微生物法在原料奶验收中的应用

对微生物法的应用进行了研究。采用对抗生素最敏感的嗜热链球菌,从产酸能力和凝乳时间两个因素来检测牛乳中抗生素的残留,并定量得出该嗜热链球菌的最低检出量为0．04单位。结果表明,该方法操作简便。     

嗜热链球菌m6A甲基化差异研究

嗜热链球菌是乳制品工业生产中最为重要的发酵剂之一，具有良好的发酵特性。DNA甲基化是DNA化学修饰的一种形式，可在不改变DNA序列的前提下影响基因表达。本文选取2株产酸表型具有较大差异的嗜热链球菌为研究对象，通过甲基化组学探究m6A甲基化与遗传表型的关系。利用PacBio SMRT 测序平台对嗜热链球菌 IMAU80278与嗜热链球菌IMAU20416进行基因组测序及m6A甲基化测定，比较2株嗜热链球菌功能基因及m6A甲基化差异。结果表明，嗜热链球菌IMAU80278与嗜热链球菌 IMAU20416基因组相似，且功能基因均以碳水化合物代谢及氨基酸代谢为主。嗜热链球菌IMAU80278与嗜热链球菌 IMAU20416 限制修饰系统，m6A甲基化位点和m6A基序均存在显著差异，特别是在碳水化合物代谢及氨基酸代谢相关功能基因的m6A甲基化存在明显差异。推测m6A甲基化的差异可能导致嗜热链球菌IMAU80278与嗜热链球菌IMAU20416产酸表型的不同。此外，从表观遗传学角度探究嗜热链球菌DNA甲基化对发酵特性的影响，为优良发酵剂的筛选及利用奠定理论基础。     

乳制品中嗜热链球菌的快速检测

利用血清学方法检测乳制品中的嗜热链球菌。首先，通过动物免疫方法制备了小鼠及家兔抗嗜热链球菌(Streptococcus thermophilus)的免疫血清。然后，用此血清经酶联免疫吸附测定法(ELISA法)对乳制品中嗜热链球菌进行检测。用间接ELISA法及双夹心ELISA法对乳制品中的嗜热链球菌进行检测。结果表明，两种方法均可测出乳制品中嗜热链球菌，其最低检测量可达10^7mE^-1。这一结果为乳制品中嗜热链球菌的血清学快速检测提供了参考。     

不同发酵剂对Mozzarella干酪品质的影响

采用嗜热链球菌和嗜热乳杆菌作为Mozzarella干酪的发酵剂，研究单一嗜热链球菌发酵剂与嗜热链球菌和嗜热乳杆菌组成的混合发酵剂对Mozzarella干酪品质的影响。结果表明，混合菌作发酵剂制得的干酪品质优于用单一嗜热链球菌作发酵剂的干酪。     

实时荧光定量PCR法鉴定发酵乳中嗜热链球菌

该研究通过系统发育树分析确定目标基因,根据目的基因设计特异性引物和探针,建立一种能够快速准确鉴定发酵乳中嗜热链球菌的实时荧光定量聚合酶链式反应（RT-fqPCR）法,通过特异性、灵敏性和抗干扰实验对所建立方法进行验证,并使用该方法对市售的60份标识含有嗜热链球菌的发酵乳样品进行检测。结果表明,recA基因具有种间特异性,种间差异率＞10%,以其为目的基因建立的RT-fqPCR方法能够特异性的检测嗜热链球菌;绝对灵敏度达1 pg/μL,相对灵敏度达103 CFU/mL;在培养物水平和基因组水平抗干扰能力良好。采用该方法从60份标识含有嗜热链球菌的发酵乳样品中均能检测出嗜热链球菌,说明实时荧光定量PCR方法能够快速、准确的对发酵乳中嗜热链球菌进行检测。     

嗜酸乳杆菌与嗜热链球菌混合培养制作保健型发酵乳的研究

对嗜酸乳杆菌和嗜热链球菌混合培养制作保健型发酵乳进行了研究，通过正交试验优化出了最佳生产工艺：加糖量为8%，嗜热链球菌和嗜酸乳杆菌比例为1∶4，接种量为4%，发酵温度为40℃.该发酵乳在4℃下贮存11天活菌数高于107cfu/ml，仍可具有良好的食疗保健作用。     

嗜热链球菌对生物活性肽QEPV吸收和利用的研究

以嗜热链球菌和生物活性肽Gln-Glu-Pro-Val(QEPV)为研究对象,探索了乳酸菌对生物活性肽的吸收和利用。首先,用添加荧光标记QEPV的M17培养基培养嗜热链球菌,采用流式细胞术检测QEPV能否被嗜热链球菌吸收。其次,测定不同剂量生物活性肽QEPV对嗜热链球菌活菌数、发酵液p H值、菌体浓度的影响,以探索嗜热链球菌对生物活性肽的利用情况。流式细胞术检测结果表明,FAM-QEPV与FITC-QEPV能够穿过嗜热链球菌细胞膜,推测QEPV能够被嗜热链球菌吸收、利用。并且0.5 mg/m L的QEPV能有效促进嗜热链球菌的生长繁殖,提高嗜热链球菌的发酵能力。     

三种乳酸菌混合培养的研究

在5L罐中对保加利亚乳杆菌、嗜酸乳杆菌和嗜热链球菌进行了单独培养及混合培养研究。实验结果表明,嗜酸乳杆菌及嗜热链球菌会抑制保加利亚乳杆菌的生长,保加亚利乳杆菌与嗜酸乳杆菌可能对嗜热链球菌的生长具有促进作用,嗜热链球菌对嗜酸乳杆菌具有促进作用,保加利亚乳杆菌对嗜酸乳杆菌的生长没有促进作用。这种实验结果为混合培养益生菌提供的实验参考。     

产PrtS蛋白酶嗜热链球菌的筛选及其生长特性研究

从商业发酵剂中初步分离出8株疑似嗜热链球菌,与实验室保存的26株嗜热链球菌,共收集到34株菌。通过PCR反应获得具有PrtS基因的一株菌,利用FSDA培养基确定该菌株具有PrtS蛋白酶活性,经生理生化实验和16S rDNA序列同源性分析该菌株为嗜热链球菌,命名为Streptococcus thermophiles 922。该菌株在LM17培养基中培养10 h时,活菌数达到9.68 log cfu/mL;在脱脂乳培养基中,产酸速度快,产酸量较高。实验结果为深入探究PrtS蛋白酶对嗜热链球菌生长和发酵性能影响提供基础。     

嗜热链球菌MN002冻干菌粉的制备工艺

为制备高活性嗜热链球菌MN002冻干菌粉,对富集培养后不同离心条件下嗜热链球菌MN002的存活率进行了研究。同时,以菌体浓缩倍数为指标,对蛋白溶解剂进行了组合筛选。进而通过单因素实验和二次正交旋转组合设计实验,考查了11种不同冻干保护剂对嗜热链球菌MN002的冻干保护效果,以获得嗜热链球菌MN002真空冷冻干燥的最佳保护剂配方。结果表明,嗜热链球菌MN002在离心转速为8 000 r/min,离心温度4℃条件下,菌体离心存活率最高。同时,以0.15%六偏磷酸钠和0.5%柠檬酸钠作为组合蛋白洗脱液,可大大提高MN002菌体浓缩倍数。试验确定嗜热链球菌MN002的最佳保护剂配方为:5%蔗糖、2%甘油、0.8%谷氨酸钠、1%抗坏血酸、5%海藻糖,在此条件下,嗜热链球菌MN002菌体存活率可达84.8%。     

用阻抗分析法快速检测牛奶中抗生素残留的研究

研究立足于牛奶中抗生素的快速检测,用嗜热乳酸链球菌作为指示菌,通过“阻抗分析仪”的自动检测、分析,根据DT时间的产生,判断牛奶中抗生素的残留情况。试验表明,方法操作简单、快捷,结果易判断,全部检测过程可在2 h之内完成。该方法的检测灵敏度与GB方法相类似,可检测牛奶中的青霉素为0.04U/mL。     

Surface Acoustic Wave (SAW) Biosensor for Rapid and Label-Free Detection of Penicillin G in Milk

Surface acoustic wave (SAW) biosensors were introduced for rapid and label-free detection of penicillin G in milk. This is the first time that the use of SAW biosensors for antibiotic detection is reported. Penicillin G belongs to the β-lactam antibiotics which are commonly used in human and veterinary medicine. Particularly because of the latter, the detection of antibiotics in foodstuffs of animal origin is essential. Current methods for specific antibiotic detection often require complex laboratory equipment and procedures. SAW biosensors, however, offer rapid detection of analyte concentrations with a minimum of experimental effort. They use label-free acoustic (gravimetric) detection. Owing to the small mass of antibiotics, detection via binding inhibition assay was preferred to direct detection in this work. Samples containing penicillin G were preincubated with the corresponding antibody, and SAW biosensor surfaces were coated with penicillin G epitopes. The antibody in the sample bound to the biosensor surface, unless it was inhibited by penicillin G. The assay allowed the detection of 2 ng/ml penicillin G in buffer and 2.2 ng/ml in low-fat milk. This is below the maximum residue limit of 4 ng/ml given by the European Commission for penicillin G and other β-lactam residues in food.     

乳酸菌发酵法检测牛乳中抗生素残留

目的建立一种简单、准确的检测牛乳中抗生素残留的方法-乳酸菌发酵法。方法以接种量和温度为变量,优化发酵条件;以青霉素为目标抗生素,建立检测方法;以发酵4 h未凝乳的样品中抗生素最低浓度为检测限,测定本方法对20种抗生素的检测限;对比本方法和GB/T 4789.27方法检测111个商品乳样品中抗生素残留检出率,验证本方法的准确性。结果建立了乳酸菌发酵法检测乳中抗生素残留的具体方法;确定了本方法对8类20种抗生素的检测限,其中4类12种抗生素的检测限低于其在牛乳中的最大残留限量(MRL);本方法和GB/T 4789.27方法检测111个商品乳样品抗生素残留检出率分别为9.0%和11.7%。结论本方法操作简单,结果准确,适合企业及基层检测单位作为初筛方法检测牛乳中的抗生素残留。     

Antibiotic resistance in gut bacteria from dairy calves: a dose response to the level of antibiotics fed in milk

Dairy calves are commonly fed milk from cows treated with antibiotics. The concentration of beta-lactam antibiotic residues found in milk from treated cows was used to determine the range of concentrations of penicillin used in a dose-regulated experiment. Thirty-one Holstein calves were randomly assigned to milk with penicillin G added at concentrations of 0, 6.25, 12.5, 25, and 50 microl/kg. Fecal swabs were taken from each calf twice weekly. Resistance to penicillin was tested by measuring the zone of inhibition in bacterial growth around a disk impregnated with the antibiotic. Inhibition was greatest for bacteria from calves fed milk with no penicillin (2.89 +/- 0.14 mm), and declined as the penicillin dose provided in the milk increased (to a low of 0.70 +/- 0.10 for the 50 microl/kg treatment group). In conclusion, resistance of gut bacteria to antibiotics increases with increasing concentrations of penicillin in the milk fed to dairy calves.     

Evaluation of a microbiological indicator test for antibiotic detection in ewe and goat milk

Antibiotics are widely used for therapeutic and prophylactic purposes in dairy animals. The presence of residual antibiotics in milk could cause potentially serious problems in human health and have technological implication in the manufacturing of dairy products. The aim of this study was to evaluate Delvotest Accelerator (DSM Food Specialties, Delft, the Netherlands), a new system for a fully automated microbial test to detect antibiotic residues in ewe and goat milk. Forty-three samples of raw, whole, refrigerated bulk-tank milk samples (22 of ewe milk and 21 of goat milk) were analyzed during the whole lactation period. Four concentrations of 4 antibiotics were diluted in milk: penicillin G at 1, 2, 3, and 4 μg/L; sulfadiazine at 25, 50, 100, and 200 μg/L; tetracycline at 50, 100, 200, and 400 μg/L; and gentamicin at 25, 50, 100, and 200 μg/L. The detection limit of the Delvotest Accelerator was calculated as the range of antibiotic concentrations within which 95% of positive result lie. The range of detection limit of penicillin G and sulfadiazine was easily detected by Delvotest Accelerator at or below the European Union maximum residue limits, both for ewe and goat milk samples. In contrast, the system showed a lower ability to detect tetracycline and gentamicin both for ewe and goat milk samples. Very low percentages of false-positive outcomes were obtained. Lactation phase did not seem to be a crucial factor affecting the ability of the Delvotest Accelerator to detect spiked milk samples. A higher detection ability was observed for goat milk samples compared with ewe milk samples. A negative correlation between the percentage of positive milk samples detected and milk fat, protein, and lactose contents was observed for gentamicin only.     

酸度计法快速检测牛奶中残留的β-内酰胺酶

为了快速、准确地检测牛奶制品中残留的β- 内酰胺酶的含量,利用β- 内酰胺酶分解青霉素产酸使牛奶pH 值下降的原理,对人工添加了β- 内酰胺酶的牛奶制品与青霉素反应后pH 值的下降规律进行研究。得到牛奶中残留的β- 内酰胺酶与青霉素反应的较适宜条件,从而获得快速检测牛奶制品中残留的β- 内酰胺酶的方法。结果确定牛奶制品中残留的β- 内酰胺酶与青霉素反应的适宜条件为温度33℃、底物质量浓度10mg/mL,检测时间仅为60min。此方法对液态纯牛奶中β- 内酰胺酶的最低检出限为8.92U/mL。     

Potential for milk containing penicillin G or amoxicillin to cause residues in calves

The potential for antibiotic residues in calves from consuming milk containing penicillin G or amoxicillin was investigated. Six calves were fed milk replacer, 6% body weight twice daily, containing 0.293, 2.92, or 5.85 microg of penicillin/ml (ppm) G or 0.25, 1.0, or 2.0 microg of amoxicillin/ml for three consecutive feedings. Urine and blood samples were collected after each feeding. Serum and urine samples were tested with a microbial receptor assay and a microbial growth inhibition assay to indicate potential drug residues. Penicillin G and amoxicillin were detected in the serum and urine of several calves 3 h after drinking spiked milk replacer. Possible violative drug residues in the calves were detected by the microbial growth inhibition assay up to 15 h after drinking spiked milk replacer. Penicillin G, but not amoxicillin, could be detected in urine 24 h after the final feeding of spiked milk replacer. Subsequently, six calves were fed milk replacer containing 11.7 microg of penicillin G/ml (ppm) twice daily, 6% body weight per feeding. Calves were slaughtered 3 h after the final feeding. Mean (+/-SD) concentrations of penicillin G measured by high-pressure liquid chromatography in liver, kidney, muscle, and serum were 0.409 (+/-0.167) microg/g, 0.031 (+/-0.012) microg/g 0.008 (+/-0.002) microg/g, and 0.013 (+/-0.006) mg/ml, respectively. This study indicates that calves fed milk with amoxicillin or penicillin G could possibly have violative residues if slaughtered within 24 h after feeding. Violative drug residues in liver tissue were found in calves slaughtered 3 h after consuming milk replacer containing 11.7 microg of penicillin G/ml (ppm).     

DETECTION OF ANTIMICROBIAL DRUG RESIDUES IN KENYAN MILK

The improved Dutch tube diffusion test was used to study the occurrence of inhibitory substances in raw bulk milk samples within the Nakuru District in Kenya. Initially the detection limits of the method were verified using milk standards spiked with selected antibiotics. Addition of penicillinase to inhibitor-positive samples was used for preliminary identification of penicillin G-type antibiotics and residue levels were estimated against a standard curve constructed by means of a B. stearothermophilus disc assay. The two-tube test was used to screen 1109 field samples of which 229 (21%) were suspect positive. The identification procedure confirmed 165 samples (14.9%) to contain penicillin G-type residues of which 118 contained levels exceeding the established EU MRL for penicillin G (4 μg/kg). This study indicates that antibiotic residues are prevalent in milk within the Nakuru district of Kenya. It suggests that the improved tube diffusion test in combination with a multiplate system could be useful for qualitative and quantitative identification of antimicrobial drug residues in milk.     

Inactivation of penicillin G in milk using hydrogen peroxide

Milk antibiotic residues have been a public concern in recent years. The Grade A Pasteurized Milk Ordinance mandates that raw Grade A milk will test negative for beta-lactam antibiotic residues before processing. The purpose of this research was to investigate the ability of various levels of peroxide and heat to inactivate penicillin G in raw milk. Whole milk spiked to a mean of 436 +/- 15.1 (standard error of the mean) ppb of potassium penicillin G was treated with hydrogen peroxide at levels of 0.0, 0.09, 0.17, and 0.34%. Samples at each peroxide level (n = 6 per treatment) were treated as follows: 1) incubated at 54.4 degrees C for 3 h, 2) pasteurized at 62.8 degrees C for 30 min, 3) incubated and pasteurized as in treatments 1 and 2, or 4) received no further treatment. A beta-lactam competitive microbial receptor assay was used for quantification of penicillin G. Concentrations of penicillin in selected samples were determined by HPLC for a comparison of test methods. Treatments were evaluated relative to their ability to reduce milk penicillin G levels to below the safe level of 5 ppb. The 0.09% hydrogen peroxide level was ineffective for all treatments. Hydrogen peroxide at 0.17% lowered the mean penicillin G (+/- SEM) from 436 +/- 15.1 to 6 +/- 1.49 ppb using the incubated and pasteurized heat treatment. The 0.34% concentration of hydrogen peroxide was the most effective, inactivating penicillin G to a level well below the safe level of 5 ppb with the pasteurized heat treatment, with or without incubation.