The prevalence and characterization of Bacillus cereus isolated from raw and pasteurized buffalo milk in southwestern China

Bacillus cereus is an important food-borne pathogenic bacteria and a putrid microorganism in the dairy industry. Raw and pasteurized buffalo milk play important roles in the dairy market in southwestern China. However, the reports on the prevalence and characterization of B. cereus strains isolated from the above sources are lacking. In this study, 150 raw buffalo milk samples and 300 pasteurized buffalo milk samples were collected from 3 provinces in southwestern China. The genotype, virulence gene distribution, antibiotic resistance, and biofilm-forming ability of isolates were analyzed. Ninety-six B. cereus strains were isolated and identified: 50 isolates (33.3%) from buffalo raw milk and 46 isolates (15.3%) from pasteurized buffalo milk. These strains were classified into 41 sequence types (ST) and 5 groups, of which ST857 was the predominant ST. The detection rates of virulence genes nheABC cluster, hblACD cluster, cytK, bceT, entFM, hlyII, and cesB were 89.6%, 13.5%, 64.6%, 71.9%, 84.4%, 62.5%, and 6.25%, respectively. The antimicrobial susceptibility testing showed that more than 90% of the isolates were susceptible to gentamicin, chloramphenicol, ciprofloxacin, erythromycin, vancomycin, and tetracycline, as well as resistant to ampicillin, cefepime, oxacillin, and rifampin. The results of biomass biofilm evaluation of the isolates on the stainless-steel tube showed that the optical density values at a wavelength of 595 nm of all strains in group I were greater than 1, with the strongest overall biofilm-forming ability among 5 groups, and the overall biofilm-forming ability of group III was the weakest. There was a relationship between the biofilm-forming ability and phylogenetic relationship of B. cereus strains. Taken together, our findings are the first to report the contamination situation and characterization of B. cereus isolated from raw and pasteurized buffalo milk in southwestern China as well as indicate the potential risk posed by this pathogen to dairy industry and public health.     

3个引进油橄榄品种初榨油多酚类化合物含量及抗氧化活性研究

以3个引进油橄榄品种初榨油为材料,测定总多酚含量和各单体酚含量并评价其抗氧化活性,同时采用SPSS应用软件分析总多酚含量、各单体酚含量与体外抗氧化活性的相关性。结果表明:3个品种总多酚含量分别为21.26、23.31、26.04μg/g,品种间无显著差异,而单体酚含量均较少;3个品种中佛奥对DPPH自由基的清除能力最强,而对羟基自由基的清除能力和Fe2+螯合能力则是豆果最强;羟基酪醇含量与DPPH自由基清除能力、羟基自由基清除能力和Fe2+螯合能力极显著负相关或显著正相关,相关系数分别为-0.956、0.713、0.721;对羟基苯甲酸含量与Fe2+螯合能力显著正相关;槲皮素含量与DPPH自由基清除能力显著负相关,与Fe2+螯合能力显著正相关;而总多酚含量、咖啡酸和表儿茶素含量与体外抗氧化能力的的相关程度较低,因此羟基酪醇、对羟基苯甲酸和槲皮素是主要的抗氧化成分。     

Bacillus cereus spores during housing of dairy cows: factors affecting contamination of raw milk

The contamination of raw milk with Bacillus cereus spores was studied during the indoor confinement of dairy cattle. The occurrence of spores in fresh and used bedding material, air samples, feed, feces, and the rinse water from milking equipment was compared with the spore level in bulk tank milk on 2 farms, one of which had 2 different housing systems. A less extensive study was carried out on an additional 5 farms. High spore concentrations of >100 spores/L in the raw milk were found on 4 of the farms. The number of spores found in the feed, feces, and air was too small to be of importance for milk contamination. Elevated spore contents in the rinse water from the milking equipment (up to 322 spores/L) were observed and large numbers of spores were found in the used bedding material, especially in free stalls with >5 cm deep sawdust beds. At most, 87,000 spores/g were found in used sawdust bedding. A positive correlation was found between the spore content in used bedding material and milk (r = 0.72). Comparison of the genetic fingerprints obtained by the random amplified polymorphic DNA PCR of isolates of B. cereus from the different sources indicated that used bedding material was the major source of contamination. A separate feeding experiment in which cows were experimentally fed B. cereus spores showed a positive relationship between the number of spores in the feed and feces and in the feces and milk (r = 0.78). The results showed that contaminated feed could be a significant source of spore contamination of raw milk if the number of spores excreted in the feces exceeded 100,000/g.     

Occurrence and characterization of Staphylococcus aureus isolated from raw milk for cheesemaking

Fifty-four samples of raw milk for cheesemaking were tested for the presence of Staphylococcus aureus . Multiplex polymerase chain reactions were performed to identify presumptive isolates and the presence of enterotoxin-coding genes sea-see . The strains were tested for antibiotic resistance. Eighty strains were identified as S. aureus and 31 of these carried one or more enterotoxin genes ( sea-see ). Resistance to eritromycin, penicillin and ampicillin was widespread among isolates . Staphylococcus aureus in raw milk for cheesemaking may constitute a risk with respect to staphylococcal food poisoning from raw milk products.     

碳酸水保藏生牛乳及其去除效果的研究

以添加不同量碳酸水的生牛乳（4℃）为研究对象,检测其冷藏期的理化指标及菌落总数,并进行碳酸水去除实验,探索一种延长生牛乳冷藏保存期的方法。结果表明:添加碳酸水可延缓生乳的酸败,且溶解的CO₂浓度越高,抑菌作用越显著;在碳酸水去除实验中,CO₂浓度为41.47 mmol/L的生乳在55℃、-0.085^-0.095 MPa条件下旋蒸15 min后,其水分含量、CO₂含量及热稳定性可恢复到对照组水平。碳酸水不会对生牛乳的理化性质造成影响且易去除,因此,可以采用添加碳酸水的方法延长生牛乳的冷藏保存期。      

原料乳中优势嗜冷菌株的确定及其微生物学特征研究

对原料乳样品进行嗜冷菌的分离筛选,结果得到4株优势嗜冷菌。本文研究了这4株优势嗜冷菌的生长特性及产脂肪酶特性,分别得到了4株优势嗜冷菌株的最佳生长条件及最佳产脂肪酶条件。      

抗菌肽cp1对蜡样芽孢杆菌的抑菌作用及在巴氏杀菌乳中的应用

目的 探究抗菌肽cp1对蜡样芽孢杆菌的抑菌作用,并进一步分析抗菌肽cp1在巴氏杀菌乳中的应用。方法 以蜡样芽胞杆菌为研究对象,通过测定抑菌圈直径(diameter of inhibition zone, DIZ)、最小抑菌浓度(minimal inhibitory concentration, MIC)、最小杀菌浓度(minimum bactericidal concentration, MBC)评价抗菌肽cp1对蜡样芽胞杆菌的抑菌效果。通过分析生长曲线、细胞内三磷酸腺苷(adenosine triphosphate, ATP)、膜电位、内容物渗出和细胞形态的变化,来揭示可能的作用机制。在此基础上,并探究抗菌肽cp1在巴氏杀菌乳贮藏中的应用。结果 当抗菌肽cp1质量浓度为10μg/mL时,其对蜡样芽胞杆菌的DIZ为(16.19±1.29) mm;抗菌肽cp1对蜡样芽孢杆菌的MIC、MBC分别为4μg/mL和8μg/mL;当抗菌肽cp1质量浓度为2 MIC时,蜡样芽胞杆菌几乎不生长;抗菌肽cp1导致细胞内ATP含量降低,细胞膜超极化,细胞液(包括核酸和蛋白质)漏出,细胞形态破坏;在巴氏杀...     

Minimizing the level of Bacillus cereus spores in farm tank milk

In a year-long survey on 24 Dutch farms, Bacillus cereus spore concentrations were measured in farm tank milk (FTM), feces, bedding material, mixed grass and corn silage, and soil from the pasture. The aim of this study was to determine, in practice, factors affecting the concentration of B. cereus spores in FTM throughout the year. In addition, the results of the survey were used in combination with a previously published modeling study to determine requirements for a strategy to control B. cereus spore concentrations in FTM below the MSL of 3 log₁₀ spores/L. The B. cereus spore concentration in FTM was 1.2 ± 0.05 log₁₀ spores/L and in none of samples was the concentration above the MSL. The spore concentration in soil (4.9 ± 0.04 log₁₀ spores/g) was more than 100-fold higher than the concentration in feces (2.2 ± 0.05 log₁₀ spores/g), bedding material (2.8 ± 0.07 log₁₀ spores/g), and mixed silage (2.4 ± 0.07 log₁₀ spores/g). The spore concentration in FTM increased between July and September compared with the rest of the year (0.5 ± 0.02 log₁₀ spores/L difference). In this period, comparable increases of the concentrations in feces (0.4 ± 0.03 log₁₀ spores/g), bedding material (0.5 ± 0.05 log₁₀ spores/g), and mixed silage (0.4 ± 0.05 log₁₀ spores/g) were found. The increased B. cereus spore concentration in FTM was not related to the grazing of cows. Significant correlations were found between the spore concentrations in FTM and feces (r = 0.51) and in feces and mixed silage (r = 0.43) when the cows grazed. The increased concentrations during summer could be explained by an increased growth of B. cereus due to the higher temperatures. We concluded that year-round B. cereus spores were predominantly transmitted from feeds, via feces, to FTM. Farmers should take measures that minimize the transmission of spores via this route by ensuring low initial contamination levels in the feeds (<3 log₁₀ spores/g) and by preventing growth of B. cereus in the farm environment. In addition, because of the extremely high B. cereus spore concentrations in soil, the contamination of teats with soil needs to be prevented.     

Results from raw milk microbiological tests do not predict the shelf-life performance of commercially pasteurized fluid milk

Analytical tools that accurately predict the performance of raw milk following its manufacture into commercial food products are of economic interest to the dairy industry. To evaluate the ability of currently applied raw milk microbiological tests to predict the quality of commercially pasteurized fluid milk products, samples of raw milk and 2% fat pasteurized milk were obtained from 4 New York State fluid milk processors for a 1-yr period. Raw milk samples were examined using a variety of tests commonly applied to raw milk, including somatic cell count, standard plate count, psychrotrophic bacteria count, ropy milk test, coliform count, preliminary incubation count, laboratory pasteurization count, and spore pasteurization count. Differential and selective media were used to identify groups of bacteria present in raw milk. Pasteurized milk samples were held at 6°C for 21 d and evaluated for standard plate count, coliform count, and sensory quality throughout shelf-life. Bacterial isolates from select raw and pasteurized milk tests were identified using 16S ribosomal DNA sequencing. Linear regression analysis of raw milk test results versus results reflecting pasteurized milk quality consistently showed low R² values (<0.45); the majority of R² values were <0.25, indicating small relationship between the results from the raw milk tests and results from tests used to evaluate pasteurized milk quality. Our findings suggest the need for new raw milk tests that measure the specific biological barriers that limit shelf-life and quality of fluid milk products.     

高效液相色谱法分析原料乳和奶粉中三聚氰胺

建立了二极管阵列检测器-高效液相色谱测定原料乳及奶粉中三聚氰胺的方法。奶粉样品经三氯乙酸超声提取,乙酸铅沉淀蛋白,三氯甲烷除去脂肪和非极性杂质,PROELUTPXCSPE阳离子交换固相萃取小柱进一步除去样品基质干扰,氨化甲醇洗脱。实验对色谱分离条件进行了优化,三聚氰胺的质量浓度在1.0～100.0μg/mL范围内与色谱峰面积呈良好的线性关系,回归方程为y=89.763x-113.72,相关系数为R2=0.9988。加标回收率为90.5%～105.0%,相对标准偏差(RSD)小于4.0%。该方法简单、快速,结果准确可靠。     

原料乳与乳制品中碳水化合物质量控制研究进展

原料乳与乳制品中碳水化合物具有极高的营养价值,对人体健康具有重要作用,但其掺伪现象较普遍,严重影响了原料乳与乳制品的品质。文章综述了原料乳与乳制品中碳水化合物及其掺伪物质检测技术研究进展,旨在为做好原料乳与乳制品中碳水化合物质量控制提供科学依据。      

Effect of leaving milk trucks empty and idle for 6 h between raw milk loads

The US Pasteurized Milk Ordinance (PMO) allows milk tanker trucks to be used repeatedly for 24 h before mandatory clean-in-place cleaning, but no specifications are given for the length of time a tanker can be empty between loads. We defined a worst-case hauling scenario as a hauling vessel left empty and dirty (idle) for extended periods between loads, especially in warm weather. Initial studies were conducted using 5-gallon milk cans (pilot-scale) as a proof-of-concept and to demonstrate that extended idle time intervals could contribute to compromised raw milk quality. Based on pilot-scale results, a commercial hauling study was conducted through partnership with a Pacific Northwest dairy co-op to verify that extended idle times of 6 h between loads have minimal influence on the microbiological populations and enzyme activity in subsequent loads of milk. Milk cans were used to haul raw milk (load 1), emptied, incubated at 30°C for 3, 6, 10, and 20 h, and refilled with commercially pasteurized whole milk (load 2) to measure cross-contamination. For the commercial-scale study, a single tanker was filled with milk from a farm known to have poorer quality milk (farm A, load 1), emptied, and refilled immediately (0 h) or after a delay (6 h) with milk from a farm known to have superior quality milk (farm B, load 2). In both experiments, milk samples were obtained from each farm's bulk tank and from the milk can or tanker before unloading. Each sample was microbiologically assessed for standard plate count (SPC), lactic acid bacteria (LAB), and coliform counts. Selected isolates were assessed for lipolytic and proteolytic activity using spirit blue agar and skim milk agar, respectively. The pilot-scale experiment effectively demonstrated that extended periods of idle (>3 h) of soiled hauling vessels can significantly affect the microbiological quality of raw milk in subsequent loads; however, extended idle times of 6 h or less would not measurably compromise milk quality in subsequent loads in commercial tankers. Current tanker sanitation practices appear to be sufficient for maintaining raw milk SPC, LAB, and coliform levels, which are important measures of milk quality.     

原料乳生产中的HACCP体系研究

通过对原料乳生产线上的工序、设备微生物污染状况的检测，分析了微生物数量的变化规律，确定了奶牛饲养、乳房清洗、设备清洗、原料奶暂存运输4个工序为关键控制点，并提出了相应的标准操作规程。     

Nisin and lacticin 481 coproduction by Lactococcus lactis strains isolated from raw ewes’ milk

Bacteriocin-producing lactococci were isolated from raw ewes’ milk samples obtained from 5 different Protected Designation of Origin Zamorano cheese manufacturers. Thirteen isolates with antimicrobial activity against Lactococcus lactis HP were selected. Eleven were identified by a PCR technique as L. lactis ssp. lactis and 2 were identified as L. lactis ssp. cremoris. They were grouped under 4 different pulsed-field gel electrophoresis patterns. The presence of structural genes of both nisin and lacticin 481 was detected in 10 L. lactis ssp. lactis isolates belonging to 2 different pulsed-field gel electrophoresis patterns. Coproduction of nisin and lacticin 481 was confirmed after semipurification by using selective indicators. The production of 2 bacteriocins by the same strain is an uncommon property, with relevance in food safety. Nisin and lacticin 481 L. lactis-producing strains might be used as adjunct cultures to the commercial starter in the manufacture of dairy products.     

控制体细胞数在优质生鲜奶生产中的应用

本文主要介绍了生鲜奶中体细胞数的作用、检测方法以及国内外的应用现状，讨论了体细胞数的影响因素及其控制措施．建议在我国开展牛群的体细胞数控制，从而提高生鲜奶的产量和质量。     

油橄榄叶提取物的抑菌活性研究

以牛津杯扩散法考察油橄榄叶提取物(OLE)对大肠杆菌、金黄色葡萄球菌等8种致病菌的抑菌活性,用稀释法分析OLE对8种细菌生长的影响,并探究最低抑菌质量浓度(MIC)和最低杀菌质量浓度(MBC)。结果表明:以盐酸小檗碱作为对照,OLE对蜡状芽孢杆菌、金黄色葡萄球菌、抗四环素大肠杆菌的抑菌作用优于盐酸小檗碱; OLE对表皮葡萄球菌、金黄色葡萄球菌、大肠杆菌的抑菌作用较强(MIC 1 mg/mL,MBC 2 mg/mL)。20 mg/mL的OLE对大肠杆菌的抑制作用最强,为极度敏感(大肠杆菌的d=(25. 07±3. 02) mm,抗四环素大肠杆菌的d=(24. 89±1. 58) mm);葡萄球菌次之(表皮葡萄球菌的d=(20. 11±1. 02) mm,金黄色葡萄球菌的d=(21. 35±2. 06) mm,金黄色葡萄球菌(SH7) d=(20. 01±1. 21) mm);而芽孢杆菌处于中度敏感状态。OLE除对铜绿假单胞菌的对数生长期无明显影响外,对其余7种细菌的对数生长期均有延缓作用。OLE具有较强的抑菌活性,可作为一种绿色安全的植物源抑菌制剂。     

巴氏牛奶中蜡样芽孢杆菌的风险评估

为了了解巴氏牛奶中蜡样芽孢杆菌的风险状况，调查研究了北京地区乳品企业巴氏牛奶的运输和销售的总体情况以及北京地区居民牛奶消费的相关情况，结合预测微生物学的方法建立了巴氏牛奶中蜡样芽孢杆菌在流通领域的暴露评估模型。从整个评估过程来看，流通领域的温度和时间是影响巴氏奶安全性的主要因素。     

原料乳危害及控制措施研究

分析了原料乳的生物性、化学性和物理性危害，针对各种危害的来源提出相应的控制措施．为原料乳HACCP体系的建立提供参考。     

原料乳微生物特性对ESL牛乳货架期影响的研究

研究了ESL乳生产过程中对原料乳质量的要求。结果表明,ESL乳的生产中,在保证原料乳细菌总数≤5×105mL-1时,原料乳中的芽孢数应控制在≤5×103mL-1,同时,应选取低温菌数量和体细胞数量都较低的原料乳。