霉菌奶酪中脂肪酸代谢及其对产品品质影响研究进展霉菌奶酪中脂肪酸代谢及其对产品品质影响研究进展 栾滨羽1,史海粟1,*,李彦博2,武俊瑞1,乌日娜1,陶冬冰1,吴 琛1,岳喜庆1 (1.沈阳农业大学食品学院,辽宁 沈阳 110866;2.辽宁省食品检验检测院,辽宁 沈阳 110015) 摘要:脂肪酸是霉菌奶酪中重要的风味物质,在奶酪成熟阶段会发生剧烈反应。本文围绕霉菌奶酪及其成熟过程中脂肪酸变化这一主题进行综述,首先介绍了各类霉菌奶酪的生产工艺流程和脂肪酸的营养功效,总结了霉菌奶酪成熟阶段脂肪酸变化途径,影响脂肪酸种类和含量的因素和提高霉菌奶酪中不饱和脂肪酸含量的方法,并对霉菌奶酪未来的发展方向进行了展望。 关键词:霉菌奶酪;脂肪酸;不饱和脂肪酸 奶酪是一种以乳、稀奶油、脱脂乳、酪乳或其混合物为原料,利用发酵剂发酵并在凝乳酶的作用下促使乳发生凝结而制成的发酵乳制品。根据发酵剂的不同,可以分为细菌型奶酪和霉菌型奶酪。霉菌奶酪根据促熟霉菌的主要生长部位分成两种:表面霉菌成熟奶酪和内部霉菌成熟奶酪[1]。其工艺技术各具特色,感官特性也有所不同[2]。 信赖天脊硝酸磷型复合肥的农民客户圈越来越大。30年来,农民朋友从认识施用硝酸磷型复合肥到今日增产增效信仰硝酸磷型复合肥,终端人数在不断增加,朋友圈在不断扩大,真正诠释了“天脊品牌、中国品质,始终如一、良心制造”的内涵。这正是天脊“再领风骚30年”的力量之源。 表面霉菌成熟奶酪主要有:软质奶酪(如Camembert[3-4])、半硬质型奶酪(如Saint Nectaire、Armada和Reblochon等)、硬质奶酪(如Salers、Rodez等[5-6])。这类奶酪的明显共同点是表面存在霉菌生长形成的菌丝体结构。例如,最为常见的Camembert奶酪,其白色外表主要由卡门贝尔青霉(Penicillium camemberti)和白地霉(Geotrichum candidum)共同作用产生;而半硬质型奶酪Saint Nectaire是经传统自然发酵制成,其霉菌种类较为复杂,较常见的霉菌有毛霉属(Mucor sp.)、粉红单端孢霉(Trichothecium roseum)等,其表面会形成灰褐色外壳;同样通过传统方法制成的硬质奶酪Salers,关于其霉菌种类的研究较少,通常有帚霉属(Scopulariopsis sp.)[6]。还有一种布里(Brie)奶酪,其感官特征和工艺流程均与Camembert奶酪相似[7],但成品尺寸与Camembert奶酪不同。 内部霉菌成熟奶酪可以分为蓝纹奶酪和挪威青霉奶酪[6]。蓝纹奶酪主要有Cabrales、Danablu、Roquefort、Stilton、Gorgonzola等[3,8]。其中Roquefort奶酪年产量大,曾是法国第二大商业化奶酪[3],其最明显的特点是遍布表面的孔洞、菌丝由内向外生长产生的青色大理石纹路及其次级发酵剂娄地青霉(Penicillium roqueforti)促使形成的独特风味[9-12]。作为一种历史悠久的奶酪,Roquefort奶酪在原料乳处理和霉菌促熟等方面仍保留了一些传统方法[3,13]。对比几种奶酪关键工艺发现,Roquefort奶酪与Camembert奶酪明显不同的工艺是人为穿孔过程。这是由于成熟过程中,一些细菌代谢产生CO2,过度累积会抑制菌丝生长,故需要通过孔洞释放[8],且其次级发酵剂可以直接加入到原料乳中,而不是单纯接种在奶酪表面[12]。 虽然其他蓝纹奶酪在工艺上与Roquefort奶酪大体相似,但产品感官特征却截然不同。Stilton奶酪产于英国,根据外观和成熟度不同可分为3 个品种,而不同品种的奶酪成熟工艺也有所差别;Danablu奶酪源于丹麦,同样是一种商业化程度很高的奶酪,很多国家以其为原型生产蓝纹奶酪[3]。Gorgonzola奶酪是一种产于意大利的传统蓝纹奶酪,但传统方法无法控制奶酪成熟过程,导致其质量特征参差不齐[14]。而挪威传统Gamalost奶酪,其生产过程中会加入高大毛霉(Mucor mucedo)和总状毛霉(Mucor racemosus),伴随着成熟过程的进行,霉菌由内至外的生长使奶酪变成黄褐色[6]。 以上是几种常见的霉菌奶酪。近几年随着生物技术的不断发展,人们对微生物特性的了解逐渐加深,一些由新菌种研发出的新奶酪也逐渐出现。Lorrungruang等[15]以脂肪含量更低的豆乳为原料,以干酪乳杆菌(Lactobacillus casei)和红曲霉(Monascus purpureus)为发酵剂,制成一种风味与表面霉菌成熟奶酪完全不同的红色奶酪。孙颜君等[16]将不产桔霉素的红曲霉用于制作Camembert奶酪,这种方法制备的奶酪与传统Camembert奶酪相比,具有不同种类的酮类、酯类和硫化物,且较低的硫化物含量使其风味更加柔和,更符合中国人饮食习惯。汪建明等[17]利用雅致放射毛霉(Actinomucor elegans)制作出一种霉菌发酵型奶酪粉。谢芳等[18-19]将中国南方特有的水牛乳作为原料,分别用A. elegans和M. purpureus作为发酵剂,制作所得干酪品质较好,表明这两种菌种用于奶酪制作具有一定的可行性。 表1、2分别为几种常见霉菌奶酪的工艺流程和特点。 40年来,伴随着改革开放的逐步深入,我国农药工业逐渐发展壮大,极大地推动了农药由弱势产业转向强势产业,促进了我国由农药大国转向农药强国的健康发展轨道。尤其是近20年来,中国农药工业突飞猛进,已形成包括原药生产、制剂加工、科研创新开发和原料中间体配套的较为完整的农用化学品工业体系,为保证我国农业可持续发展、粮食安全和国家稳定作出了巨大贡献。 表 1 几种霉菌奶酪的工艺流程[3,20-21]
Table 1 Flow chart of the production process of several mold-fermented cheeses[3,20-21] Camembert奶酪生牛乳→标准化处理→加入嗜温乳酸菌(Mesophilic lactobacilli)、凝乳酶凝乳→垂直切分→装模沉积→排乳清→盐渍或盐水浸泡→表面喷涂霉菌孢子→成熟→包装→贮存Roquefort奶酪 生羊乳→30 ℃加入凝乳酶→28~34 ℃加入M. lactobacilli和P. roqueforti孢子→切分→排乳清→盐渍→穿孔→成熟→包装丹麦蓝纹奶酪 牛乳→标准化→热化处理、加入CaCl2→加入发酵剂和P. roqueforti孢子→加入凝乳酶凝乳→切分→热烫→排乳清→装模→盐水腌制→穿孔→成熟→贮存Stilton奶酪 牛乳→标准化→巴氏杀菌→21 ℃加入M. lactobacilli和P. roqueforti孢子→30 ℃凝乳→切分→盐渍装模→成熟→分级→包装Gorgonzola奶酪 牛乳→巴氏杀菌→30 ℃接种天然发酵剂→接种P. roqueforti孢子→加入凝乳酶→切分凝乳→穿孔→盐渍→4~6 ℃促熟83 d→包装
表 2 几种霉菌奶酪的工艺特点[3,20,22-23]
Table 2 Processing characteristics of several mold-fermented cheeses[3,20,22-23] 注:—.文献未提供相关信息。 奶酪 Camembert奶酪 Roquefort奶酪 丹麦蓝纹奶酪 Stilton奶酪 Gorgonzola奶酪外观images/BZ_221_1407_1784_1552_1884.pngimages/BZ_221_1595_1763_1710_1905.pngimages/BZ_221_1779_1764_1870_1910.pngimages/BZ_221_1950_1771_2044_1915.pngimages/BZ_221_2121_1771_2219_1916.png奶酪分类 软质奶酪 软质奶酪 半硬质奶酪 半软质奶酪 软质奶酪原料乳种类 生牛乳 羊乳 牛乳 牛乳 牛乳原料乳处理热化、巴氏杀菌及膜微过滤处理除过滤外无任何物理操作热化处理、脱脂乳与奶油混合 巴氏杀菌 巴氏杀菌后冷却至30 ℃标准化处理脂肪与蛋白质质量比1∶1 — 将奶油均质化 需根据季节标准化处理 —凝乳条件 30 ℃凝乳10~20 min 凝乳酶凝乳 29~32 ℃凝乳50~70 min 30 ℃下采用牛凝乳酶凝乳30~34 ℃凝乳10~15 min排乳清过程凝乳放入特定容器内,通过挤压旋转等方式排乳清10~20 ℃排乳清48 h 18 ℃下自然排乳清20~24 h控温环境下不断上下倒转装奶酪的模具以促进排乳清18 ℃下翻转凝乳4 次,持续10 h盐渍过程 干盐腌制 10 ℃干盐腌制5 d 18 ℃盐水腌制48 h 干盐腌制 22 ℃干盐腌制40 h发酵剂种类P. camemberti、G. candidum、M. lactobacilli P. roqueforti明串珠菌属(Leuconostoc)、乳酪链球菌(Streptococcus diacetilactis)、P. roqueforti M. lactobacilli、P. roqueforti S. thermophiles、P. roqueforti等天然发酵剂菌株成熟条件奶酪放在10~13 ℃、相对湿度大于95%环境成熟初次成熟在8~10 ℃、相对湿度95%环境中持续2 周,再在低温下用密封箔包裹后二次成熟相对湿度90%~95%,温度8~11 ℃下成熟3~5 周将奶酪表面密封,防止氧气进入;在特定相对湿度和温度环境下放置6 周后穿刺处理相对湿度85%~90%、4~6 ℃环境中成熟83 d包装/贮存环境用透湿、透气的纸张包装奶酪密封箔包裹在2~6 ℃下贮存12 周White Stilton奶酪成熟7 d后可包装销售,Blue Stilton、Vintage Blue Stilton奶酪需分级包装销售—
1 霉菌奶酪中脂肪酸的营养功效脂肪酸是细胞生长繁殖不可缺少的物质,其主要作用有供给能量、构成细胞各种膜结构[24]、促进细胞间信息转导和影响基因表达[25]。不饱和脂肪酸按碳链长度和饱和程度不同可分为有1 个双键的单不饱和脂肪酸(monounsaturated fatty acids,MUFAs)和有2 个及以上双键的多不饱和脂肪酸(polyunsaturated fatty acids,PUFAs),而根据双键所在位置又可以将其分为ω-3、ω-6和ω-9 3 种[12]。霉菌奶酪中营养物质含量丰富,其中脂质约占30%[3],饱和脂肪酸含量可达600 g/kg、MUFAs和PUFAs含量分别为235 g/kg和46 g/kg[26-27]。饱和脂肪酸中棕榈酸(C16:0)、肉豆蔻酸(C14:0)、硬脂酸(C18:0)含量较多[26],不饱和脂肪酸中油酸(C18:1 c9)较为常见。不同脂肪酸的营养价值也不相同。 ω-3脂肪酸中的二十碳五烯酸(eicosapentaenoic acid,EPA)(C20:5,n-3)和二十二碳六烯酸(docosahexaenoic acid,DHA)(C22:6,n-3)具有降血压[28]、预防心血管疾病[29]、抑制炎症[30]、抑制肿瘤[31]和促进胎儿大脑发育[32]等功效。但人体中缺乏关键酶无法大量合成[33],所以需要通过饮食直接补充或补充其前体物质α-亚麻酸(α-linolenic acid,ALA)(C18:3,n-3)。ω-6脂肪酸中亚油酸(C18:2,n-6)和花生四烯酸(C20:4,n-6)具有降血压[34]、促进细胞间信息传递过程[35]、维持皮肤结构[36]、促进免疫底物反应[25]等作用。而近几年一些研究表明不饱和脂肪酸还会对肌肉生长[37]、认知功能[38]和精神疾病的缓解[39]具有影响。EPA和DHA在鱼类中含量丰富[40],但一些鱼类由于环境问题导致体内汞污染物积累而不适合作为补充来源[41]。ALA则在一些植物组织中含量丰富[34],但植物同样缺乏关键酶而不能产生EPA和DHA[42]。亚油酸在植物种子、坚果和植物油中大量存在[34]。 由于IEET工程认证首先通过评估教学目标、设计或改进学生应具备的核心能力,并考核学生的核心能力。课程规划与教学须符合产业需求,并能培养学生将所学应用于实务技术的能力。 由于霉菌奶酪中存在反式脂肪酸,其被认为是引发冠心病的原因,导致消费者对霉菌奶酪的营养价值存在争议[43]。然而,霉菌奶酪中存在的共轭亚油酸(conjugated linoleic acid,CLA)是天然形成的反式脂肪酸,是对人体健康有益的功能活性物质,这种特殊的反式脂肪酸具有抗癌[26]、控制体脂和预防心血管疾病等作用[44]。 106 MCGS与西门子PLC在大齿圈润滑控制系统中的应用 ………………………………… 段谟喜,孙益敏 综上所述,霉菌奶酪中的不饱和脂肪酸对人体健康有促进作用,但由于其脂肪酸仍以饱和脂肪酸居多,因此需要调整脂肪酸比例,提高不饱和脂肪酸含量,对影响脂肪酸转化的因素进行人为控制。 从目前的教学实践来看,不少任课教师无法全面实现上述培养目标,存在一些亟待解决的细节问题,主要集中在以下五个方面: 2 霉菌奶酪成熟过程中脂肪酸转化规律及其对感官品质的影响成熟是霉菌奶酪加工工艺中的重要环节。在发酵剂菌株、凝乳酶及原料乳耐热酶的作用下[45],原料乳中的营养物质产生不同程度的降解、转化,逐渐形成奶酪特有的质地和风味[46],其中,霉菌奶酪中脂肪酸的转化对感官质量具有重要影响[47]。 教学大纲是实现课程人才培养的主体,关系到培养的学生能否真正成为工程实践能力强、创新能力强、具备国际竞争力的新工科人才。它是本课程教学改革的重点。 2.1 霉菌奶酪成熟过程中的脂肪酸转化脂肪酸是霉菌的一种代谢产物[48]。在霉菌奶酪成熟过程中,不饱和脂肪酸的合成有多种途径,而最为常见的途径是饱和脂肪酸在脂肪酸延长酶(fatty acid elongase,FAE)和脂肪酸脱饱和酶(fatty acid desaturase,FAD)的作用下生成长链不饱和脂肪酸[49]。一般以硬脂酸(C18:0)为底物引入双键,在细胞内质网上完成碳链的延长和脱饱和过程,最终形成ω-9、ω-6、ω-3 3 种不饱和脂肪酸[48,50-51]。图1详细地介绍了不饱和脂肪酸的合成途径。由于不同生物所含FAE和FAD的种类有差别,因此,出于工业目的,一些实验针对能合成PUFAs的微生物进行了筛选及改良。 图 1 不饱和脂肪酸的合成过程[52]
Fig. 1 Synthesis process of unsaturated fatty acids during mold-fermented cheese ripening[52]
目前,对霉菌奶酪成熟过程中不饱和脂肪酸转化的研究较少。霉菌奶酪辅助发酵剂中FAD功能的鉴定对提高霉菌奶酪不饱和脂肪酸含量具有重要意义。至今仅有G. candidum中的Δ12 FAD功能被鉴定[53]。 在式(2)中,Ra为增强体单桩竖向承载力特征值,按JGJ 79—2012《建筑地基处理技术规范》计算: 除上述过程外,三酰甘油的降解是霉菌奶酪中产生脂肪酸的主要途径。在奶酪中多种脂肪酶的作用下,三酰甘油中脂肪酸和甘油之间的酯键被水解,从而释放出游离脂肪酸[54-55]。根据反应底物的不同,脂肪酶主要可以分成两种:水解短链酰酯(C2~C8)的酯酶和水解长链酰酯的脂肪酶[56]。 2.2 脂肪酸对霉菌奶酪感官品质的影响甘油三酯水解产生的游离脂肪酸中,长链脂肪酸(碳原子数大于12)的嗅觉阈值较高,对风味贡献较小,而短链及中链、且具有偶数碳原子(C4~C12)和挥发性的脂肪酸对风味影响较大,具有较低的嗅觉阈值和各自独特的气味[57-59]。例如,具有典型醋味的醋酸和丙酸,具有酸臭奶酪气味的丁酸和具有明显羊膻味的4-乙基辛酸[58]。脂肪酸的挥发性气味会受到奶酪pH值的影响。在高pH值环境下,脂肪酸被转化为非挥发性盐,挥发性降低;而环境pH值较低时,脂肪酸处于游离状态,虽然利于挥发,但浓度过高易于造成嗅觉疲劳[60-61]。 脂肪酸还对奶酪的质地有影响,脂肪可以使食物的质地更加柔滑。除此之外,脂质还对奶酪微观结构的形成起重要作用[62]。脂质主要以球体的形式分散在奶酪蛋白凝胶中[63-64],通过填补蛋白凝胶中的空隙,阻止蛋白质发生进一步聚集。因此,一些霉菌奶酪制作工艺需要通过均质化处理防止局部脂质浓度过高阻碍均匀蛋白质结构的形成或者脂质与蛋白质无法充分混合[64]。除这种作用外,脂质中的单甘油酯还被认为是一种可以减小脂肪球大小的良好乳化剂[65]。虽然脂质赋予了霉菌奶酪不同寻常的口感,但由于其衍生物质的多样性间接导致霉菌奶酪微观结构复杂而难以观测。 2.3 脂肪酸衍生物对霉菌奶酪风味的影响脂肪酸还是其他风味物质,如甲基酮、仲醇、酯类等的重要前体物质[57,66]。图2简略描述了脂肪酸代谢的条件及反应途径。由此可以证明脂肪酸对于霉菌奶酪的风味形成具有重要意义。将脱脂乳奶酪与全脂乳奶酪进行对比,脱脂乳奶酪更低的感官评分也从侧面证明了这一结论[67]。通过固相微萃取和同时蒸馏萃取等方法提取霉菌奶酪中的挥发性物质,进而确定其特征风味[68-69]。表3列举了Camembert奶酪中几种典型的脂肪酸和关联风味物质的嗅觉阈值和风味特征。酮类、酯类、醇类和含硫化合物等是霉菌奶酪主要的特征风味成分,但这些物质在奶酪中含量较低。 2.3.1 甲基酮 游离脂肪酸可通过β-氧化作用脱去羧基,进而形成风味化合物甲基酮[70-71]。甲基酮在Camembert奶酪和蓝纹奶酪中含量较多,前者每100 g脂肪中约含25~60 mmol甲基酮[57,72]。与脂肪酸相比,甲基酮对奶酪感官品质的影响相对较小,但其独特的风味及其在霉菌奶酪中含量较多,均表明其对霉菌奶酪风味的形成必不可少。如具有果香味的2-壬酮,具有花香味的2-癸酮和具有发霉味的2-十一烷酮[73]。 有天晚上我在龙田村的大榕树下吃夜宵,碰到了大发厂的同事刘建。刘建腆着脸,眼里流露出羡慕,说课长,听说你现在当厂长了,太牛了。我说啥鸡巴厂长,还不是打工一个。刘建说,打工也分三六九等嘛,你是高级打工啦。我说,高级打工也是打工,打工就要受人管,不过呢,手里的权力比以前大了一些,安排一两人没问题。刘建好像就等着我这句话似的,说课长,我正想找你呢。我说你找我干啥呀。刘建说景花厂现在要人么?我有个亲戚从老家刚出来,想进厂。 奶酪中甲基酮的产率受pH值、温度、霉菌的生理状态及脂肪酸浓度影响。P. roqueforti的菌丝和孢子都可以将脂肪酸转化为甲基酮,其孢子可氧化含有2~12 个碳原子的脂肪酸,而菌丝则可以在pH 5~7范围内氧化脂肪酸,此pH值范围接近于蓝纹奶酪成熟时的表面pH值[45]。不同霉菌对脂肪酸的敏感度也不相同。例如P. camemberti菌丝相对P. roqueforti菌丝对高浓度脂肪酸更敏感[74]。 2.3.2 内酯 霉菌奶酪中存在的内酯类主要有γ-癸内酯、δ-癸内酯、γ-十二内酯和δ-十二内酯,都呈现果香味[72]。它们可由氢化脂肪酸通过酯交换反应形成内酯类[70]。而氢化脂肪酸既可以通过加热过程产生,也可以由脂肪酸正常分解产生,或者由不饱和脂肪酸通过微生物脂氧合酶和水合酶产生[70,72,75-76]。 表 3 几种典型霉菌奶酪中脂肪酸及其关联风味物质的风味特征和嗅觉阈值
Table 3 Thresholds and flavor characteristics of fatty acids and related flavor compounds in some typical mold-fermented cheeses 注:a.在蒸馏水中;b.在油或黄油中;c.在牛奶中;d.在空气中。Gorgonzola奶酪风味物质嗅觉阈值单位为μg/kg;蓝纹奶酪和Camembert奶酪风味物质嗅觉阈值单位为mg/kg。 奶酪脂肪酸 酮类 醇类 酯类 内酯类 含硫化合物 参考文献主要特征风味典型风味物质及其嗅觉阈值主要特征风味典型风味物质及其嗅觉阈值主要特征风味典型风味物质及其嗅觉阈值主要特征风味典型风味物质及其嗅觉阈值主要特征风味典型风味物质及其嗅觉阈值主要特征风味典型风味物质嗅觉阈值Camembert奶酪醋味、辛辣味、酸臭、干酪味、果香味、羊膻味丁酸0.3~7.0a丙酮味、果香味、花香味、果香味、发霉味2-丁酮30b蘑菇味、果香味、酒精味、花香味、发霉味、泥土味辛-2-醇0.018a菠萝味、香蕉味、花香味、蜂蜜味乙酸乙酯6.6a椰子味、酒香味、牛奶味、桃味、黄油味、麝香味δ-辛内酯0.57a卷心菜味、大蒜味、成熟奶酪味甲硫醇2a[57,69]异戊酸0.07a 2-戊酮22d 1-辛烯-3-醇0.01a 丙酸乙酯0.009 9a δ-癸内酯0.14~0.16a 二甲基三硫化物2.5b 4-甲基辛酸0.02a 3-辛酮0.05a 辛-1,5-二烯-3-醇0.01a 已酸乙酯0.000 13a γ-十二内酯1b 甲硫基丙醛0.2b蓝纹奶酪2-甲基丙酸0.019 5d 2-庚酮3a 戊醇4a 十六烷酸甲酯>2a γ-癸内酯0.011~0.09a 甲硫醚19c[68]丁酸0.038 9d~0.200 0a 2-辛酮0.05a 己醇2.5a 丁酸乙酯0.016c γ-十二内酯0.007a甲硫醇2a己酸0.012 6d 2-戊酮0.5c 辛烯-3-醇0.048d 己酸乙酯0.85b 2-苯乙基乙酸0.137b Gorgonzola奶酪 乙酸0.145d 2-庚酮9a 1-戊醇730a 丁酸乙酯1a γ-癸内酯0.011~0.090a 甲硫基丙醛0.05b [22,69]2-壬酮 20a 2-壬醇75a 醋酸戊酯1a γ-十二内酯2-十一烷酮 7a 2-庚醇5a 己酸乙酯1a 0.007a
图 2 脂肪酸的代谢及风味物质的产生途径[22,72]
Fig. 2 Fatty acid metabolism and production pathways of flavor substances[22,72]
3 影响霉菌奶酪中脂肪酸种类和含量的因素霉菌奶酪的成熟过程是各种生化反应的动态集合,这些生化反应的细微改变可能间接造成产品质量的巨大差异。这也导致霉菌奶酪的品种相对于其他乳制品更加多样。现代工业化的生产模式基本保证了产品品质的一致性,但为新品种奶酪的研发,影响奶酪质量因素的作用机理仍需要深入研究。脂肪酸作为奶酪风味的关键成分,其质量同样受到多种工艺参数的交叉影响。 3.1 脂肪酶奶酪中脂肪酶的来源主要有原料乳脂肪酶、发酵剂菌株释放的脂肪酶和非发酵剂菌株产生的脂肪酶[45]。这些脂肪酶对三酰甘油的作用位点不同,因此产生的不同种类的脂肪酸是造成霉菌奶酪风味差异的主要原因。 3.1.1 原料乳脂肪酶 奶牛血液中含有一种脂蛋白脂肪酶(lipoprotein lipase,LPL),这种脂肪酶可通过乳腺细胞膜渗出进入原料乳中[45],但由于原料乳中的脂质多以脂肪球的形式存在,被脂蛋白膜包裹,因此通常LPL不能与脂质直接接触发生反应,但在搅拌和均质化等外力作用下,脂蛋白膜被破坏,造成LPL与脂质反应产生臭味。LPL对单/二/三酰基甘油的sn-1和sn-3位点具有位置特异性[77],其最适反应条件为37 ℃、pH 7,在78 ℃下保持10 s才会被完全灭活;因此,巴氏杀菌有助于使LPL部分失活,防止原料乳在加工中产生异味[45]。 3.1.2 发酵剂菌株释放的脂肪酶 多种挥发性脂肪酸及其衍生物构成了霉菌奶酪特有的风味,而遍布奶酪内外的微生物产生的脂肪酶是造成这种差异的主要原因,因此排除原料乳种类和加工方式等外在因素影响,采用不同微生物发酵成熟的奶酪,其脂肪酸和其他香味物质的含量也会有所差别。 霉菌奶酪中部分脂肪酶来源于乳酸菌,它可以产生活性较弱的胞内脂肪酶[78],通过成熟过程中菌体的自溶作用被释放到奶酪中[55]。除此之外,霉菌奶酪中作为辅助发酵剂的各种霉菌菌株也提供了多种活性较强的脂肪酶[79]。表4对比了几种奶酪脂肪酸含量的差异,可以直观地看出霉菌奶酪相对细菌型奶酪有更剧烈的脂肪分解反应[72]。 G. candidum能产生一系列对长链不饱和脂肪酸具有特异性的脂肪酶,其脂肪酶的催化活性已有广泛研究,但关于其特异性的结论并不一致[80-83]。事实上,G. candidum会产生多种具有不同特异性的脂肪酶[84],按照其优先水解甘油三酯酯键的位置可以分成3 种:sn-1/3[72,82]、sn-2[83]和无特异性位点[85]。这些脂肪酶的最适pH值(6.0~7.0)、最适温度(20~40 ℃)和等电点(pI 4.0~5.0)等性质基本相似[86-88],且都会优先水解含有cis-Δ9双键结构的脂肪酸[81,84]。 蓝纹奶酪中常见的P. camemberti和P. roqueforti 也是脂肪酶的高效生产者[89]。由P. camemberti产生的脂肪酶相对特殊,主要体现在其只在酶活性位点丝氨酸残基附近区域,表现出与G. candidum和米黑毛霉(Rhizomucor miehei)脂肪酶存在序列同源性,但整体上,三者三维结构相似,且均具有脂肪酶常见的由Asp、His和Ser构成的催化三联体结构[90]。其对单酰基甘油和二酰基甘油的sn-1/3位点存在位置特异性[91-92]。Isobe等[90]通过测定P. camemberti脂肪酶的晶型结构,发现这种特异性的不同主要是由于酶与底物结合区域的表面结构存在差异。 表 4 几种奶酪的脂肪酸组成
Table 4 Free fatty acid compositions of several cheeses mg/kg 注:a.C18:0的同系物;b.测定结果为脂肪酸在萃取物中的相对含量/%。HPLC.高效液相色谱(high performance liquid chromatography);GC-MS.气相色谱-质谱(gas chromatography-mass spectrometry)。 脂肪酸 C2:0 C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C18:0饱和脂肪酸总计 C18:1 C18:2 C18:3不饱和脂肪酸总计 总计 测定方法参考文献细菌型奶酪 Cheddar奶酪476 952 143 175 159 571 952 1 556 794 5 778 2 841 635 238 3 714 9 492 HPLC[45,101]3.08b 9.10 b 8.46 b 2.43 b 1.78 b 24.85 b 24.85 GC-MS霉菌型奶酪Camembert奶酪 361 287 160 225 298 622 1 442 303 4 023 1 043 1 043 5 066 GC[45]Roquefort奶酪 992 751 715 2 104 1 403 2 632 6 452 17 404a 32 453 32 453 GC 961 626 707 2 280 1 295 3 185 6 230 2 241 18 791 6 282 896 7 178 25 969 GC蓝纹奶酪 1 146 777 546 1 275 1 835 4 147 11 416 14 088a 35 230 35 230 GC丹麦蓝纹奶酪 0.56±0.16b14.90±0.75b13.92±0.72b12.51±1.34b14.82±1.67b4.74±1.00b1.19±0.21b 62.76 b 0.12±0.03b 62.81b GC-MS [68]
P. roqueforti会产生至少两种胞外脂肪酶[71]和一种胞内脂肪酶[6,93-94],均能降解脂质。P. roqueforti的孢子和菌丝体能产生脂肪氧化酶,且在相同干质量时两者的脂肪酸氧化率相同[95-97]。但孢子的细胞壁结构使其产生的脂肪氧化酶能耐受环境中高浓度的脂肪酸,且对底物具有强渗透性[74,97],因此,孢子的脂肪代谢效果好于菌丝体。 3.1.3 非发酵剂菌株释放的脂肪酶 受加工条件影响,一些环境中的微生物会污染奶酪,其中异发酵M. lactobacilli最为常见[98],其最初被认为是奶酪中的腐败微生物。近几年的研究表明,异发酵M. lactobacilli不仅能促进奶酪风味形成,还可产生抗菌化合物,其脂肪酶活性弱于奶酪中霉菌和酵母的脂肪酶,且由于其较强的菌株依赖性,所以多作为奶酪加工的一种辅助发酵剂[98-100]。 (1)个人情况:问卷重点调查海员当前的健康状况和睡眠质量,这些都是疲劳的直接表现。作为社会关系,收集员工婚姻状况和家庭关系的信息; 3.2 高压处理用高压处理来延长食品保质期的方法早在19世纪就已出现[101]。在奶酪工业中,高压处理的目的是缩短成熟时间[102],从而降低生产成本。除此之外,霉菌奶酪的营养成分使其比其他品种奶酪更适合病原微生物生长,尤其是单核细胞李斯特菌(Listeria monocytogenes),因此,有必要对霉菌奶酪进行一定程度的灭菌[103]。 Wachowska等[104]提出一种方法,将奶酪于50 MPa、25 ℃条件下处理,能将奶酪的成熟期由6 个月缩短至3 d。Voigt等[105]将蓝纹奶酪在20 ℃分别用400 MPa和600 MPa压力处理,再在室温下成熟28 d,结果表明,高压处理对蓝纹奶酪脂肪酸组成无较大影响,但能明显观察到高压处理后脂肪酸含量下降的速率要低于对照组,经高压处理后奶酪的风味化合物含量明显低于对照组,这与其他实验结果相似[106-108]。Calzada等[109]为抑制蓝纹奶酪由于货架期过长导致的过熟,将蓝纹奶酪在成熟第21、42或63天进行400 MPa或600 MPa的高压处理,再于不同温度条件下成熟长达360 d;结果表明,奶酪中的脂肪酸含量同时受压强和成熟时间的影响,成熟第21天经600 MPa处理的奶酪脂肪酸含量最低,而成熟第42天经400 MPa处理的奶酪脂肪酸含量最高。高压处理可对微生物的酶活性产生抑制,导致酯解速度变慢。Martinez-Rodriguez等[110]研究了高压对P. roqueforti菌丝生长、孢子萌发和酯酶活性的影响,结果表明,菌丝质量和孢子活性均随压强增加而不同程度的降低,当压强超过400 MPa时两者均发生永久性损害,脂质分解活动也随着压强的增加而减缓。 3.3 包装方式奶酪的包装方式一般有两种:铝箔包装和真空包装;主要区别在于真空包装阻止了奶酪中CO2和水分的释放[55]。Duval等[111]研究用不同渗透性薄膜的真空包装和铝箔包装对蓝纹奶酪脂肪水解的影响,结果表明两种方法均不会改变蓝纹奶酪脂肪酸的含量,但能明显改变脂肪酸的代谢速率,间接影响奶酪货架期的品质变化。包装方式对霉菌奶酪脂肪酸含量以及风味的影响仍需深入研究。 当年,明朝的边将李成梁,在抚顺一带守边。明朝朝廷呢,挺好,在东北设挺多马市,当中就有清原马市、抚顺关马市。那时候马市,每个月定期开放两次,让女真人和汉人交换物品,就像现在的自由市场。朝廷呢,在边关收税,明朝朝廷的规定挺好,都正常交税。可是守关的明军,欺负、勒索女真人,女真人野性,不服他们勒索,就跟守关的明军干仗,仇也越积越深。 4 霉菌奶酪中不饱和脂肪酸含量的提高4.1 改变原料乳成分在反刍动物的饲料中添加植物油是一种改善乳中脂质状况的常用方法[112]。在反刍动物饲料中添加不同来源脂肪酸对牛奶脂肪酸具有影响,不同植物油脂肪酸成分不同,对乳的影响也不同[113]。同样,这种方式对奶酪不饱和脂肪酸含量也有影响[114]。Bodas等[115]将棕榈油、橄榄油、大豆油、亚麻籽油按一定比例混入母羊饲料中并制作羊奶奶酪,结果表明这几种植物油的加入改变了奶酪脂肪酸的比例, cis-9,trans-11 C18:2含量都有不同程度的上升。Mele[116]和Vargas-Bello-Pérez[117]等分别将亚麻籽油和橄榄油加入羊乳,结果表明,亚麻籽油能使奶酪饱和脂肪酸比例降低,而橄榄油使奶酪ω-6/ω-3降低,间接提高了奶酪脂肪酸的营养价值。此外,在饲料中添加芝麻油同样可以使奶酪cis-9,trans-11 C18:2含量上升[112]。 牧场环境因素对奶酪脂肪酸同样有影响。Chion等[118]研究季节变化对奶酪脂肪酸特性的影响,表明与冬季相比,夏季奶酪中饱和脂肪酸含量较低,MUFAs、PUFAs和CLA含量较高。 4.2 改变发酵剂菌种除改变原料,还可以通过改变发酵剂菌种组成来提高奶酪不饱和脂肪酸含量。根据发酵剂种类,一般将霉菌奶酪发酵剂分成两类:霉菌发酵剂和乳酸菌发酵剂[119]。 大多数霉菌发酵剂没有自身合成大量PUFAs的能力,通过基因工程等手段使其具备合成PUFAs的能力是未来的研究方向。Luo Xue等[53]成功将G. candidum GcFADS12基因在酿酒酵母中表达,并确定其具有编码Δ12 FAD并促进油酸转化为亚油酸再转化为α-ALA的功能特性。此外,也有研究使G. candidum的脂肪酶基因在毕赤酵母、耶氏解脂酵母等酵母中得到很好表达[120-121]。 乳酸菌发酵剂中,益生菌可依靠其特异性同分异构酶将亚油酸转化成CLA,这一过程主要受氧气浓度和益生菌种类的影响[122-125]。Barbosa等[126]将嗜酸乳杆菌(Lactobacillus acidophilus La-05)、双歧杆菌(Bifidobacterium animalis subsp. lactis BB-12)和菊粉用于制作羊奶奶酪,结果显示奶酪中短链脂肪酸含量减少,中链脂肪酸和长链脂肪酸含量均高于普通奶酪,特别是CLA含量增加,且奶酪的感官质量变化较小。Albenzio等[127]则采用嗜酸乳杆菌、长双歧杆菌(Bifidobacterium longum)和乳双歧杆菌(Bifidobacterium lactis)制作羊奶奶酪,其CLA含量可达传统奶酪的2 倍。此外,乳酸菌发酵剂保持肠道菌群平衡和增强对病原体抵抗力的作用也可提高奶酪的营养价值[128]。 很多父母说:“宝宝病了以后,好像长大懂事了。”正如老人说的:“生病的宝宝长见识。”这是因为父母能够利用疾病这个特殊事件,比较理性地教育宝宝:“要做一个勇敢的宝宝!爸爸妈妈喜欢勇敢的宝宝!”于是,宝宝在大人的鼓励下,战胜了疾病带来的生理痛苦和心理压力,变得坚强起来。 5 结 语霉菌奶酪虽然营养丰富,但其饱和脂肪酸含量较高,会引发冠心病等心血管疾病[43]。若使对人体有益的不饱和脂肪酸取代部分饱和脂肪酸,则会显著提高霉菌奶酪的营养价值,更会提高奶酪的市场价值。目前市场上富含PUFAs的产品已形成一定规模[129],但富含PUFAs的霉菌奶酪仍在起步阶段[130]。需要对影响奶酪不饱和脂肪酸的因素进行更深入的研究,尤其是针对发酵剂菌株代谢所需环境条件的研究,还需考虑改变脂肪酸组成对奶酪风味的影响。 参考文献: [1] COPETTI M V. 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A Review of Free Fatty Acid Catabolism in Mold-Fermented Cheese and Its Effect on Product Quality LUAN Binyu1, SHI Haisu1,*, LI Yanbo2, WU Junrui1, WU Rina1, TAO Dongbing1, WU Chen1, YUE Xiqing1
(1. College of Food Science, Shenyang Agricultural University, Shenyang 110866, China;2. Liaoning Institute for Food Control, Shenyang 110015, China) Abstract: Free fatty acids are important volatile flavor compounds in mold-fermented cheeses, which can react violently during cheese ripening. In this paper, free fatty acid catabolism during cheese ripening is reviewed. We firstly introduce the production process of various types of mold-fermented cheese and the nutritional properties of fatty acid in mold-fermented cheese. The transformation of fatty acids during the maturation process of mold-fermented cheese, the factors affecting the types and levels of fatty acids and currently available methods to increase the level of unsaturated fatty acids in moldfermented cheese are also summarized. Finally, we discuss future development directions. Keywords: mold-fermented cheese; fatty acid; unsaturated fatty acid
收稿日期:2019-04-30 基金项目:国家自然科学基金青年科学基金项目(31801567);辽宁省教育厅一般项目(LSNQN20124); 沈阳农业大学博士引进人才项目(880417040) 第一作者简介:栾滨羽(1998—)(ORCID: 0000-0002-1722-8699),男,本科生,研究方向为食品生物技术。E-mail: lby149157@163.com*通信作者简介:史海粟(1983—)(ORCID: 0000-0002-7268-5171),男,副教授,博士,研究方向为食品生物技术、功能性生物脂质、乳品微生物及生物技术。E-mail: shihaisu@syau.edu.cnDOI:10.7506/spkx1002-6630-20190430-394 中图分类号:TS252.1 文献标志码:A 文章编号:1002-6630(2020)09-0211-11 引文格式: 栾滨羽, 史海粟, 李彦博, 等. 霉菌奶酪中脂肪酸代谢及其对产品品质影响研究进展[J]. 食品科学, 2020, 41(9): 211-221.DOI:10.7506/spkx1002-6630-20190430-394. http://www.spkx.net.cnLUAN Binyu, SHI Haisu, LI Yanbo, et al. A review of free fatty acid catabolism in mold-fermented cheese and its effect on product quality[J]. Food Science, 2020, 41(9): 211-221. (in Chinese with English abstract) DOI:10.7506/spkx1002-6630-20190430-394. http://www.spkx.net.cn
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