良好的潛力[9]。乳酸菌作為酸菜發(fā)酵的主導菌株,既能正常發(fā)酵酸菜又能抑制酸菜腐敗菌的生長,對酸菜的標準化生產(chǎn)具有重要意義。酸的產(chǎn)生被認為是乳酸菌抑制腐敗菌最重要的機制,有機酸的抑制作用是由未解離的分子引起的,這些分子在低pH環(huán)境下占主導地位,有機酸滲入細胞膜導致細胞內pH降低[10]。此外,乳酸菌能產(chǎn)生抑菌物質,如低分子量代謝物、抗真菌化合物和細菌素,有效抑制食品中腐敗菌的生長[11];二氧化碳能促進厭氧環(huán)境的形成,抑制對氧氣具有偏好性的腐敗菌,二氧化碳溶于細胞液中使pH下降,導致細胞內酶活性下降,影響細胞代謝,降低細胞膜的傳遞能力[12];雙乙酰對革蘭氏陽性和革蘭氏陰性細菌均有抗菌作用[12];細菌素能夠影響細胞膜的完整性,對敏感細菌細胞的殺菌作用主要是細胞質膜功能不穩(wěn)定的結果[13]。從植物乳桿菌中提取的有機酸、脂肪酸、細菌素、環(huán)二肽和酚類化合物已被證明有抑制腐敗菌的作用[14—15]。Li等[16]從植物乳桿菌CCFM8724的無細胞上清液中分離出兩個環(huán)二肽,發(fā)現(xiàn)它們能降低白色念珠菌毒力基因的表達,減少生物膜的形成。Yang等[8]研究發(fā)現(xiàn)Lactobacillus fermentum SHY10代謝物可以抑制泡菜的白膜生成。Dong等[17]發(fā)現(xiàn)乳酸鏈球菌的細菌素抑制了即食火腿和生菜上單核細胞增生李斯特菌。
東北酸菜作為酸菜的典型代表,以其獨特的風味和潛在的健康益處在我國北方被廣泛食用,但也存在腐敗菌污染酸菜的問題。篩選具有抑制東北酸菜腐敗菌的乳酸菌,既能有效控制東北酸菜腐敗的發(fā)生,又能改善產(chǎn)品品質,這將對酸菜的標準化生產(chǎn)具有重要意義。但迄今為止,關于對腐敗菌庫德里阿茲威畢赤酵母具有抑制作用的乳酸菌研究較少,對東北酸菜腐敗的影響尚不清楚。本研究針對傳統(tǒng)東北酸菜的腐敗問題,通過篩選能夠抑制庫德里阿茲威畢赤酵母的乳酸菌,再將乳酸菌與腐敗菌共發(fā)酵,通過酸菜發(fā)酵過程中pH、腐敗菌活菌數(shù)、生物膜形成量、有機酸含量和生物胺含量的變化,研究酸菜腐敗是否受到抑制。本研究可為開發(fā)生物法抑制東北酸菜腐敗提供方法參考。
1材料與方法
1.1材料與試劑
酸菜汁來源:將實驗室自然發(fā)酵成熟的、無腐敗菌污染的酸菜榨汁,離心、膜過濾后取上清液備用。
腐敗菌(1株):分離自哈爾濱市道里區(qū)自然發(fā)酵的腐敗酸菜樣品,Pichia kudriavzevii HSDF-pks-2022(pks)。
乳酸菌(7株):分離自哈爾濱市阿城區(qū)農家自然發(fā)酵成熟的酸菜樣品,Lactiplantibacillus paraplantarum HSDF-lpo-2022(lpo)、Pediococcus parvulus HSDF-ppo-2022(xp)、Levilactobacillus brevis HSDF-lbn-2022(lbn)、Lactobacillus sakei HSDF-lso-2022(qj)、Leuconostoc mesenteroides HSDF-lmo-2022(cm)、Loigolactobacillus coryniformis HSDF-lco-2022(rs)、Lactiplantibacillus xiangfangensis HSDF-lxo-2022(xf)。
PDA培養(yǎng)基:用于真菌腐敗菌的計數(shù);YPD液體培養(yǎng)基:以1%酵母膏、2%蛋白胨、2%葡萄糖配制,用于真菌的活化和培養(yǎng);MRS肉湯:用于乳酸菌的活化;生物胺標準溶液為鹽酸β-苯乙胺、鹽酸腐胺、鹽酸尸胺、鹽酸組胺、鹽酸酪胺、鹽酸三精胺、鹽酸四精胺:同路生物制藥股份有限公司;其他試劑均為國產(chǎn)分析純。
1.2儀器與設備
YXQ-70A立式壓力蒸汽滅菌器上海博迅醫(yī)療生物儀器股份有限公司;BSA223S電子天平賽多利斯科學儀器(北京)有限公司;THZ98AB恒溫振蕩器、LRH-250生化培養(yǎng)箱上海一恒科學儀器有限公司;DL-CJ-1ND1L超凈工作臺北京東聯(lián)哈爾儀器制造有限公司;DK-98-ⅡA電熱恒溫水浴鍋天津市泰斯特儀器有限公司;Vortex-2漩渦振蕩儀長沙米淇儀器設備有限公司;PH-20 pH計杭州杰源儀器科技有限公司;LC-2030 Plus高效液相色譜儀島津企業(yè)管理(中國)有限公司。
1.3方法
1.3.1抑制庫德里阿茲威畢赤酵母的乳酸菌篩選
1.3.1.1乳酸菌無細胞上清液的制備
將冷凍保存的乳酸菌活化后,按0.1%的接種量接種到MRS肉湯中,37 ℃培養(yǎng)24 h,8 000 r/min離心5 min,取無細胞上清液備用[18]。
1.3.1.2庫德里阿茲威畢赤酵母菌液的制備
將冷凍保存的pks按1%的接種量接種到YPD液體培養(yǎng)基中,置于28 ℃、150 r/min恒溫搖床中振蕩培養(yǎng)24 h活化后備用。
1.3.1.3抑菌圈的測定
參考敖曉琳等[19]的方法測定乳酸菌上清液對pks的抑制效果。將pks懸浮液稀釋至105 CFU/mL后與50 ℃的PDA培養(yǎng)基混合,再將混合物(20 mL)倒入PDA平板中,用鑷子將牛津杯置于平板上,加入50 μL乳酸菌上清液。以MRS肉湯作為對照組,置于28 ℃培養(yǎng)箱中培養(yǎng)24 h,用游標卡尺測量孔周圍不透明圓的直徑,試驗重復3次,用抑菌圈大小反映乳酸菌抑制pks的效果。
1.3.1.4共發(fā)酵篩選抑制庫德里阿茲威畢赤酵母的乳酸菌
無腐敗菌污染的東北酸菜經(jīng)過榨汁、離心和膜過濾后,以2%接種量pks作為對照組,以2%接種量乳酸菌和pks作為試驗組,12 ℃密封發(fā)酵7 d。發(fā)酵結束后,通過測定酸菜發(fā)酵液pH、pks活菌數(shù)、生物膜形成量、有機酸含量和生物胺含量,篩選抑制pks的乳酸菌。
rs-pks、lbn-pks、qj-pks、cm-pks和xf-pks分別代表乳酸菌Loigolactobacillus coryniformis(rs)、Levilactobacillus brevis(lbn)、Lactobacillus sakei(qj)、Leuconostoc mesenteroides(cm)、Lactiplantibacillus xiangfangensis(xf)和腐敗菌Pichia kudriavzevii(pks)在酸菜中共發(fā)酵。
1.3.2腐敗菌活菌數(shù)的測定
按照GB 4789.15—2016[20]規(guī)定,利用平板計數(shù)法對pks在PDA培養(yǎng)基上生長的菌落進行計數(shù)。
1.3.3腐敗菌生物膜形成量的測定
采用楊露等[21]的方法,觀察酸菜發(fā)酵液液面是否有生物膜形成,利用生物膜干重法測定。
1.3.4pH的測定
酸菜樣品的pH直接使用PH-20 pH計進行測定。
1.3.5有機酸含量的測定
按照Luo等[22]的方法,酸菜樣品中有機酸含量采用高效液相色譜法測定。
1.3.6生物胺含量的測定
按照Jin等[23]的方法,酸菜樣品中生物胺含量采用高效液相色譜法測定。
1.4數(shù)據(jù)分析
采用Origin 2021軟件進行直方圖分析。采用IBM SPSS 25.0軟件進行Spearman相關系數(shù)分析,所有試驗重復3次。
2結果與討論
2.1不同乳酸菌無細胞代謝物對腐敗菌的抑制
采用牛津杯法抑菌試驗可以從大量試驗菌中快速篩選出對腐敗菌有抑制作用的有效菌,提高效率。本研究采用牛津杯法對7株乳酸菌進行了抑制腐敗菌試驗,結果見表1。
由表1可知,7株乳酸菌無細胞代謝物均對pks有抑菌效果,其中5株乳酸菌rs、lbn、qj、cm、xf對pks產(chǎn)生的抑菌圈較大,說明這5株乳酸菌均對pks具有較好的抑制能力。乳酸菌通過自身的生長繁殖和代謝產(chǎn)物(有機酸、細菌素等)的作用抑制腐敗菌[11]。為了確定乳酸菌抑制腐敗菌效果是代謝物起到抑菌作用,研究人員將乳酸菌懸浮液離心,將腐敗菌與乳酸菌無細胞代謝物共培養(yǎng),然后在抑菌試驗中測試無細胞上清液粗提取物[12,24],證實了乳酸菌代謝物能夠抑制腐敗菌。例如,Song等[25]研究發(fā)現(xiàn)L. plantarum Y42無細胞上清液通過中斷單核細胞增生李斯特菌的代謝,對單核細胞增生李斯特菌生物膜形成有抑制作用。Rahmati-Joneidabad等[26]發(fā)現(xiàn)Levilactobacillus brevis具有明顯的抗菌活性,Levilactobacillus brevis無細胞上清液可有效抑制Listeria monocytogenes生物膜的形成,本研究結果與上述研究結果類似,初步確定乳酸菌代謝物對腐敗菌具有抑菌效果,不同乳酸菌的抑菌效果有較大差異。牛津杯法操作簡單,可直觀地判定乳酸菌代謝物對pks的抑菌效果,但是不能具體反映出乳酸菌抑制pks的發(fā)酵差異,因此利用乳酸菌和pks共同發(fā)酵酸菜來檢驗所篩選的乳酸菌抑制腐敗菌的實際效果。
2.2不同乳酸菌與腐敗菌共發(fā)酵對腐敗菌的抑制作用
2.2.1不同乳酸菌對酸菜腐敗菌活菌數(shù)的影響
乳酸菌通過抑制腐敗菌的活菌數(shù)減少生物膜的形成,不同乳酸菌對pks活菌數(shù)的影響見圖1。
由圖1可知,試驗組與對照組相比,pks活菌數(shù)均減少,與牛津杯抑菌圈法的試驗結果一致,表明5株乳酸菌均對pks的活菌數(shù)有抑制效果,其中qj-pks試驗組的pks活菌數(shù)最少,為5.00×106 CFU/mL,其他乳酸菌抑制pks活菌數(shù)由低到高為xf-pkslt;lbn-pkslt;cm-pkslt;rs-pks。pks在發(fā)酵后期利用殘留的糖或有機酸等代謝物大量繁殖,導致酸菜出現(xiàn)成膜、沉淀、渾濁等變質現(xiàn)象。pks的活菌數(shù)與酸菜是否產(chǎn)膜醭和鹵水的渾濁度具有相關性,活菌數(shù)越多,酸菜發(fā)酵液表面形成膜醭的程度和鹵水的渾濁度越嚴重[19]。pks活菌數(shù)與乳酸菌的種類和數(shù)量有關,乳酸菌抗菌代謝物在發(fā)酵食品中作為天然防腐劑,可抑制腐敗菌的生長[27-28]。例如,Mauriello等[29]發(fā)現(xiàn)Lactobacillus curvatus 32Y產(chǎn)生的細菌素通過降低豬肉中單核細胞增生李斯特菌的數(shù)量抑制腐敗菌的生長。Zhang等[30]研究發(fā)現(xiàn)在低鹽香腸中接種植物乳桿菌發(fā)酵顯著抑制了多種腐敗菌(如環(huán)絲菌屬和假單胞菌屬)的活菌數(shù)。Choi等[31]研究發(fā)現(xiàn)Leconostoc mesenteroides 13374和Lactobacillus plantarum 33133代謝物對大腸桿菌具有抗菌活性、乳酸菌快速生長和代謝產(chǎn)物的作用,導致泡菜中大腸桿菌活菌數(shù)急劇減少。Wang等[32]研究發(fā)現(xiàn)接種清酒乳桿菌能夠抑制改良氣調包裝(MAP)煙熏雞腿中常見腐敗菌如變形沙雷氏菌的活菌數(shù),使肉制品的貨架期延長。Park等[33]研究發(fā)現(xiàn)泡菜中的清酒乳桿菌NR28能夠有效抑制大腸埃希氏菌ATCC-43894的活菌數(shù)。Strm等[34]研究發(fā)現(xiàn)清酒乳桿菌酶解產(chǎn)生的環(huán)肽可有效抑制真菌腐敗菌的活菌數(shù)。
本研究得到不同乳酸菌抑制pks的活菌數(shù)不同,尤其是qj使pks活菌數(shù)最少,推測原因是乳酸菌代謝物不同,具有抑菌活性的代謝物抑制pks的生長,使食品的保質期延長[35]。
2.2.2不同乳酸菌對酸菜腐敗菌生物膜形成量的影響
膜的形成是東北酸菜產(chǎn)品腐敗變質最典型的特征,成膜通過軟化東北酸菜的質地和產(chǎn)生異味,顯著降低了東北酸菜的感官品質。pks在東北酸菜中形成生物膜,成膜量越高,對東北酸菜生產(chǎn)的危害越大。不同乳酸菌對pks生物膜形成量的影響見圖2。
由圖2可知,試驗組與對照組pks相比,生物膜形成量都減少,表明5株乳酸菌均對pks的生物膜形成量有抑制效果,其中qj-pks的生物膜形成量最小,為0.021 g,其他乳酸菌抑制pks生物膜形成量由低到高為xf-pkslt;lbn-pkslt;cm-pkslt;rs-pks。發(fā)酵后的蔬菜在形成生物膜后會變軟、腐爛、發(fā)臭,最終影響發(fā)酵蔬菜的可食性和安全性[8]。pks的生物膜形成量與乳酸菌的種類和數(shù)量有關,乳酸菌的代謝產(chǎn)物如有機酸、細菌素和抗菌肽通過抑制產(chǎn)膜菌株的數(shù)量和代謝從而抑制生物膜形成量,乳酸菌抑制腐敗菌成膜也有報道,如乳酸菌通過分泌黏蛋白增強自身黏附定植能力,競爭性地排斥腐敗菌,有效抑制腐敗菌生物膜的形成及對細胞表面的黏附作用,產(chǎn)生抗菌物質,抑制腐敗菌的生長和增殖[36]。Woo等[37]研究發(fā)現(xiàn)嗜酸乳桿菌KACC 12419通過競爭、排斥和置換作用,有效減少了腸炎沙門氏菌亞種生物膜的形成。Lin等[38]從安徽雞西酸豇豆中篩選的發(fā)酵乳桿菌AJS2-4可抑制酸豇豆中嗜水氣單胞菌的群體感應和生物膜形成。Yang等[39]從酸菜中分離的植物乳桿菌4-10產(chǎn)生細菌素,抑制單核細胞增生李斯特菌膜運輸相關基因crr、agrB和dltC的表達,破壞細胞膜的完整性,限制生物膜的形成,抑制單核細胞增生李斯特菌的生長。Lian等[40]研究發(fā)現(xiàn)Limmosilactobacillus fermentum SHY10代謝物誘導細胞膜凹陷,導致產(chǎn)膜菌株死亡,抑制泡菜生物膜的形成。Settier-Ramírez等[41]研究發(fā)現(xiàn)在低pH環(huán)境下,清酒乳桿菌TH1對未解離的乳酸有抑菌作用,抑制真空包裝香腸中單增李斯特菌的生長。Bredholt等[42]研究發(fā)現(xiàn)清酒乳桿菌能夠抑制真空包裝的切片熟火腿中大腸桿菌O157∶H7的生長,抑制生物膜的形成。
本研究得到了不同乳酸菌抑制pks生物膜形成量的結果,尤其是qj使pks的生物膜形成量最少,為生物法抑制酸菜腐敗提供了良好的菌株。
2.2.3不同乳酸菌對酸菜pH的影響
pH的變化與酸菜腐敗有關,也是衡量酸菜腐敗的重要參數(shù)[43]。當酸菜發(fā)酵鹵水的pH降至4.0以下時,被認為酸菜發(fā)酵成熟,此時酸菜適宜食用[5]。在本研究中,酸菜發(fā)酵液的初始pH為3.82,是發(fā)酵正常、未發(fā)生腐敗的酸菜。Kim等[44]、Johanningsmeier等[45]和Franco等[46]研究發(fā)現(xiàn),pH 4.6~5.0是酸菜腐敗惡化的敏感區(qū)域,腐敗菌大量繁殖。不同乳酸菌對酸菜pH的影響見圖3。
由圖3可知,試驗組與對照組pks相比,pH都降低,但程度不同,其中qj-pks的pH最低,為3.34,其他乳酸菌降低酸菜pH由低到高為xf-pkslt;lbn-pkslt;cm-pkslt;rs-pks。對照組pks的pH大于4.6,酸菜腐敗變質,驗證了Kim等[41]、Johanningsmeier等[45]和Franco等[46]的結論,推測pks在發(fā)酵后期利用殘留的糖或有機酸等代謝物生長繁殖,使pH升高,引起酸菜腐敗變質[4]。不同乳酸菌的產(chǎn)酸種類和含量不同導致pH不同。pH的降低會影響腐敗菌細胞膜的結構和通透性,導致細胞內部代謝物和蛋白質合成等細胞活動泄漏,并對非嗜酸菌物種產(chǎn)生選擇性屏障[13]。Hwanhlem等[47]證實了乳酸菌KT2W2L通過降低pH抑制腐敗菌的生長;腸膜明串珠菌L124和彎曲乳桿菌L442能夠產(chǎn)生細菌素,細菌素與低pH環(huán)境協(xié)同抑制腐敗菌的生長[48-49]。
本研究中qj發(fā)酵酸菜的pH最低,抑菌效果最好,其他發(fā)酵蔬菜研究中也有報道清酒乳桿菌該特點,主要歸結于清酒乳桿菌pH低、活菌數(shù)高,如Yang等[50]研究發(fā)現(xiàn),清酒乳桿菌在復雜的厭氧和酸性條件下一直處于絕對優(yōu)勢地位。
2.2.4不同乳酸菌對酸菜有機酸含量的影響
乳酸菌產(chǎn)生有機酸是抑制腐敗菌生長的主要機制之一,未解離的有機酸分子在低pH環(huán)境下占主導地位,通過競爭作用、增加腐敗菌細胞膜的通透性、改變細胞內滲透壓、抑制大分子的合成等方式來抑制腐敗菌生長[51]。乳酸菌發(fā)酵產(chǎn)生的乳酸、檸檬酸和乙酸等有機酸具有特定的抑制腐敗菌活性,被用作食品防腐劑[52]。不同乳酸菌對酸菜中有機酸含量的影響見圖4。
由圖4可知,試驗組與對照組pks相比,有機酸含量均增加,其中qj-pks的有機酸含量最多,為62.10 mg/mL,其他乳酸菌增加酸菜中有機酸含量由低到高為xf-pkslt;rs-pkslt;cm-pkslt;lbn-pks。與對照組pks相比,乳酸、檸檬酸和乙酸比例大的試驗組抑菌效果較好。腐敗菌能夠利用乳酸和乙酸,導致pH上升和發(fā)酵底物還原,使酸菜發(fā)生腐敗變質,但從試驗數(shù)據(jù)來看,pks只有在pH適中、有機酸總量不高和乳酸、檸檬酸、乙酸含量不高的情況下才能利用某些有機酸作為能源進行繁殖代謝[4]。圖4可以充分證明有機酸的種類和數(shù)量與乳酸菌的種類有關,乳酸在低pH下有較強的抗菌作用,對細胞膜具有滲透性,從而增強其他抗菌物質的作用[53];檸檬酸在低pH下使腐敗菌細胞膜電荷改變,干擾腐敗菌細胞中酶的某些功能,通過抑制能量代謝中磷酸化酶的功能從而達到抑菌的作用[54];乙酸是異型乳酸發(fā)酵和乙醇發(fā)酵的副產(chǎn)物,對真菌腐敗菌具有抑制活性。同型發(fā)酵乳酸菌(如植物乳桿菌)發(fā)酵產(chǎn)生乳酸[4],異型發(fā)酵乳酸菌(如腸膜明串珠菌)代謝產(chǎn)物包括乳酸、乙酸、CO2和乙醇[55]。乳酸、檸檬酸和乙酸是qj主要的抗菌代謝產(chǎn)物,通過降低環(huán)境pH,破壞細胞內微環(huán)境和代謝途徑,抑制腐敗菌生長[56]。例如,Guo等[56]研究發(fā)現(xiàn)乳酸、檸檬酸和乙酸3種有機酸對沙門氏菌代謝的影響無顯著性差異,能有效降低黃瓜中沙門氏菌的數(shù)量,破壞細胞膜,降低環(huán)境pH,抑制沙門氏菌的生長;在新鮮農產(chǎn)品中,乳酸、乙酸和檸檬酸能夠抑制腐敗菌生長[57]。
本研究中qj所產(chǎn)的有機酸含量最高,乳酸、檸檬酸和乙酸含量也最高,抑制pks的效果最好,推測主要原因是清酒乳桿菌具有更廣泛的代謝碳水化合物產(chǎn)酸、產(chǎn)細菌素的能力,從而具有潛在的生存優(yōu)勢[58]。例如,Kim等[59]研究發(fā)現(xiàn)清酒乳桿菌能夠使糖完全酵解,具有6個磷酸葡萄糖酸鹽/磷酸酮醇酶途徑、乳酸脫氫酶基因和1個不完整的三羧酸循環(huán),表明清酒乳桿菌同時參與同型發(fā)酵和異型發(fā)酵,具有產(chǎn)生大量有機酸的能力。
2.2.5不同乳酸菌對酸菜生物胺含量的影響
腐敗菌可以將酸菜中的氨基酸脫羧為生物胺(BAs),飲食中攝入高水平BAs的食物與許多不利的健康影響有關,如偏頭痛、血壓升高和心跳加速[60-62]。不同乳酸菌對生物胺含量的影響見圖5。
由圖5可知,試驗組與對照組pks相比,xf-pks生物胺含量和種類最少,分別為酪胺31.17 mg/L、組胺5.39 mg/L、尸胺6.66 mg/L和腐胺7.56 mg/L,其他組生物胺含量由低到高為qj-pkslt;cm-pkslt;rs-pkslt;lbn-pks。試驗組xf-pks、qj-pks和cm-pks的酪胺、組胺和總生物胺含量均未超過推薦限量(組胺和酪胺含量lt;100 mg/kg;總生物胺含量lt;1 000 mg/kg)[63-64]。研究結果表明,不同乳酸菌導致pks生物胺的種類和含量不同,推測不同乳酸菌抑制pks代謝產(chǎn)生物胺的能力不同或者對生物胺的降解能力不同。從對照組試驗得出pks可以將氨基酸脫羧為生物胺,主要包括組胺、尸胺、腐胺或酪胺;選擇能夠抑制脫羧腐敗菌生長和活性的乳酸菌是抑制發(fā)酵食品中生物胺積累的主要策略[65]。利用乳酸菌降低生物胺的應用也比較常見,例如,Alvarez等[66]研究發(fā)現(xiàn)乳酸菌具有通過胺降解酶(如氨基氧化酶)的作用降解生物胺的能力。Peas等[67]在酸菜中接種腸膜明串珠菌和植物乳桿菌進行發(fā)酵,發(fā)現(xiàn)接種腸膜明串珠菌的酸菜生物胺含量明顯降低。Zhang等[68]發(fā)現(xiàn)含有胺氧化酶的乳酸菌發(fā)酵劑使發(fā)酵香腸中的生物胺含量降低38.79%~50.29%。
本研究中qj能有效抑制酸菜中生物胺含量,雖然效果不如xf,但綜合考慮其他發(fā)酵指標,明顯提高了酸菜的安全性。清酒乳桿菌常用于泡菜生產(chǎn),同時發(fā)現(xiàn)具有明顯降低生物胺含量的效果,例如,Lee等[69]從泡菜中分離清酒乳桿菌,該菌產(chǎn)生的3-磷酸甘油醛脫氫酶能夠降解組胺的含量。Kim等[70]研究發(fā)現(xiàn)清酒乳桿菌接入泡菜發(fā)酵后,能夠顯著降低生物胺含量。
3結論
本研究通過牛津杯抑菌圈法研究不同乳酸菌無細胞代謝物對pks的抑制作用,從7株乳酸菌中篩選出5株抑制pks明顯的乳酸菌,利用乳酸菌和pks共同發(fā)酵酸菜來檢驗所篩選乳酸菌抑制腐敗菌的實際效果。結果顯示,根據(jù)發(fā)酵液pH、有機酸含量、腐敗菌活菌數(shù)、生物膜形成量和生物胺含量情況,從5株乳酸菌中篩選出的乳酸菌qj抑制pks的效果顯著。試驗組與對照組相比,乳酸菌qj與腐敗菌pks共發(fā)酵的酸菜發(fā)酵液的pH、pks活菌數(shù)、生物膜形成量均最低,生物胺含量顯著降低,酪胺、組胺和總生物胺含量均未超過推薦限量;有機酸含量最高,主要的有機酸為乳酸、乙酸和檸檬酸,抑制pks的效果最好。綜上表明Lactobacillus sakei HSDF-lso-2022(qj)作為生物防腐劑通過多種機制抑制酸菜腐敗菌庫德里阿茲威畢赤酵母的生長,能夠改善酸菜的發(fā)酵特性和感官品質,有效延長食品的保質期,提高食品的安全性,本研究為發(fā)酵食品生物法抑制東北酸菜腐敗提供了借鑒。
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