Clostridium tyrobutyricum has been identified as a major species associated with the late blowing defect (LBD) of semi-hard and hard cheeses, due to undesirable butyric acid fermentation. To find new strategies to control this spoilage bacterium, we investigated the delivery of a bacteriophage endolysin by a cheese starter culture. The nisin producer Lactococcus lactis subsp. lactis INIA 415 was engineered to produce the CTP1L endolysin, encoded by the virulent bacteriophage ΦCTP1 of C. tyrobutyricum and with a demonstrated lytic activity in vitro, to the cheese matrix. The presence of the nisRK two-component regulatory system in the host strain allowed constitutive expression of the endolysin under the control of the nisA promoter (P_nisA), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. Engineered lysins with a second cell wall biding domain were also tested and shown to have improved lytic activity. Transformation of L. lactis subsp. lactis INIA 415 with endolysin delivery plasmids had a detrimental effect on its ability to produce nisin in milk, but did not affect its acidifying capacity. Transformed L. lactis subsp. lactis INIA 415 were evaluated as starters in cheeses contaminated with spores of C. tyrobutyricum. Evolution of microbiological parameters, pH and dry matter of cheeses were studied, and Clostridium metabolism and LBD in cheeses were monitored by sensory and instrumental analyses during ripening. Cheese made with the parental strain L. lactis subsp. lactis INIA 415 delayed LBD by one month, attributable to the activity of the nisin, but it was not sufficient to arrest the growth of C. tyrobutyricum during ripening completely. The use of the endolysin-producing strains in cheese manufacture as single cultures also delayed the appearance of LBD by one month, attributable to the activity of the endolysin produced in situ during ripening, because nisin activity in these cheeses was very low at day 1 and undetectable from 15 days onwards. Endolysin was more effective than nisin in inhibiting Clostridium growth, since cheeses made with the CTP1L or the chimeric derivative producers only as starters showed lower LBD symptoms, higher lactic acid levels and lower concentrations of propionic and butyric acids (associated with off-flavours) than cheese made with the parental strain. Investigation of different promoters to maximise endolysin production may help to implement CTP1L as a tool to control C. tyrobutyricum by L. lactis cheese starter and reduce LBD even further.

由于不希望的丁酸发酵，酪酸梭状芽胞杆菌已被鉴定为与半硬干酪和硬干酪的后期吹气缺陷（LBD）有关的主要种类。为了找到控制这种变质细菌的新策略，我们研究了通过奶酪发酵剂培养物递送噬菌体内溶素的过程。乳链菌肽生产者乳酸乳球菌亚种。球菌INIA 415被改造以产生所述细胞内溶素CTP1L通过的有毒噬菌体ΦCTP1编码酪丁酸梭菌和带证实裂解活性在体外，对干酪基质。nisRK的存在宿主菌株中的两组分调节系统允许在nisA启动子（P _nisA）的控制下组成型表达溶素，而信号肽（SLPmod）的使用导致活性溶素成功分泌到周围介质中。还测试了具有第二个细胞壁结合域的工程化溶素，并显示出改善的裂解活性。乳酸乳杆菌亚种的转化。球菌INIA 415内溶交付质粒对其能否在牛奶生产乳酸链球菌素产生不利的影响，但不影响其酸化能力。转化乳球菌亚种 乳酸菌INIA 415被评估为被干酪孢子污染的奶酪中的起子酪丁酸梭菌。研究了奶酪的微生物学参数，pH和干物质的演变，并通过感官和仪器分析对奶酪中梭状芽胞杆菌的代谢和LBD进行了监测。用亲代乳酸乳球菌亚种制成的奶酪。乳酸菌INIA 415将LBD延迟了一个月，这归因于乳链菌肽的活性，但不足以完全阻止酪丁酸梭菌的生长。在干酪生产中使用产生内溶素的菌株作为单一培养物也将LBD的出现延迟了一个月，这归因于原位产生的内溶素的活性在成熟过程中，因为这些奶酪中的乳链菌肽活性在第1天就非常低，从15天开始就无法检测到。内溶素比乳链菌肽在抑制梭状芽胞杆菌的生长上更有效，因为与CTP1L或嵌合衍生物生产商生产的奶酪相比，初学者显示出更低的LBD症状，更高的乳酸水平以及更低的丙酸和丁酸浓度（与异味相关）用亲本菌株制成的奶酪。不同的启动子的研究，以最大限度地提高生产的内溶素可帮助实现CTP1L作为控制工具酪丁酸梭菌由L.乳干酪起动，减少LBD更进一步。