Use of microbial beta-lactamase to destroy penicillin added to milk

A simple method is described for destruction of penicillin residues in bulk milk to an undetectable amount (less than .003 U/ml) with commercially available crude beta-lactamase enzyme. Milk containing .1 or .5 U/ml penicillin G was treated with .01 to 1.0 mU/ml of beta-lactamase (Bacillus cereus) for up to 96 h. The Bacillus stearothermophilus var. calidolactis assay was used to quantify penicillin in milk between .003 to 1.0 U/ml. The .5 U/ml of penicillin G was reduced to an undetectable amount within 18 h at 4 degrees C by 1.0 mU/ml of beta-lactamase. The development of titratable acidity over 5 to 6 h in contaminated milks treated with beta-lactamase and inoculated with Streptococcus thermophilus GH, Streptococcus cremoris, Streptococcus lactis, or a commercial starter culture was the same as for control milk samples containing no additives or only enzyme. Pilot-scale manufacture of Swiss and Cheddar cheeses from contaminated milks treated with beta-lactamase yielded cheeses of comparable quality, to control cheeses produced from penicillin-free milk. There were no delays in acid production as judged from pH measurements during production and ripening of the cheeses. About 50% of beta-lactamase activity added to milk remained after pasteurization at 63 degrees C for 30 min. The safety for human consumption of cheese containing small quantities of penicillin degradation products from milk treated with beta-lactamase remains to be established.     

固定化精氨酸脱亚胺酶的制备与性质研究

从7种大孔型离子交换树脂中筛选出固定化效果最好的弱碱性苯乙烯系阴离子交换树脂D301-G,通过先吸附后交联的方法对精氨酸脱亚胺酶进行固定化条件及固定化酶性质研究。经单因素实验,结果表明,最佳固定化条件为每克树脂加入156 U精氨酸脱亚胺酶液,p H4.0,28℃条件下吸附4 h后,在4℃冷却,加入戊二醛溶液至体系内戊二醛体积分数为0.07%,4℃下交联4 h,最优条件下固定化酶活回收率可达85%以上。固定化酶的最适温度和p H分别为50⁶0℃和5.0⁵.5,较游离酶具有更高的温度稳定性,同时固定化酶的米氏常数Km值比游离酶高。固定化酶在重复使用8次后仍保留57.7%的酶活,表明该固定化酶具有较好的操作稳定性,可为连续生产瓜氨酸提供技术依据。      

Performance of a rapid ELISA for penicillin G in milk

A rapid enzyme-linked immunosorbent assay (ELISA) for the detection of penicillin G in milk at concentrations of 6 ng/ml (0.01 IU/ml) was used to screen 1651 off-farm milk samples previously reported as containing no detectable levels of antimicrobial substances. Using a single-well per test format and a percent inhibition cut-off for the determination of positive/negative endpoint, 3.1% positives were obtained. Comparison with intra-assay standards lowered the numbers of positives to 0.42%. Analysis of 170 milk samples positive for antimicrobials confirmed 92.4% as containing penicillin G using the prescribed cut-off level and 87.6% when compared to standards. The ELISA, now available commercially in kit form, analyses between 1 and 48 samples in 15 minutes. It will detect 0.01 IU/ml to a level of confidence of 99% and 0.005 IU/ml with between 75 and 95% confidence.     

乳中青霉素热稳定性和耐酸性的研究

以新鲜牛乳为原料,采用Benedict法对乳中青霉素的热稳定性和耐酸性进行研究。结果表明,当达到灭菌温度后,青霉素的清除率为77.4%～87.1%。一定温度条件下,随着体系pH值的减小,青霉素的清除率增加,当体系pH值为5.4时,青霉素清除率高达96.8%～100%。     

牛奶中青霉素含量的快速检测

在1 ml待检牛奶中加入3滴Benedict试剂,煮沸5 min.根据试样中颜色变化,判断牛奶中青霉素含量.青霉素含量为(80～100)×10-6显红色,(50～70)×10-6显红黄色,(30～40)×10-6显橘黄色,(10～20)×10-6显浅橘黄色,(5～9)×10-6显正黄色,(0～4)×10-6显黄色.     

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.     

Thermal destruction of bacterial spores immobilized in food/alginate particles

Spores of Clostridum sporogenes PA 3679 (NCIB 8053) and Bacillus stearothermophilus (NCIB 8919) were immobilized in large (0·8–2·4 cm) cubes consisting of alginate mixed with pureed potatoes, pea or meat. These were heated in experimental heating chambers at temperatures between 120 and 150°C. Theoretical predictions of the efficiency of the sterilization process were then compared with experimental data obtained from spore survival levels. The results indicate that this method may be used for the experimental substantiation of thermal processes received by large particulate foods.     

分子印迹技术应用于牛乳青霉素残留的检测

应用分子印迹方法合成了对青霉素有特异性吸附能力的印迹聚合物PenG-MIP,与本尼迪特（Benedict）试剂结合分析确定了PenG-MIP在牛乳中的吸附平衡时间为6 h,选择性吸附实验表明在牛乳中青霉素印迹聚合物对青霉素表现出高特异性。用一定量颗粒吸附处理含有青霉素的牛乳,再进行发酵活性实验,通过颗粒的用量及发酵情况确定乳中青霉素的残留量,实现分子印迹技术用于乳中青霉素残留的快速定量检测,检测下限为5μg/L或以下,结果准确度高,操作简便,且PenG-MIP颗粒可以反复使用,极大的降低了检测的成本。     

Fluorescent immunoassay for determination of penicillin and sulphonamide residues in milk using carboxylic magnetic nanoparticles

A fluorescent immunoassay for separate and simultaneous determination of penicillin (PEN) and sulphadimethoxine (SDM) in milk was developed. Monoclonal antibodies immobilised on carboxylic magnetic nanoparticles (MNPs) were used. It was proved that the protein A‐oriented immobilisation method (OI) provided a 1.5‐times more sensitive assay than the random immobilisation one (RI). The activation of MNPs with a mixture of N‐(3‐dimethylaminopropyl)‐N‐ethylcarbodiimide (EDC) and N‐hydroxysuccinimide (NHS) ensured a five‐times higher sensitivity of immunoassay than the activation using EDC. The linearity of standard curves in milk was: 3/10 ng/mL PEN (OI and RI), 50/250 ng/mL SDM (OI), and 50/500 ng/mL SDM (RI).     

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).     

固定化脂肪酶的制备及其酶学性质的研究

以大孔树脂为载体进行脂肪酶的固定化,通过优化固定化条件得到高活性固定化脂肪酶并对其酶学性质进行研究。结果表明:弱碱性大孔阴离子交换树脂D301R是最佳的固定化载体;最优固定化条件为加酶量10%(以大孔树脂质量计),加水量30%(以大孔树脂质量计),4℃下在异辛烷中固定5 h。与游离脂肪酶相比,固定化脂肪酶具有良好的热稳定性、储存稳定性、吸附稳定性和对有机溶剂的抗逆性,循环使用6次,水解橄榄油的酶活回收率仍可达到50%。     

Removal of penicillin G from contaminated milk by ultrafiltration

Penicillin G purposedly added to whole raw milk at concentrations from .05 to .20 IU/ml was removed to undetectable levels by a combination of ultrafiltrations and permeate washes. After penicillin removal, whole milk 3:1 vol/vol concentrated retentates were reconstituted to milk with antibiotic-free permeate with little change in composition and with very good to excellent flavor. Washing 3:1 vol/vol concentrated retentates from penicillin-contaminated milks with water also removed penicillin to an undetectable level but changed the composition of product, which displayed a flat flavor.     

超声波对牛奶中青霉素的降解研究

在不同的温度、pH、超声功率和作用时间下,利用超声波技术对牛奶中青霉素进行降解,并用高效液相色谱(HPLC)法对牛奶中青霉素残留量进行检测,最终得到最佳降解条件为25℃、pH7、超声功率150W、作用时间35min。此时残留量小于1μg/L,符合相关规定。     

固定化乳糖酶制备低乳糖牛奶的研究进展

不同来源的β-半乳糖苷酶已经用于水解牛乳或乳清中的乳糖。酶水解乳糖的基本产物为葡萄糖和半乳糖。经水解后的乳糖增加了产品的甜度并使得牛乳适于那些患乳糖不耐症的人群。由于用游离乳糖酶会使牛奶掺入外来蛋白以及使用游离乳酶酶提高了生产成本,从而使乳糖酶的应用受到限制。将乳糖酶固定化后既可以重复使用,又能连续操作,且缩短了处理时间,从而明显降低了使用成本,因此对于乳糖酶的固定化受到了酶学专家的关注。本文简要介绍了乳糖和乳糖酶及其分类、乳糖酶的固定化方法及其应用,包括包埋法、交联法、吸附法、结合法及多种方法的混合使用及国内外的研究现状,并简要介绍了固定化乳糖酶的清洗。     

An evaluation of the Charm test--a rapid method for the detection of penicillin in milk

Using the Charm radioactive assay technique as a rapid test for the determination of penicillin in milk in experiments with milks containing known concentrations of penicillin, the coefficient of variation was found to be 15 and 11% for raw whole and reconstituted skim-milks respectively. Storage of reagents for up to 4 d increased the variance of the test by more than 20% and the estimated penicillin concentration 0.4%/d of storage up to 25 d. In comparison with the Bromocresol Purple Test, which was capable of 100% accuracy in defining a threshold of 0.02 i.u. penicillin/ml milk, the Charm Test falsely classified 15.6% of the samples.     

低乳糖奶的研究进展及应用

乳制品富含优质蛋白质、乳脂、乳糖等营养成分和钙、磷、钾等矿物质及多种维生素,同时含有多种免疫物质、酶及激素等具有生理活性调节功能的生物活性物质,且消化率和吸收率极高,被成为人体的"白色血液",是目前发现的最理想的天然食品。近年来,我国乳业发展非常迅速,但与世界平均水平仍存在巨大的差距,制约我国乳业发展的一个重要原因就是乳糖不耐症。如利用β-半乳糖苷酶对牛奶进行水解,可通过水解反应生成易被人体吸收的葡萄糖和半乳糖,其中部分半乳糖在β-半乳糖苷酶的作用下合成6%8%的"双歧因子"低聚半乳糖。低乳糖牛奶不仅提高了牛奶的营养价值,缓解乳糖不耐症的症状,提高钙的吸收利用率,而且含有的低聚半乳糖还可以促进体内益生菌群的增殖,减少肠道内有害物质的生成。     

纯芝麻汁饮料研制

通过研究乳化剂和增稠剂的种类和添加量,发现当添加0.2%由单脂肪酸甘油酯和蔗糖脂肪酸酯(HLB11)以1∶3组成的复合乳化剂及0.15%由黄原胶和海藻酸钠以1∶2组成的复合增稠剂时,可以获得稳定的芝麻饮料,而且当去除50%的油脂及磨浆水量为芝麻重量的15倍时,制备的芝麻饮料乳液稳定,口感醇厚。     

奶啤的研制

以牛奶、麦芽和酒花为主要原料,通过二次生物发酵研制出具有奶香和啤酒风味的奶啤饮料。通过实验确定奶啤的最佳配方及生产工艺流程并对奶啤的稳定性进行探讨,从而研制出即有啤酒的低酒精度、泡沫丰富和适度的二氧化碳,又具有酸乳饮料的酸甜可口、营养全面的奶啤饮料。     

沙蒿胶磁性微球固定化脂肪酶及部分理化性质的研究

以沙蒿多糖-壳聚糖复合磁性微球为载体,采用物理吸附法固定化脂肪酶,对固定化过程中对酶活力有影响的各种因素做了研究,同时对固定化酶的部分理化性质、最适pH、最适温度、酶的热稳定性以及表现米氏常数与游离酶做了比较.固定化酶的Km小于游离酶的Km,其最适pH和最适温度分别为8.0和50℃,而且固定化脂肪酶具有良好的热稳定性、可应用性和重复使用性.