Occurrence of foodborne pathogens and characterization of Staphylococcus aureus in cheese produced on farm-dairies

The objective of this study was to address knowledge gaps identified in an earlier risk assessment of Staphylococcus aureus and raw milk cheese. A survey of fresh and short-time ripened cheeses produced on farm-dairies in Sweden was conducted to investigate the occurrence and levels of S. aureus, Listeria monocytogenes and Escherichia coli, to characterize S. aureus isolates with special emphasis on enterotoxin genes, antibiotic resistance, bio-typing and genetic variation, and to collect information related to production practices. In general, the hygienic quality of farm-dairy cheeses appeared to be of an acceptable microbiological quality, e.g. L. monocytogenes and staphylococcal enterotoxin were not detected in cheese samples. However, E. coli and enterotoxigenic S. aureus were frequently found in raw milk cheeses and sometimes at levels that are of concern, especially in fresh cheese. Interestingly, levels in raw milk fresh cheese were significantly lower when starter cultures were used. Up to five S. aureus colonies per cheese, if possible, were characterized and about 70% of isolates carried one or more enterotoxin genes, most common were sec and sea. The Ovine biotype (73%) was most common among isolates from goat milk cheese and the Human biotype (60%) from cow milk cheese. Of all isolates, 39% showed decreased susceptibility to penicillin, but the proportion of isolates from cows' cheese (66%) compared to isolates from goats' cheese (27%) was significantly higher. S. aureus isolates with different properties were detected in cheese from the same farm and, sometimes even the same cheese. Isolates with the same pulsed-field gel electrophoresis (PFGE)-pattern were detected on geographically distant dairies. This indicates that multiple sources and routes of contamination are important. To improve the safety of these products efforts to raise awareness of the importance of hygiene barriers and raw milk quality as well as improved process control can be suggested, e.g. use of starter cultures and monitoring of fermentation with a pH-meter. For future safety assessments, a better understanding of factors determining toxin production in these cheeses is needed.     

Phage inactivation of foodborne pathogens on cooked and raw meat

Phages infecting Salmonella Typhimurium PT160 and Campylobacter jejuni were added at a low or high (10 or 10(4)) multiplicity of infection (MOI) to either low or high (<100 or 10(4)cm(-2)) densities of host bacteria inoculated onto raw and cooked beef, and incubated at 5 and 24 degrees C to simulate refrigerated and room temperature storage. Counts of host bacteria were made throughout the incubation period, with phages being counted at the first and last sampling times. Host inactivation was variable and depended on the incubation conditions and food type. Significant host inactivations of the order of 2-3 log(10)cm(-2) at 5 degrees C and >5.9 log(10)cm(-2) at 24 degrees C were achieved compared to phage-free controls using the Salmonella phage under optimal conditions (high host cell density and MOI). These results alongside those already published indicate that phages may be useful in the control for foodborne pathogens.     

一起多所幼儿园食源性疾病暴发事件的溯源分析

目的 对一起涉及多所幼儿园的食源性疾病暴发事件的可疑食品和致病因子进行溯源调查,为今后类似事件的防控和处置提供参考依据。方法 采用实时荧光PCR法、质谱技术等快速检测技术,结合分离培养、酶联免疫法（ELISA）等传统鉴定方法对3所幼儿园采集的32份样品开展病原检测。使用描述性流行病学调查方法对事件进行调查,脉冲场凝胶电泳（PFGE）方法对致病因子进行分子溯源分析。结果 3所幼儿园共有幼儿568名,62名病例,患病率为10.92%。临床症状主要为呕吐、腹泻等。从10份生物标本和某配餐公司统一配送的2份留存的拔丝肉松蛋糕检出金黄色葡萄球菌,蛋糕中金黄色葡萄球菌量分别为2.0×10⁷ CFU/g、1.4×10⁷ CFU/g,11株分离自病例和蛋糕的金黄色葡萄球菌同时检出葡萄球菌A型肠毒素基因（sea）和A型肠毒素（SEA）。PFGE指纹图谱为同一带型,提示病例和食品的分离株为同一来源。结论 本起暴发事件是由配餐公司统一配送的拔丝肉松蛋糕污染金黄色葡萄球菌产生肠毒素导致的3所幼儿园的食物中毒,应进一步加强对学校配餐食材的监管。     

Surface enhanced Raman scattering (SERS) with biopolymer encapsulated silver nanosubstrates for rapid detection of foodborne pathogens

A biopolymer encapsulated with silver nanoparticles was prepared using silver nitrate, polyvinyl alcohol (PVA) solution, and trisodium citrate. It was deposited on a mica sheet to use as SERS substrate. Fresh cultures of Salmonella Typhimurium, Escherichia coli, Staphylococcus aureus and Listeria innocua were washed from chicken rinse and suspended in 10 ml of sterile deionized water. Approximately 5 μl of the bacterial suspensions was placed on the substrate individually and exposed to 785 nm HeNe laser excitation. SERS spectral data were recorded over the Raman shift between 400 and 1800 cm^− 1 from 15 different spots on the substrate for each sample; and three replicates were done on each bacteria type. Principal component analysis (PCA) model was developed to classify foodborne bacteria types. PC1 identified 96% of the variation among the given bacteria specimen, and PC2 identified 3%, resulted in a total of 99% classification accuracy. Soft Independent Modeling of Class Analogies (SIMCA) of validation set gave an overall correct classification of 97%. Comparison of the SERS spectra of different types of gram-negative and gram-positive bacteria indicated that all of them have similar cell walls and cell membrane structures. Conversely, major differences were noted around the nucleic acid and amino acid structure information between 1200 cm^− 1 and 1700 cm^− 1 and at the finger print region between 400 cm^− 1 and 700 cm^− 1. Silver biopolymer nanoparticle substrate could be a promising SERS tool for pathogen detection. Also this study indicates that SERS technology could be used for reliable and rapid detection and classification of food borne pathogens.     

快速检测技术在食源性沙门氏菌检测中的应用研究进展

沙门氏菌是一种广泛存在于自然界中的食源性致病菌,能导致人食物中毒,引发胃肠炎、败血症等疾病,严重时危及生命。建立快速、准确的沙门氏菌检测方法是预防和控制沙门氏菌疾病的关键,发展快速灵敏的检测方法对于保障食品质量与安全具有重要意义。本文通过对免疫学检测技术、分子生物学技术和生物传感器检测技术等在食源性沙门氏菌的检测方面的应用进行综合分析,阐明各种食源性沙门氏菌快速检测技术的原理、优缺点及研究进展,为优化食源性沙门氏菌快速检测方法提供参考。     

Feasibility of colloidal silver SERS for rapid bacterial screening

This study reports the feasibility of citrate-reduced colloidal silver surface-enhanced Raman scattering (SERS) for differentiating three important food borne pathogens, E. coli, Listeria, and Salmonella. FT-Raman and SERS spectra of both silver colloids and colloid-K₃PO₄ mixtures were collected and analyzed to evaluate the reproducibility and stability of silver colloids fabricated in a batch-production process. The results suggest that the reproducibility of the colloids over the batch process is high and that their binding effectiveness remains consistent over a 60-day storage period. Two specific SERS bands at 712 and 390 cm⁻¹ were identified and used to develop simple 2-band ratios for differentiating E. coli-, Listeria-, and Salmonella-colloid mixtures with a 100% success. These results indicate that colloidal silver SERS technique may be a practical alternative method suitable for routine and rapid screening of E. coli, Listeria, and Salmonella bacteria.     

基于全基因组测序技术的食品分离致泻大肠埃希菌的分子特征及耐药性研究

目的 了解上海市肉品和水产品中分离致泻大肠埃希菌（DEC）的生物学特征及耐药性,为防控由该菌引起的食源性疾病提供科学依据。方法对食品分离DEC菌株进行全基因组测序及药物敏感性试验,利用BioNumerics软件对全基因组测序数据进行拼接,利用拼接序列开展多位点序列分型、毒力基因和耐药基因分析。结果本研究中56株DEC菌株中肠道聚集性大肠埃希菌（EAEC）占比最高,达73.2%,且以astA基因为主（90.2%）。56株DEC分为37个ST型,显示高度多样性。DEC菌株对喹诺酮类抗生素耐药性较高（64.3%）,其次是对氨基糖苷类、β-内酰胺类抗生素和四环素类抗生素耐药,且多重耐药性菌株占48.2%,耐药基因相应的以喹诺酮类、氨基糖苷类、β-内酰胺类抗生素耐药基因为主,且大多数对抗生素耐药的菌株均携带其相应的耐药基因。结论 EAEC是上海市食品中分离DEC菌株的主要型别,对喹诺酮类、β-内酰胺类、氨基糖苷类抗生素的耐药率较高,且携带相应的耐药基因,全基因组测序技术可应用于DEC的分子生物学监测中,为疾病预防控制提供科学依据。     

Loss of viability of Listeria monocytogenes in contaminated processed cheese during storage at 4, 12 and 22 °C

The behaviour of Listeria monocytogenes in a processed cheese product was evaluated over time by inoculating the product with three different L. monocytogenes strains (Scott A, CA and a strain isolated from processed cheese) at three different inoculation levels (ca. 6 × 10⁵, ca. 6 × 10³ and 10² CFU/g of cheese or less) and after storage of the contaminated products at 4, 12 or 22 °C. Growth of L. monocytogenes was not observed in any of the experimental trials (experiments involving different combinations of strain, inoculum level and storage temperature) throughout the storage period. L. monocytogenes populations decreased over time with a rate that was strain- and storage temperature-dependent. Nonetheless, for cheeses that had been inoculated with the higher inoculum and stored at 4 °C viable populations of L. monocytogenes could be detected for up to nine months post-inoculation. The L. monocytogenes survival curves obtained from the different trials were characterised by a post-inoculation phase during which the populations remained essentially unchanged (lag phase) followed by a phase of logarithmic decline. The duration of the lag phase and the rate of inactivation of L. monocytogenes in the different trials were estimated based on data from the linear descending portions of the survival curves. In addition, a non-linear Weibull-type equation was fitted to the data from each survival curve with satisfactory results. The results of the present study emphasize that, according to the definition laid down in the European Union Regulation 1441/2007, the processed cheese product tested in this work should be considered and classified as one that does not support the growth of L. monocytogenes under reasonable foreseeable conditions of distribution and storage. However, post-processing contamination of the product should be austerely avoided as the pathogen can survive in the product for extended periods of time, particularly under refrigerated storage (4 °C).     

Microbial characterisation of the traditional Spanish blue-veined Cabrales cheese: identification of dominant lactic acid bacteria

The investigation included six batches of artisan Cabrales cheese manufactured at different times of the year by two different producers and followed over a 90-day ripening period. Profound variations were found between batches due to the different mixtures of milk used from cow, goat and sheep and due to differences in temperature and humidity during ripening. Lactococci became dominant early after manufacture reaching approximately 4.0×10⁹ cfu g⁻¹ by day 3 and remained so throughout the ripening period. Lactobacilli remained at a lower level corresponding to about 3.2×10⁸ cfu g⁻¹ by day 3. Dextran-producing Leuconostoc were present in numbers of 1.0×10⁶ to 1.0×10⁷ cfu g⁻¹. Large populations of coliforms (1×10⁷ cfu g⁻¹), enterococci (1×10⁶ cfu g⁻¹) and staphylococci (1×10⁶ cfu g⁻¹) were present throughout manufacture in all batches, but only the latter continued to grow during ripening, and mostly on the surface (up to 1.6×10⁷ cfu g⁻¹). Filamentous fungi, among which P. roqueforti was a majority, reached their maximum (around 5.0×10⁸ cfu g⁻¹) between day 15 and day 30. By molecular methods, all lactococcal isolates were identified as Lactococcus lactis subsp. lactis. Fifty two percent of the lactobacilli were classified as Lactobacillus plantarum or Lactobacillus paraplantarum and a further 27% as Lactobacillus casei or Lactobacillus paracasei. Dextran-producing Leuconostoc mesenteroides (58%), Leuconostoc citreum (24%) and Leuconostoc pseudomesenteroides (12.5%) were identified from the MSE agar plates, although strains of non-producing Leuconostoc lactis were also isolated from MRS.     

Factors affecting growth of foodborne pathogens on minimally processed apples

Escherichia coli O157:H7, Salmonella and Listeria innocua increased by more than 2 log₁₀ units over a 24 h period on fresh-cut ‘Golden Delicious’ apple plugs stored at 25 and 20 °C. L. innocua reached the same final population level at 10 °C meanwhile E. coli and Salmonella only increased 1.3 log₁₀ units after 6 days. Only L. innocua was able to grow at 5 °C. No significant differences were observed between the growth of foodborne pathogens on fresh-cut ‘Golden Delicious’, ‘Granny Smith’ and ‘Shampion’ apples stored at 25 and 5 °C. The treatment of ‘Golden Delicious’ and ‘Granny Smith’ apple plugs with the antioxidants, ascorbic acid (2%) and NatureSeal^® (6%), did not affect pathogen growth. The effect of passive modified atmosphere packaging (MAP) on the growth of E. coli, Salmonella and L. innocua on ‘Golden Delicious’ apple slices was also tested. There were no significant differences in growth of pathogens in MAP conditions compared with air packaging of ‘Golden Delicious’ apple plugs, but the growth of mesophilic and psychrotrophic microorganisms was inhibited. These results highlight the importance of avoiding contamination of fresh-cut fruit with foodborne pathogens and the maintenance of the cold chain during storage until consumption.     

2022年上海市生禽肉和调理肉制品中主要食源性致病菌监测

目的 分析上海市市售生禽肉和调理肉制品中沙门菌、单核细胞增生李斯特菌及小肠结肠炎耶尔森氏菌等食源性致病菌的污染情况。方法 2022年1～8月,从上海市农贸市场、超市、餐饮店等环节采集样品348份,其中生禽肉240份,调理肉制品108份。按照食品安全国家标准分别对样品中的沙门菌、单核细胞增生李斯特菌和小肠结肠炎耶尔森氏菌的检测。采用VITEK2全自动微生物鉴定仪对可疑菌株进行生化鉴定,并对沙门菌分离株进行血清学分型。结果 生禽肉中沙门菌、单核细胞增生李斯特菌和小肠结肠炎耶尔森氏菌的检出率分别为28.33%(68/240)、5.00%(12/240)和0.83%(2/240);调理肉制品中沙门菌、单核细胞增生李斯特菌和小肠结肠炎耶尔森氏菌的检出率分别为5.56%(6/108)、28.70%(31/108)和1.85%(2/108);血清学分型结果显示,生禽肉中68株沙门菌分布于14种不同血清型,其中科瓦利斯沙门菌（35.29%）、鼠伤寒沙门菌（16.18%）和肠炎沙门菌（13.24%）占比较高,调理肉制品中6株沙门菌分布于3种不同血清型,分别为肠炎沙门菌（66.67%）、肯塔基沙门菌（16...     

Microbiology and foodborne pathogens in honey

Honey has been considered a relatively safe foodstuff due to its compositional properties, with infant botulism caused by Clostridium botulinum being the most prominent health risk associated with it. Our review is focused on the honey microflora along the food chain and evaluates the pathogenic potential of those microorganisms found in honey. This product may contain a great variety of bacteria and, particularly, fungi that eventually entered the food chain at an early stage (e.g., via pollen). For many of these microorganisms, opportunistic infections in humans have been recorded (e.g., infections by Staphylococcus spp., Citrobacter spp., Escherichia coli, Hafnia alvei, Aspergillus spp., Fusarium spp., Trichoderma spp., Chaetomium spp.), although direct infections via honey were not registered.     

我国食源性金黄色葡萄球菌耐药及遗传特征情况研究

目的 探究我国2020年食源性金黄色葡萄球菌的耐药性、毒力因子及分子分型特征。方法 采用微量肉汤稀释法对菌株进行药物敏感性试验,基于全基因序列分析多位点序列分型及重要耐药基因（mecA）和毒力基因。结果 224株食源性金黄色葡萄球菌对12类抗菌药物的整体耐药率为87.9%（197/224）,对青霉素的耐药率最高为82.6%（185/224）。多重耐药率为23.2%（52/224）,多重耐药菌中ST398占比最高为26.9%（14/52）,耐甲氧西林金黄色葡萄球菌检出率为8.0%（18/224）。葡萄球菌肠毒素基因整体携带率为52.2%（117/224）,其中sea的携带率最高为24.6%（55/224）,携带肠毒素基因种类最多的ST型为ST1。共检测到31种ST型,其中ST7最多（12.9%,29/224）,ST398次之（10.7%,24/224）。中毒性休克综合征毒素编码基因（tsst-1）和杀白细胞素编码基因（lukF-PV和lukS-PV）的携带率分别为6.3%（14/224）和4.5%（10/224）。结论 我国食源性金黄色葡萄球菌耐药率和肠毒素携带率均较高,且检出了临床感染中重要的毒力基因,提示食品中金黄色葡萄球菌的潜在危害不容忽视。ST型与食品类别、耐药性和致病性存在一定相关性,可为了解我国食源性金黄色葡萄球菌的流行特征和风险防控提供数据支撑。     

Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese

The objective of this study was to characterize Staphylococcus aureus isolates from Swiss raw milk cheeses that had been found to be contaminated with coagulase-positive staphylococci and to estimate the frequency of the various genotypes, in particular the mastitis-associated Staph. aureus genotype B (GTB). The isolates were also tested for staphylococcal enterotoxin (SE) genes and other virulence factors. From 623 coagulase-positive staphylococci isolated from 78 contaminated raw milk cheeses, 609 were found to be Staphylococcus aureus. Genotyping of all Staph. aureus isolates was performed by PCR amplification of the 16S–23S rRNA intergenic spacer region, as this method was used previously to differentiate between mastitis subtypes associated with their clinical outcome. In total, 20 different genotypes were obtained and the 5 most frequently occurring genotypes were distributed in 6.4% or more of the samples. The enterotoxin-producing Staph. aureus GTB, known for its high contagiousness and increased pathogenicity in Swiss mastitis herds, was found to be the most abundant subtype at the sample level (71.8%) as well as among the isolates (62.0%). A subset of 107 isolates of the different genotypes were analyzed for the presence of SE genes and revealed 9 different SE gene patterns, with sed being most frequently detected and 26% being PCR-negative for SE genes. Almost all isolates of the major contaminant GTB contained the SE gene pattern sed, sej, ser, with half of them additionally carrying sea. Production of SE in vitro was consistent with the SE genes detected in most of the cases; however, some isolated GTB did not produce SEA. Staphylococcus aureus Protein A (spa) typing revealed 30 different subtypes and most GTB isolates belonged to the bovine spa type t2953; GTB/t2953 was linked among other subtypes to SE production in cheese and staphylococcal intoxication cases. Furthermore, 1 of the 623 isolates was a methicillin-resistant Staph. aureus, which was an seh-carrying Staph. aureus spa type tbl 0635 (non-GTB). We conclude that control and reduction of enterotoxigenic Staph. aureus GTB in dairy herds in Switzerland will not only prevent economic losses at the farm level but also improve the safety of raw milk cheeses; distribution of methicillin-resistant Staph. aureus via raw milk cheese is of less concern.     

PCR技术检测食品有害微生物的应用

介绍了PCR技术的原理及其在食品有害微生物检测中的应用，同时提出了尚存在的问题，并对其应用前景作了展望。     

Control of food spoiling bacteria in cooked meat products with nisin,lacticin 3147, and a lacticin 3147-producing starter culture

Nisin, in the form of the commercial product Nisaplin, and lacticin 3147 in whey powdered form were added to minced pork-meat in amounts of 0.15% (w/w) and 1.5% (w/w), respectively. The meat was cooked and inoculated with a Staphylococcus aureus strain of meat origin and a Listeria innocua strain at a level of 10⁷ or 10⁵ CFU g^–1. The batches were stored vacuum-packaged for 21 days at 8 °C. Nisin and lacticin 3147 immediately reduced the L. innocua population at the time of inoculation. Nisin showed higher inhibitory activity than lacticin 3147. During the storage period, a slight L. innocua growth was observed in the batches inoculated with the larger inoculum, and a bacteriostatic effect was observed against Listeria in the batches inoculated with 10⁵ CFU g^–1. Nisin maintained a constant S. aureus population in the cooked batch inoculated with 10⁷ CFU g^–1, although the bacteriocin was capable of reducing the amount of S. aureus by 90% in the batch inoculated with 10⁵ CFU g^–1. On the other hand, lacticin 3147 did not show an inhibitory effect against S. aureus in the cooked meat. The starter culture Lactococcus lactis DPC 303-T4 (containing the conjugative plasmid encoding production of lacticin 3147) was inoculated in a portion of a Longissimus dorsi pork muscle with brine. L. lactis DPC 303-T4 performed a good fermentation, but lacticin 3147 production was not found after 7 days at 12 °C of storage.     

Fate of Staphylococcus aureus in cheese treated by ultrahigh pressure homogenization and high hydrostatic pressure

We evaluated the influence of ultrahigh pressure homogenization (UHPH) treatment applied to milk containing Staphylococcus aureus CECT 976 before cheese making, and the benefit of applying a further high hydrostatic pressure (HHP) treatment to cheese. The evolution of Staph. aureus counts during 30 d of storage at 8°C and the formation of staphylococcal enterotoxins were also assessed. Milk containing approximately 7.3 log₁₀ cfu/mL of Staph. aureus was pressurized using a 2-valve UHPH machine, applying 330 and 30 MPa at the primary and the secondary homogenizing valves, respectively. Milk inlet temperatures (T_in) of 6 and 20°C were assayed. Milk was used to elaborate soft-curd cheeses (UHPH cheese), some of which were additionally submitted to 10-min HHP treatments of 400 MPa at 20°C (UHPH+HHP cheese). Counts of Staph. aureus were measured on d 1 (24 h after manufacture or immediately after HHP treatment) and after 2, 15, and 30 d of ripening at 8°C. Counts of control cheeses not pressure-treated were approximately 8.5 log₁₀ cfu/g showing no significant decreases during storage. In cheeses made from UHPH treated milk at T_in of 6°C, counts of Staph. aureus were 5.0 ± 0.3 log₁₀ cfu/g at d 1; they decreased significantly to 2.8 ± 0.2 log₁₀ cfu/g on d 15, and were below the detection limit (1 log₁₀ cfu/g) after 30 d of storage. The use of an additional HHP treatment had a synergistic effect, increasing reductions up to 7.0 ± 0.3 log₁₀ cfu/g from d 1. However, for both UHPH and UHPH+HHP cheeses in the 6°C T_in samples, viable Staph. aureus cells were still recovered. For samples of the 20°C T_in group, complete inactivation of Staph. aureus was reached after 15 d of storage for both UHPH and UHPH+HHP cheese. Staphylococcal enterotoxins were found in controls but not in UHPH or UHPH+HHP treated samples. This study shows a new approach for significantly improving cheese safety by means of using UHPH or its combination with HHP.     

Occurrence of Escherichia coli O157, O111 and O26 in raw ewe's milk and performance of two enrichment broths and two plating media used for its assessment

The occurrence of Escherichia coli O157, O111 and O26 in 159 raw ewe's milk samples was examined. Sample-aliquots were incubated simultaneously in TSB added with yeast extract (YETSB) and mTSB with novobiocin (N-mTSB). Serogroup-specific immunomagnetic separation (IMS) was then used and IMS beads were plated in a cefixime tellurite (CT)-containing media (CT-SMAC, CT-SBMAC and CT-RMAC for E. coli O157, O111 and O26, respectively) and E. coli O157:H7 chromogenic ID agar. A sweep of confluent growth from each medium was examined for the presence of E. coli O157 and O111 using PCR, and for E. coli O26 using a latex agglutination test. Enumeration of E. coli O157 and O111 was performed in the samples tested positive for the correspondent serogroup using the most probable number (MPN) method combined with PCR. Percentage occurrences of E. coli O157, O111 and O26 were 18.2, 8.2 and 5.7, respectively. Mean E. coli O157 and O111 levels were 0.22 and < 0.04 MPN/mL, respectively. Enrichment in YETSB resulted in higher detection rates of E. coli O157 and O26 than in N-mTSB. When YETSB was used as enrichment broth and for these last two serogroups, the analysis of the confluent growth from the CT-media gave more positive results than that from E. coli O157:H7-ID medium.     

Lysis of Lactobacillus casei 5Mn 373 accelerates Grana cheese ripening

The influence of the adjunct of a peptidolytic Lactobacillus casei strain on Grana cheese ripening was studied. Strain erythromycin resistance enabled the monitoring of its growth and death kinetics during cheese maturation. Cell lysis was estimated by the dosage of intracellular X-prolyl-dipeptidyl aminopetidase in cheese extracts. L. casei growth reached a maximum level after the second month of cheese ripening when the initial added cell level was 5×10⁵ cfu/g cheese, while L. casei counts decreased from the beginning of the ripening period when the initial added cell level was 4.5×10⁷ cfu/g cheese. The maximum death rate occurred two months after the maximal cell growth, and bacterial lysis was observed approximately two months later. The characteristic amino acid pattern of control Grana cheese was obtained for all of the mature experimental cheeses independently of the inoculum size, and more rapidly when higher amounts of inocula were used due to L. casei cell lysis. The adjunct of the L. casei strain to cheese-milk substantially shortened the ripening time with no negative effects on cheese-making, chemical gross composition or flavour in the mature experimental cheeses compared to the control.