【作者】 袁磊；

【导师】 何国庆；

【作者基本信息】 浙江大学 ， 食品科学， 2020， 博士

【副题名】基于腐败特性及其生物被膜形成的角度

【摘要】 牛奶因具有极高的营养价值而深受消费者喜爱,但同时也为微生物的生长代谢提供了良好的介质。低温冷链技术能抑制原料奶中微生物的生长,而嗜冷菌在低温下仍能生长繁殖,并逐渐演变成原料奶中的优势菌群。目前,嗜冷菌的分泌耐热腐败酶能力和生物被膜形成能力是制约乳制品行业发展的两大重要因素。虽然嗜冷菌经常规热处理后能被杀死,但嗜冷菌所分泌的耐热腐败酶仍有酶活残留,进而影响乳制品的品质和货架期。生物被膜对乳品行业的危害主要有:食源性疾病的传播、乳制品的腐败变质、对加工设备的损耗及能耗的升高。本论文对我国16个地区/牧场采集的原料奶样品中的嗜冷菌进行分离鉴定,分别从产腐败酶和生物被膜形成两个方面探讨嗜冷菌对乳品工业的潜在危害。同时,探究AHLs信号分子对嗜冷菌生长代谢、分泌腐败酶以及生物被膜形成的调控机制。采用分离培养结合RAPD-PCR技术,将16个地区/牧场的原料奶样品中的480株嗜冷菌聚类成85个RAPD组,且经16S rRNA鉴定后发现优势菌属依次为假单胞菌属(58.8%)、不动杆菌属(13.3%)、黄杆菌属(6.0%)、鞘氨醇杆菌属(4.2%)和沙雷氏菌属(3.1%);而在种水平上的优势菌种为Pseudomonas fluorescens(15.8%)、Pseudomonas fragi(7.1%)、Pseudomonas psychrophila(5.4%)、Acinetobacter johnsonii(5%)、Pseudomonas putida(4.6%)和 Pseudomonas azotoformans(3.8%)。同种、不同株的嗜冷菌(如P.fluorescens,P.fragi,P.psychrophila等)RAPD分子指纹图谱存在种内差异。此外,Acinetobacter harbinensis,Acinetobacter oryzae等嗜冷菌首次在原料奶中被分离鉴定到。假单胞菌属、不动杆菌属、黄杆菌属、产吲哚金黄杆菌属、沙雷氏菌属和产气单胞菌属不仅是原料奶中的优势菌属,也是重要的产腐败酶来源,它们在室温和低温下都有较强的产腐败酶的能力。大多数蛋白酶和脂肪酶经热处理(70℃、80℃和90℃)后仍有酶活残留,代表性嗜冷菌株所分泌的蛋白酶和脂肪酶的酶灭活热力学参数也证实这些酶具有较高的热稳定性。嗜冷菌产腐败酶是一个依赖于生长温度的过程,生长温度对其分泌腐败酶的能力影响显著,但对腐败酶的耐热性并无显著影响。大多数嗜冷菌在低温下(7℃)具有生物被膜形成能力(OD595值的范围为0.087-1.324),72个RAPD组中有25株、10株和19株菌分别呈弱、中等和强生物被膜形成能力,且假单胞菌属菌株的生物被膜形成能力显著强于其它属的菌株。不同培养基对嗜冷菌生物被膜的形成有影响,与脱脂牛奶相比(1.04-6.1 log CFU/cm2),嗜冷菌在TSB中更容易在不锈钢表面形成生物被膜(1.15-6.63 log CFU/cm2)。96孔板法和不锈钢片表面吸附法这两种方法之间呈现出中等相关性(r=0.42),证明采用这两种方法评估微生物生物被膜形成的可靠性。嗜冷菌的生物被膜形成能力存在异质性,即同种、不同株的嗜冷菌形成生物被膜的能力存在差异。嗜冷菌之间的相互作用对生物被膜的形成和消毒剂的抗性影响显著。Pseudomonas libanensis 能提高 Chryseobacterium oncorhynchi 和 Aeromonas hydrophila生物被膜的形成能力,且P.libanensis与C.oncorhynchi呈协同效应关系。与单一菌落形成的生物被膜相比,二元混合菌生物被膜模型(P.libanessis+A.hydrophila、P.libanesnsi+C.oncorhynchi)对过氧乙酸、过氧化氢和次氯酸钠这三种消毒剂抗性均显著提高。过氧乙酸对单菌、混合菌形成的生物被膜清除率最高,其次为过氧化氢和次氯酸钠。平板计数法和CLSM观察分析法均能有效评估不同消毒剂处理对生物被膜的影响,这两种方法间相关性好。采用报告平板法筛选能产AHLs信号分子的嗜冷菌,阳性检出率为71.43%;进一步使用LC-MS鉴定代表菌株P.azotoformans(a)和Serrita liquefaciens产AHLs 的种类,发现P.azotoformans(a)产 C6-HSL,而 S.liquefaciens 产 C8-HSL。外源性添加 C6-HSL 和 C8-HSL 对 P.azotoformans(a)和 S.liquefaciens的生长、分泌腐败酶以及生物被膜的形成均有显著的促进作用。外源性添加C6-HSL和C8-HSL也加速P.azotoformans(a)和S.liquefaciens对牛奶的腐败作用,其中牛奶的粒径大小显著增大、牛奶中的酸性风味物质显著增加。转录组学的研究结果发现,外源性添加AHLs能显著调控P.azotoformans(a)和S.liquefaciens这两株菌相关基因的表达,且上调基因明显多于下调基因。AHLs对这两株菌在生长代谢、产腐败酶和生物被膜形成这三大方面均起到调控作用。编码与细菌生长代谢密切相关的基因(核糖体、氧化磷酸化、糖酵解、TCA循环、蛋白质转运、氨基酸代谢、脂肪酸代谢、嘌呤代谢、抗胁迫等)上调后,促进细菌的生长代谢,并导致环境中菌液的浓度更高;环境中菌液浓度的升高加速细菌生物被膜的形成,此外,编码与生物被膜形成(主要为细菌鞭毛结构)密切相关的基因上调后,细菌的生物被膜形成能力提高;环境中菌液浓度的升高也会提高环境中腐败酶的总量,且编码与分泌腐败酶相关的基因上调后也会提高细菌产腐败的能力。此外,AHLs对菌株的调控作用呈现出信号分子种类、菌株的依赖性,即不同AHLs对不同株菌的基因和具体的代谢途径的调控作用存在差异。更多还原

【Abstract】 Milk is one of the ideal foods among consumers due to its high nutritional value,while it also serves as a good medium for the growth of a broad spectrum of microorganisms.In order to inhibit the growth of microorganisms,the cold chain system is widely used in the dairy industry.However,psychrotrophic bacteria can still grow at low temperatures and gradually become the dominant microflora in raw milk.In general,the secretion of heat-resistant spoilage enzymes and the formation of biofilms are two vital factors that restrain the development of dairy industry.Although psychrophilic bacteria can be killed by conventional heat treatment,the heat-resistant proteases and lipases produced by psychrophilic bacteria may still remain activity,which potentially affect the quality and shelf-life of dairy products.Biofilms formed by psychrophilic bacteria may lead to foodborne diseases,dairy spoilage,the loss of processing equipment and an increase in energy consumption.In this study,480 psychrophilic bacterial isolates from 16 raw milk samples were clustered and identified,and their potential risks based on the production of spoilage enzymes and biofilms formation were studied.Meanwhile,the regulation of AHLs on the growth,secretion of spoilage enzymes and biofilm formation of of psychrophilic bacteria was also explored.A number of 480 psychrophilic bacterial isolates in raw milk samples from 16 different regions were clustered into 85 different RAPD groups based on different RAPD patterns.The 16S rRNA results showed that Pseudomonas(58.8%),Acinetobacter(13.3%),Flavobacterium(6.0%),Sphingomonas(4.2%)and Serratia(3.1%)were the dominant genus.At the species level,Pseudomonas fluorescens(15.8%)proved to be the most dominant species,followed by Pseudomonas fragi(7.1%),Pseudomonas psychrophila(5.4%),Acinetobacter johnsonii(5%),Pseudomonas putida(4.6%)and Pseudomonas azotoformans(3.8%).Isolates belonging to the same species,such as P.fluorescens,P.fragi and P.psychrophila,have different RAPD profiles.In addition,some psychrotrophic bacteria have been identified as potential novel species in raw milk.Isolates of Pseudomonas,Acinetobacter,Flavobacterium,Chryseobacterium,Serratia,and Aeromonas proved to be the strong enzyme producers either at the room temperature or the low temperature.Proteases and lipases produced by psychrotrophic bacteria retained activity after heat treatment at 70,80,or 90℃,and further thermodynamic parameters determination of proteases and lipase also confirmed that these enzymes have high thermal stabilities.The production of spoilage enzymes is a temperature-dependent process,as the incubation temperature siginificantly affected the enzymes production,while no significant effect on the heat resistance of enzymes was observed.Most psychrophilic bacterial isolates have the ability to form biofilms at 7℃,with the OD595 values ranging from 0.087 to 1.324.Results showed that biofilm formation is a strain-dependent trait,and 25,10,19 RAPD groups were weak,moderate and high biofilm formers,respectively,based on results of the crystal violet staining.The biofilm formation ability of isolates belonging to Pseudomonas were significantly higher than that isolates of other genera.The composition of media is a key factor that affect the biofilm formation by bacteria.Psychrophilic bacteria were more likely to form biofilm on the surface on stainless steel in TSB(1.15-6.63 log CFU/cm2)than in skim milk(1.04-6.llog CFU/cm2).A moderate correlation(r=0.42)between the crystal violet assay of polystyrene and methods based on cell enumeration on coupons was observed,indicating the reliability of the two methods for evaluating the biofilm formation by bacteria.A marked variation in biofilm-forming capacity was observed within the strains belonging to the same species.Bacterial interactions significantly affected the spatial properties and the resistance to disinfectants of the formed biofilms.Pseudomonas libanensis enhanced the biofilm formation by Chryseobacterium oncorhynchi and Aeromonas hydrophila,and a synergistic effect was observed between P.libanensis and C.oncorhynchi.The resistance to three kinds of disinfectants(peroxyacetic acid,hydrogen peroxide and sodium hypochlorite)of the two dual-species biofilms(P.libanensis+A.hydrophila,P.libanensis+C.oncorhynchi)was significantly increased when compared to each single-species biofilm.Peracetic acid proved to be the most effective disinfectant to remove biofilms,followed by hydrogen peroxide and sodium hypochlorite.Both the plate counting method and the CLSM observation method effectively evaluated the disinfection treatment on the biofilms.The capacity of producing AHLs were screened by reporter plate method,and the positive detection rate of 28 isolates was 71.43%.LC-MS was further applied to identify the structure of AHLs produced by P.azotoformans(a)and Serrita liquefaciens,and it was found that P.azotoformans(a)produced C6-HSL,while S.liquefaciens produced C8-HSL.Exogenous addition of C6-HSL and C8-HSL significantly promoted the growth,spoilage enzymes production and biofilm formation of P.azotoformans(a)and S.liquefaciens.Meanwhile,exogenous addition of C6-HSL and C8-HSL also accelerated the spoilage of milk caused by P.azotoformans(a)and S.liquefaciens.The particle size of milk was significantly increased,and the acid flavor in milk is significantly increased.RNA sequencing results revealed that the addition of AHLs significantly regulated genes expressions of P.azotoformans(a)and S.liquefaciens,and up-regulated genes were more than down-regulated genes.AHLs play a regulatory role in the growth,production of spoilage enzymes and biofilm formation of psychrotrophic bacteria.Up-regulated genes(ribosomes,oxidative phosphorylation,glycolysis,TCA cycle,protein transport,amino acid metabolism,fatty acid metabolism,purine metabolism and resistant to stress)that closely relating to bacterial growth promotes bacterial growth and leads to higher concentrations of bacteria.Up-regulated genes relating to the biofilm formation(bacterial flagellar structure)and enzymes production also improved capacity to form biofilms and produce enzymes by psychrotrophic bacteria,respectively.In addition,the regulation of QS showed a dependence on types of AHLs and strains.更多还原