摘要:庫(kù)德里阿茲威畢赤酵母(Pichia kudriavzevii)是一種常見(jiàn)的東北酸菜腐敗菌,導(dǎo)致東北酸菜成膜、變軟和產(chǎn)生異味等質(zhì)量缺陷。從自然發(fā)酵的東北酸菜中分離出7株乳酸菌,對(duì)Pichia kudriavzevii HSDF-pks-2022(pks)進(jìn)行抑制和發(fā)酵影響的研究。通過(guò)對(duì)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L(zhǎng)evilactobacillus 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(guò)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(jī)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(jī)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(lèi)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(guān)鍵詞:清酒乳桿菌;腐敗菌;庫(kù)德里阿茲威畢赤酵母;酸菜
中圖分類(lèi)號(hào):TS201.3文獻(xiàn)標(biāo)志碼:A文章編號(hào):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(jiǎn)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(guò)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(guò)程不可控,導(dǎo)致發(fā)酵時(shí)間延長(zhǎng)和產(chǎn)品質(zhì)量不穩(wěn)定,甚至發(fā)生腐敗變質(zhì),進(jìn)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(jī)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(xì)胞壁降解酶使酸菜組織軟化,導(dǎo)致酸菜腐敗變質(zhì)[4]。酸菜中常見(jiàn)的腐敗菌有畢赤酵母、白地霉和芽孢桿菌等,特別是庫(kù)德里阿茲威畢赤酵母(Pichia kudriavzevii)會(huì)導(dǎo)致酸菜成膜、變軟和產(chǎn)生異味等,從而導(dǎo)致酸菜發(fā)生腐敗變質(zhì)現(xiàn)象[5]。Moon等[6]研究發(fā)現(xiàn)從過(guò)熟泡菜中分離出的庫(kù)德里阿茲威畢赤酵母(Pichia kudriavzevii)導(dǎo)致成膜和產(chǎn)生異味等質(zhì)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(dǎo)致泡菜質(zhì)地軟化和產(chǎn)生異味。腐敗菌存在于酸菜中會(huì)造成潛在的食品安全問(wèn)題,并導(dǎo)致重大的經(jīng)濟(jì)損失[8]。
目前控制酸菜腐敗菌的方法主要有化學(xué)法、物理法和生物法,化學(xué)法和物理法因操作簡(jiǎn)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(xué)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(diǎn),如限制了某些正常發(fā)酵菌的生長(zhǎng),從而抑制獨(dú)特風(fēng)味的形成;物理法中,熱處理是最常用的方法,但該方法也會(huì)導(dǎo)致酸菜的感官特性和營(yíng)養(yǎng)特性損失。近年來(lái),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(xué)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(lǐng)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(dǎo)菌株,既能正常發(fā)酵酸菜又能抑制酸菜腐敗菌的生長(zhǎng),對(duì)酸菜的標(biāo)準(zhǔn)化生產(chǎn)具有重要意義。酸的產(chǎn)生被認(rèn)為是乳酸菌抑制腐敗菌最重要的機(jī)制,有機(jī)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(dǎo)地位,有機(jī)酸滲入細(xì)胞膜導(dǎo)致細(xì)胞內(nèi)pH降低[10]。此外,乳酸菌能產(chǎn)生抑菌物質(zhì),如低分子量代謝物、抗真菌化合物和細(xì)菌素,有效抑制食品中腐敗菌的生長(zhǎng)[11];二氧化碳能促進(jìn)厭氧環(huán)境的形成,抑制對(duì)氧氣具有偏好性的腐敗菌,二氧化碳溶于細(xì)胞液中使pH下降,導(dǎo)致細(xì)胞內(nèi)酶活性下降,影響細(xì)胞代謝,降低細(xì)胞膜的傳遞能力[12];雙乙酰對(duì)革蘭氏陽(yáng)性和革蘭氏陰性細(xì)菌均有抗菌作用[12];細(xì)菌素能夠影響細(xì)胞膜的完整性,對(duì)敏感細(xì)菌細(xì)胞的殺菌作用主要是細(xì)胞質(zhì)膜功能不穩(wěn)定的結(jié)果[13]。從植物乳桿菌中提取的有機(jī)酸、脂肪酸、細(xì)菌素、環(huán)二肽和酚類(lèi)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(wú)細(xì)胞上清液中分離出兩個(gè)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(dá),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(xì)菌素抑制了即食火腿和生菜上單核細(xì)胞增生李斯特菌。
東北酸菜作為酸菜的典型代表,以其獨(dú)特的風(fēng)味和潛在的健康益處在我國(guó)北方被廣泛食用,但也存在腐敗菌污染酸菜的問(wèn)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(zhì),這將對(duì)酸菜的標(biāo)準(zhǔn)化生產(chǎn)具有重要意義。但迄今為止,關(guān)于對(duì)腐敗菌庫(kù)德里阿茲威畢赤酵母具有抑制作用的乳酸菌研究較少,對(duì)東北酸菜腐敗的影響尚不清楚。本研究針對(duì)傳統(tǒng)東北酸菜的腐敗問(wèn)題,通過(guò)篩選能夠抑制庫(kù)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(guò)酸菜發(fā)酵過(guò)程中pH、腐敗菌活菌數(shù)、生物膜形成量、有機(jī)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(kāi)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(lái)源:將實(shí)驗(yàn)室自然發(fā)酵成熟的、無(wú)腐敗菌污染的酸菜榨汁,離心、膜過(guò)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(nóng)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(jì)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(biāo)準(zhǔn)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(guó)產(chǎn)分析純。
1.2儀器與設(shè)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(xué)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(xué)儀器有限公司;DL-CJ-1ND1L超凈工作臺(tái)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(zhǎng)沙米淇?jī)x器設(shè)備有限公司;PH-20 pH計(jì)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(guó))有限公司。
1.3方法
1.3.1抑制庫(kù)德里阿茲威畢赤酵母的乳酸菌篩選
1.3.1.1乳酸菌無(wú)細(xì)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(wú)細(xì)胞上清液備用[18]。
1.3.1.2庫(kù)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Y(jié)PD液體培養(yǎng)基中,置于28 ℃、150 r/min恒溫?fù)u床中振蕩培養(yǎng)24 h活化后備用。
1.3.1.3抑菌圈的測(cè)定
參考敖曉琳等[19]的方法測(cè)定乳酸菌上清液對(duì)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(duì)照組,置于28 ℃培養(yǎng)箱中培養(yǎng)24 h,用游標(biāo)卡尺測(cè)量孔周?chē)煌该鲌A的直徑,試驗(yàn)重復(fù)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(kù)德里阿茲威畢赤酵母的乳酸菌
無(wú)腐敗菌污染的東北酸菜經(jīng)過(guò)榨汁、離心和膜過(guò)濾后,以2%接種量pks作為對(duì)照組,以2%接種量乳酸菌和pks作為試驗(yàn)組,12 ℃密封發(fā)酵7 d。發(fā)酵結(jié)束后,通過(guò)測(cè)定酸菜發(fā)酵液pH、pks活菌數(shù)、生物膜形成量、有機(jī)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(cè)定
按照GB 4789.15—2016[20]規(guī)定,利用平板計(jì)數(shù)法對(duì)pks在PDA培養(yǎng)基上生長(zhǎng)的菌落進(jìn)行計(jì)數(shù)。
1.3.3腐敗菌生物膜形成量的測(cè)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(cè)定。
1.3.4pH的測(cè)定
酸菜樣品的pH直接使用PH-20 pH計(jì)進(jìn)行測(cè)定。
1.3.5有機(jī)酸含量的測(cè)定
按照Luo等[22]的方法,酸菜樣品中有機(jī)酸含量采用高效液相色譜法測(cè)定。
1.3.6生物胺含量的測(cè)定
按照J(rèn)in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(cè)定。
1.4數(shù)據(jù)分析
采用Origin 2021軟件進(jìn)行直方圖分析。采用IBM SPSS 25.0軟件進(jìn)行Spearman相關(guān)系數(shù)分析,所有試驗(yàn)重復(fù)3次。
2結(jié)果與討論
2.1不同乳酸菌無(wú)細(xì)胞代謝物對(duì)腐敗菌的抑制
采用牛津杯法抑菌試驗(yàn)可以從大量試驗(yàn)菌中快速篩選出對(duì)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(duì)7株乳酸菌進(jìn)行了抑制腐敗菌試驗(yàn),結(jié)果見(jiàn)表1。
由表1可知,7株乳酸菌無(wú)細(xì)胞代謝物均對(duì)pks有抑菌效果,其中5株乳酸菌rs、lbn、qj、cm、xf對(duì)pks產(chǎn)生的抑菌圈較大,說(shuō)明這5株乳酸菌均對(duì)pks具有較好的抑制能力。乳酸菌通過(guò)自身的生長(zhǎng)繁殖和代謝產(chǎn)物(有機(jī)酸、細(xì)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(wú)細(xì)胞代謝物共培養(yǎng),然后在抑菌試驗(yàn)中測(cè)試無(wú)細(xì)胞上清液粗提取物[12,24],證實(shí)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(wú)細(xì)胞上清液通過(guò)中斷單核細(xì)胞增生李斯特菌的代謝,對(duì)單核細(xì)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(wú)細(xì)胞上清液可有效抑制Listeria monocytogenes生物膜的形成,本研究結(jié)果與上述研究結(jié)果類(lèi)似,初步確定乳酸菌代謝物對(duì)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(jiǎn)單,可直觀地判定乳酸菌代謝物對(duì)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(lái)檢驗(yàn)所篩選的乳酸菌抑制腐敗菌的實(shí)際效果。
2.2不同乳酸菌與腐敗菌共發(fā)酵對(duì)腐敗菌的抑制作用
2.2.1不同乳酸菌對(duì)酸菜腐敗菌活菌數(shù)的影響
乳酸菌通過(guò)抑制腐敗菌的活菌數(shù)減少生物膜的形成,不同乳酸菌對(duì)pks活菌數(shù)的影響見(jiàn)圖1。
由圖1可知,試驗(yàn)組與對(duì)照組相比,pks活菌數(shù)均減少,與牛津杯抑菌圈法的試驗(yàn)結(jié)果一致,表明5株乳酸菌均對(duì)pks的活菌數(shù)有抑制效果,其中qj-pks試驗(yàn)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(jī)酸等代謝物大量繁殖,導(dǎo)致酸菜出現(xiàn)成膜、沉淀、渾濁等變質(zhì)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(guān)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(yán)重[19]。pks活菌數(shù)與乳酸菌的種類(lèi)和數(shù)量有關(guān),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(zhǎng)[27-28]。例如,Mauriello等[29]發(fā)現(xiàn)Lactobacillus curvatus 32Y產(chǎn)生的細(xì)菌素通過(guò)降低豬肉中單核細(xì)胞增生李斯特菌的數(shù)量抑制腐敗菌的生長(zhǎng)。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(duì)大腸桿菌具有抗菌活性、乳酸菌快速生長(zhǎng)和代謝產(chǎn)物的作用,導(dǎo)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?xì)庹{(diào)包裝(MAP)煙熏雞腿中常見(jiàn)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(zhǎng)。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(cè)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(zhǎng),使食品的保質(zhì)期延長(zhǎng)[35]。
2.2.2不同乳酸菌對(duì)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(zhì)最典型的特征,成膜通過(guò)軟化東北酸菜的質(zhì)地和產(chǎn)生異味,顯著降低了東北酸菜的感官品質(zhì)。pks在東北酸菜中形成生物膜,成膜量越高,對(duì)東北酸菜生產(chǎn)的危害越大。不同乳酸菌對(duì)pks生物膜形成量的影響見(jiàn)圖2。
由圖2可知,試驗(yàn)組與對(duì)照組pks相比,生物膜形成量都減少,表明5株乳酸菌均對(duì)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(huì)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(lèi)和數(shù)量有關(guān),乳酸菌的代謝產(chǎn)物如有機(jī)酸、細(xì)菌素和抗菌肽通過(guò)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(bào)道,如乳酸菌通過(guò)分泌黏蛋白增強(qiáng)自身黏附定植能力,競(jìng)爭(zhēng)性地排斥腐敗菌,有效抑制腐敗菌生物膜的形成及對(duì)細(xì)胞表面的黏附作用,產(chǎn)生抗菌物質(zhì),抑制腐敗菌的生長(zhǎng)和增殖[36]。Woo等[37]研究發(fā)現(xiàn)嗜酸乳桿菌KACC 12419通過(guò)競(jìng)爭(zhēng)、排斥和置換作用,有效減少了腸炎沙門(mén)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(yīng)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(xì)菌素,抑制單核細(xì)胞增生李斯特菌膜運(yùn)輸相關(guān)基因crr、agrB和dltC的表達(dá),破壞細(xì)胞膜的完整性,限制生物膜的形成,抑制單核細(xì)胞增生李斯特菌的生長(zhǎng)。Lian等[40]研究發(fā)現(xiàn)Limmosilactobacillus fermentum SHY10代謝物誘導(dǎo)細(xì)胞膜凹陷,導(dǎo)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(duì)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(zhǎng)。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(zhǎng),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(jié)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(duì)酸菜pH的影響
pH的變化與酸菜腐敗有關(guān),也是衡量酸菜腐敗的重要參數(shù)[43]。當(dāng)酸菜發(fā)酵鹵水的pH降至4.0以下時(shí),被認(rèn)為酸菜發(fā)酵成熟,此時(shí)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(duì)酸菜pH的影響見(jiàn)圖3。
由圖3可知,試驗(yàn)組與對(duì)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(duì)照組pks的pH大于4.6,酸菜腐敗變質(zhì),驗(yàn)證了Kim等[41]、Johanningsmeier等[45]和Franco等[46]的結(jié)論,推測(cè)pks在發(fā)酵后期利用殘留的糖或有機(jī)酸等代謝物生長(zhǎng)繁殖,使pH升高,引起酸菜腐敗變質(zhì)[4]。不同乳酸菌的產(chǎn)酸種類(lèi)和含量不同導(dǎo)致pH不同。pH的降低會(huì)影響腐敗菌細(xì)胞膜的結(jié)構(gòu)和通透性,導(dǎo)致細(xì)胞內(nèi)部代謝物和蛋白質(zhì)合成等細(xì)胞活動(dòng)泄漏,并對(duì)非嗜酸菌物種產(chǎn)生選擇性屏障[13]。Hwanhlem等[47]證實(shí)了乳酸菌KT2W2L通過(guò)降低pH抑制腐敗菌的生長(zhǎng);腸膜明串珠菌L124和彎曲乳桿菌L442能夠產(chǎn)生細(xì)菌素,細(xì)菌素與低pH環(huán)境協(xié)同抑制腐敗菌的生長(zhǎng)[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(bào)道清酒乳桿菌該特點(diǎn),主要?dú)w結(jié)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(fù)雜的厭氧和酸性條件下一直處于絕對(duì)優(yōu)勢(shì)地位。
2.2.4不同乳酸菌對(duì)酸菜有機(jī)酸含量的影響
乳酸菌產(chǎn)生有機(jī)酸是抑制腐敗菌生長(zhǎng)的主要機(jī)制之一,未解離的有機(jī)酸分子在低pH環(huán)境下占主導(dǎo)地位,通過(guò)競(jìng)爭(zhēng)作用、增加腐敗菌細(xì)胞膜的通透性、改變細(xì)胞內(nèi)滲透壓、抑制大分子的合成等方式來(lái)抑制腐敗菌生長(zhǎng)[51]。乳酸菌發(fā)酵產(chǎn)生的乳酸、檸檬酸和乙酸等有機(jī)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(duì)酸菜中有機(jī)酸含量的影響見(jiàn)圖4。
由圖4可知,試驗(yàn)組與對(duì)照組pks相比,有機(jī)酸含量均增加,其中qj-pks的有機(jī)酸含量最多,為62.10 mg/mL,其他乳酸菌增加酸菜中有機(jī)酸含量由低到高為xf-pkslt;rs-pkslt;cm-pkslt;lbn-pks。與對(duì)照組pks相比,乳酸、檸檬酸和乙酸比例大的試驗(yàn)組抑菌效果較好。腐敗菌能夠利用乳酸和乙酸,導(dǎo)致pH上升和發(fā)酵底物還原,使酸菜發(fā)生腐敗變質(zhì),但從試驗(yàn)數(shù)據(jù)來(lái)看,pks只有在pH適中、有機(jī)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(jī)酸作為能源進(jìn)行繁殖代謝[4]。圖4可以充分證明有機(jī)酸的種類(lèi)和數(shù)量與乳酸菌的種類(lèi)有關(guān),乳酸在低pH下有較強(qiáng)的抗菌作用,對(duì)細(xì)胞膜具有滲透性,從而增強(qiáng)其他抗菌物質(zhì)的作用[53];檸檬酸在低pH下使腐敗菌細(xì)胞膜電荷改變,干擾腐敗菌細(xì)胞中酶的某些功能,通過(guò)抑制能量代謝中磷酸化酶的功能從而達(dá)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(duì)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(guò)降低環(huán)境pH,破壞細(xì)胞內(nèi)微環(huán)境和代謝途徑,抑制腐敗菌生長(zhǎng)[56]。例如,Guo等[56]研究發(fā)現(xiàn)乳酸、檸檬酸和乙酸3種有機(jī)酸對(duì)沙門(mén)氏菌代謝的影響無(wú)顯著性差異,能有效降低黃瓜中沙門(mén)氏菌的數(shù)量,破壞細(xì)胞膜,降低環(huán)境pH,抑制沙門(mén)氏菌的生長(zhǎng);在新鮮農(nóng)產(chǎn)品中,乳酸、乙酸和檸檬酸能夠抑制腐敗菌生長(zhǎng)[57]。
本研究中qj所產(chǎn)的有機(jī)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(cè)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(xì)菌素的能力,從而具有潛在的生存優(yōu)勢(shì)[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(gè)磷酸葡萄糖酸鹽/磷酸酮醇酶途徑、乳酸脫氫酶基因和1個(gè)不完整的三羧酸循環(huán),表明清酒乳桿菌同時(shí)參與同型發(fā)酵和異型發(fā)酵,具有產(chǎn)生大量有機(jī)酸的能力。
2.2.5不同乳酸菌對(duì)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(guān),如偏頭痛、血壓升高和心跳加速[60-62]。不同乳酸菌對(duì)生物胺含量的影響見(jiàn)圖5。
由圖5可知,試驗(yàn)組與對(duì)照組pks相比,xf-pks生物胺含量和種類(lèi)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(yàn)組xf-pks、qj-pks和cm-pks的酪胺、組胺和總生物胺含量均未超過(guò)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(jié)果表明,不同乳酸菌導(dǎo)致pks生物胺的種類(lèi)和含量不同,推測(cè)不同乳酸菌抑制pks代謝產(chǎn)生物胺的能力不同或者對(duì)生物胺的降解能力不同。從對(duì)照組試驗(yàn)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(zhǎng)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(yīng)用也比較常見(jiàn),例如,Alvarez等[66]研究發(fā)現(xiàn)乳酸菌具有通過(guò)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(jìn)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(biāo),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(shí)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(jié)論
本研究通過(guò)牛津杯抑菌圈法研究不同乳酸菌無(wú)細(xì)胞代謝物對(duì)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(lái)檢驗(yàn)所篩選乳酸菌抑制腐敗菌的實(shí)際效果。結(jié)果顯示,根據(jù)發(fā)酵液pH、有機(jī)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(yàn)組與對(duì)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(guò)推薦限量;有機(jī)酸含量最高,主要的有機(jī)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(guò)多種機(jī)制抑制酸菜腐敗菌庫(kù)德里阿茲威畢赤酵母的生長(zhǎng),能夠改善酸菜的發(fā)酵特性和感官品質(zhì),有效延長(zhǎng)食品的保質(zhì)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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