Effect of psychrotrophic bacteria and of an isolated protease from Pseudomonas fluorescens M3/6 on the plasmin system of fresh milk

In the present study, we investigated the effect of Pseudomonas spp. growth on the plasmin enzymatic system in casein and whey fractions of fresh milk. Two bacterial strains, Pseudomonas spp. SRM28A and Pseudomonas fluorescens M3/6, were inoculated at a level of approximately 10(3) cfu/ml into fresh milk and incubated at 7 degrees C for 3 d. Bacterial counts were approximately 10(8) cfu/ml by d 3. Samples collected every 24 h were treated to separate the casein from the whey fraction. Casein and whey fractions were subjected to electrophoresis to visualize protein breakdown and plasmin activity and to colorimetric assays to quantify plasmin-related activities. With psychrotrophic bacterial growth, plasmin levels in casein fractions decreased significantly and in whey fractions increased then decreased significantly. Fresh milk results were similar for the two strains and were similar to earlier results with reconstituted nonfat dry milk. A transmission electron microscopy study by immunocytochemistry showed the presence of plasmin in casein micelles and its disappearance upon microbial growth in the milk. We hypothesized that extracellular microbial proteases produced by psychrotrophic microorganisms are responsible for this effect. To confirm this, an extracellular bacterial protease was isolated from Pseudomonas fluorescens M3/6 by ammonium sulfate fractionation and ion-exchange chromatography and incubated with fresh milk. Milk samples analyzed during incubation with the protease had significantly increased plasmin and plasminogen activities in the whey fraction within 5 h of incubation, while differences in activities in the casein fraction occurred at time 7.5 h for plasmin activity and 10 h of incubation for plasminogen activity. These quantitative data were supported by plasmin activity as visualized by casein-SDS-PAGE. These results suggest that growth of the Pseudomonas strains in fresh milk, and particularly their production of extracellular proteases, may be a causative factor in the release of plasmin from the casein micelle. Such plasmin release could affect the quality of cheeses and other food products that utilize dairy ingredients.     

Toxin production by Clostridium botulinum in pasteurized milk treated with carbon dioxide.

The addition of carbon dioxide to milk at levels of <20 mM inhibits the growth of selected spoilage organisms and extends refrigerated shelf life. Our objective was to determine if the addition of CO2 influenced the risk of botulism from milk. Carbon dioxide was added to pasteurized 2% fat milk at approximately 0, 9.1, or 18.2 mM using a commercial gas-injection system. The milk was inoculated with a 10-strain mixture of proteolytic and nonproteolytic Clostridium botulinum spore strains to yield 10(1) to 10(2) spores/ml. Milk was stored at 6.1 or 21 degrees C for 60 or 6 days, respectively, in sealed glass jars or high-density polyethylene plastic bottles. Milk stored at 21 degrees C curdled and exhibited a yogurt-like odor at 2 days and was putrid at 4 days. Botulinal toxin was detected in 9.1 mM CO2 milk at 4 days and in all treatments after 6 days of storage at 21 degrees C. All toxic samples were grossly spoiled based on sensory evaluation at the time toxin was detected. Although botulinal toxin appeared earlier in milk treated with 9.1 mM CO2 compared to both the 18.2 mM and untreated milk, gross spoilage would act as a deterrent to consumption of toxic milk. No botulinal toxin was detected in any treatment stored at 6.1 degrees C for 60 days. At 6.1 degrees C, the standard plate counts (SPCs) were generally lower in the CO2-treated samples than in controls, with 18.2 mM CO2 milk having the lowest SPC. These data indicate that the low-level addition of CO2 retards spoilage of pasteurized milk at refrigeration temperatures and does not increase the risk of botulism from treated milk stored at refrigeration or abuse temperatures.     

Volatile organic compounds associated with milk spoilage by psychrotrophic bacteria

The volatile organic compounds of milk contaminated with psychrotrophic bacteria were studied by HS‐SPME and GC/MS. Pseudomonas fluorescens PS14, Pseudomonas fragi PS55, Pseudomonas mosselii PS39, Pseudomonas rhodesiae PS62 and Serratia marcescens S92 were inoculated in sterilised milk (2.5% fat) stored at either 5 °C or 10 °C. A total of 47 volatile organic compounds (VOCs) belonging to seven chemical groups were identified in the spoiled milk. Volatile organic compound patterns peculiar to the inoculate bacterial strains were highlighted. 3‐Methylbutan‐1‐ol, 2 methylpropan‐1‐ol, 3‐hydroxybutan‐2‐one, butan‐2,3‐dione, butanoic and hexanoic acids were revealed as potential chemical spoilage indexes of milk spoilage due to the activity of the five psychrotrophic strains studied.     

冷鲜牛肉贮藏中菌群结构及优势菌致腐性的分析

为探究牛肉在0?℃冷藏下微生物菌群变化和优势腐败菌,采用培养依赖的16S rRNA结合高通量测序技术分析牛肉样品的微生物多样性变化。通过定期测定接种牛肉汁的生长、感官、蛋白酶活性、pH值和挥发性盐基氮（total volatile basic nitrogen,TVB-N）值,分析分离株的致腐特征。结果表明,牛肉冷藏中感官品质保持良好,15?d出现异味,18?d有腐臭味。而样品中菌落总数第3天快速上升,15?d菌落总数为8.73（lg（CFU/g））,之后呈现稳定,假单胞菌（Pseudomonas spp.）、热死环丝菌（Brochothrix thermosphacta）、肠杆菌（Enterobacter）和乳酸菌4 种分离培养基中细菌与菌落总数增长趋势相似,其中假单胞菌增长最快,肠杆菌数和乳酸菌数生长最慢。两种菌群鉴定结果显示,冷鲜牛肉初始微生物构成复杂,包括热死环丝菌、不动杆菌属（Acinetobacter spp.）、气单胞菌属（Aeromonas spp.）和假单胞菌等多种菌属构成,而腐败末期菌群构成趋于单一,假单胞菌和热死环丝菌为优势腐败菌,特别是莓实假单胞菌（P. fragi）。将腐败分离株10?株假单胞菌、4?株热死环丝菌和1?株蜂房哈夫尼菌（Hafnia alve）接种于冷藏的牛肉汁,发现假单胞菌和蜂房哈夫尼菌的接种组感官评分、pH值和TVB-N值高于热死环丝菌,且假单胞菌有较强的蛋白酶活性。研究表明,结合感官和微生物评价冷鲜牛肉贮藏期为15?d,且菌群多样性下降,假单胞菌和热死环丝菌是优势腐败菌,其中假单胞菌致腐性较强。     

Incidence, radioresistance, and behavior of Psychrobacter spp. in rabbit meat

The relative incidence of Psychrobacter spp. in rabbit meat, the radioresistance of these bacteria, and the growth of nonirradiated and irradiated psychrobacter isolates, alone and in coculture, during chilled storage of inoculated sterile rabbit meat was investigated. Psychrobacter spp. accounted for 4.2% of the storage psychrotrophic flora of 30 rabbit carcasses. The radiation D10-values of 10 Psychrobacter isolates, irradiated at 4 degrees C in minced rabbit meat, ranged from 0.8 to 2.0 kGy, with significant (P < 0.05) differences among strains. Over 12 days of storage at 4 degrees C, pure cultures of two nonirradiated psychrobacter strains (D10 = 2 kGy) were capable of substantial increases (up to 3 log CFU/g) in sterile rabbit meat, but when the fastest growing strain was cocultured with Pseudomonas fluorescens and Brochothrix thermosphacta isolates, maximum cell densities and growth rates were significantly (P < 0.01) lower. After irradiation (2.5 kGy) of pure cultures in sterile rabbit meat, surviving cells of both Psychrobacter strains decreased for a period of 5 to 7 days and then resumed multiplication that, at day 12, resulted in a similar increase (1.6 to 1.7 log CFU/g) over initial survivor numbers. When irradiated in combination with the spoilage bacteria, one of the strains required 12 days to reach initial numbers. In conclusion, Psychrobacter spp. are radioresistant nonsporeforming bacteria with a low relative incidence among the storage flora of rabbit meat, unable to compete with food spoilage bacteria in this ecosystem and apparently not a major contributor to the spoilage of rabbit meat after irradiation.     

Identification of lactic acid bacteria in traditional fermented yak milk and evaluation of their application in fermented milk products

In this study, 53 strains of lactic acid bacteria (LAB) isolated from Xueo, a traditional fermented yak milk in the western Sichuan Plateau of China, were identified and their use in fermented milk was evaluated. All gram-positive and catalase-negative strains were divided into 6 groups at the level of 87% similarity using amplified ribosomal DNA restriction analysis. These groups were identified as 6 species using 16S rDNA sequence analysis and atpA gene analysis. The dominant LAB strains in Xueo were Enterococcus durans, Lactobacillus fermentum, and Lactobacillus paracasei, accounting for 45.3, 22.6, and 17.0% of isolates, respectively. Milk fermented with most of the representative strains was high in quality, exhibiting relatively high viscosity, moderate acidity, good sensory quality, and high counts of viable LAB. Fermented milk of E. durans SCA16 and L. fermentum SCA52 achieved the highest scores for overall sensory quality. Most strains displayed antimicrobial activity against at least 1 of 9 spoilage microorganisms. Lactic acid was the main factor inhibiting the growth of spoilage bacteria, and H(2)O(2) was also inhibitory to some extent. Excluding the influence of acid and H(2)O(2), strains SCA52 (L. fermentum) and SCA7 (Lactobacillus plantarum) were antagonistic against some of the indicators, suggesting that the 2 strains may produce a bacteriocin-like substance. Therefore, the development of superior strains isolated from Xueo to ferment milk with similar flavor and texture to Xueo is expected.     

Modelling the relation between bacterial growth and storage temperature in pasteurized milks of varying hygienic quality

The shelf-life of pasteurized milk was mainly determined by the level of contamination with Gram-negative psychrotrophic bacteria. The length of lag phase of the bacteria was also important, although the generation times of the naturally contaminating flora seemed to be of little relevance except for milks where the shelf-life exceeded 10 days at 6°C. The effect of temperature on growth of the contaminants could be accurately determined by the 'square root' plot but the conceptual minimum temperature for growth (T_o) varied. The variation was related to the quality of the pasteurized milk. Effects of temperature on generation time, length of lag phase and shelf-life were most marked at temperatures below 15°C.
The microflora of pasteurized milk varied significantly with storage temperature. At refrigeration temperatures, spoilage was mainly due to the growth of Pseudomonas spp. Enterobacteriaceae and Gram-positive bacteria assumed greater importance in the spoilage of milks stored at temperatures above 10°C. Milks of good quality also contained Bacillus spp and this group of bacteria were not detectable in milks with short shelf-lives.     

冷藏金枪鱼优势腐败菌致腐败能力

以冷藏金枪鱼货架期终点筛选、纯化、鉴定所得优势腐败菌假单胞菌（Pseudomonas spp.）、不动杆菌 （Acinetobacter spp.）和热死环丝菌（Brochothrix thermosphacta）作为研究对象,通过分析接种优势腐败菌的冷藏 金枪鱼中各腐败微生物生长动态以及感官品质、挥发性盐基氮（total volatile basic nitrogen,TVB-N）含量、生物 胺含量的变化,以生长动力学参数和腐败物质的产量因子YTVB-N/CFU为评价标准,分析评价冷藏金枪鱼优势腐败菌假 单胞、不动杆菌和热死环丝菌的致腐败能力。结果表明：4 ℃冷藏金枪鱼中假单胞菌、不动杆菌和热死环丝菌的生 长曲线拟合效果最好的方程分别为修正Gompertz模型、Baranyi-Roberts模型和Logistic模型,分析所得生长动力学 参数发现,冷藏金枪鱼中假单胞菌、不动杆菌和热死环丝菌生长延滞时间和最大比生长速率分别为10.85、83.93、 79.11 h和0.045 7、0.182 9、0.111 1 h－1;接种假单胞菌、不动杆菌和热死环丝菌的冷藏金枪鱼感官货架期分别为 96、108 h和105 h,此时TVB-N含量分别为27.32、22.80 mg/100 g和24.85 mg/100 g;分析冷藏过程中金枪鱼生物胺 变化情况发现,假单胞菌产生物胺能力强于不动杆菌和热死环丝菌,贮藏96 h时接种假单胞菌组的生物胺指数已达 155.84,超过规定阈值100;冷藏金枪鱼致腐败能力分析结果显示,假单胞菌、不动杆菌和热死环丝菌的产量因子 YTVB-N/CFU分别为7.36×10－8、1.85×10－8 mg TVB-N/CFU和5.07×10－8 mg TVB-N/CFU,研究表明4 ℃冷藏金枪鱼贮 藏过程中假单胞菌致腐败能力较强,其次为热死环丝菌和不动杆菌。     

冷藏卵形鲳优势腐败菌的分离鉴定及致腐能力分析

为研究卵形鲳鲹冷藏末期的优势腐败菌及其致腐能力,采用传统选择性培养结合16S rDNA序列分析法,对卵形鲳鲹4℃贮藏期间腐败菌分别进行分离纯化、菌相分析及菌属确定,并以腐败物质的产量因子Y_TVB-N/CFU为评价标准,定量分析了贮藏末期优势菌的致腐能力。结果得出,卵形鲳鲹4℃贮藏过程中共分离纯化出16株细菌,其中革兰氏阴性菌10株,革兰氏阳性菌6株,其分属6个菌属。贮藏初期（0 d）时菌相丰富,希瓦氏菌属（Shewanella sp.）、假单胞菌属（Pseudomonas sp.）、葡萄球菌属（Staphylococcus sp.）、芽孢杆菌属（Bacillus sp.）、乳酸杆菌属（Lactobacillus sp.）与肠杆菌属（Enterobacter sp.）等均有出现;中期（3 d）菌群种类有所减少,以希瓦氏菌属（Shewanella sp.）与假单胞菌属（Pseudomonas sp.）所占比例较高,后期（6 d）种类进一步减少,只有3株菌占较高,分别是2号腐败希瓦氏菌（Shewanella putrefaciens 48%）、5号奥奈达希瓦氏菌（Shewanella oneidensis 26.6%）与16号霍氏肠杆菌（Enterobacter hormaechei 19.4%）;致腐能力由高至低依次为腐败希瓦氏菌 > 奥奈达希瓦氏菌 > 霍氏肠杆菌,其中腐败希瓦式菌致腐能力显著强于另两株菌;综合贮藏末期所占比例及致腐能力结果,最终确定腐败希瓦氏菌为4℃贮藏下卵形鲳鲹的优势腐败菌。     

Detection of the apr gene in proteolytic psychrotrophic bacteria isolated from refrigerated raw milk

Bacteria of the genus Pseudomonas have been associated with the spoilage of raw milk and dairy products due to the production of thermostable proteolytic enzymes. The apr gene encodes for alkaline metalloprotease in Pseudomonas and other related bacteria. Its presence in psychrotrophic proteolytic bacteria isolated from raw milk collected from cooling tanks was verified. A polymerase chain reaction (PCR) technique was used with degenerate primers. Total DNA from 112 isolates was pooled in different groups and then used as template for the amplification reactions. Controls consisted of DNA extracted from 26 cultures. An expected DNA fragment of 194 bp was detected in groups that contained bacteria identified as Pseudomonas. The PCR product was observed only when DNA from control cultures of Pseudomonas aeruginosa, Pseudomonas fluorescens, Serratia marcescens and Aeromonas hydrophila were used. A detection limit assay indicated that the apr gene could be directly amplified from pasteurized milk inoculated with 10(8) CFU/ml of P. fluorescens. With this method it was possible to detect proteolytic bacteria at 10(5) CFU/ml in reconstituted skim milk powder if cells were recovered for DNA extraction before amplification.     

肉制品中亚硝酸盐测定能力验证

为加强肉制品安全监督管理,进一步提升检验检测机构对肉制品中亚硝酸盐含量的检测能力,确保提供的检测数据准确、可靠,组织实施肉制品中亚硝酸盐含量检测能力验证计划。制备含亚硝酸钠的鸡肉火腿肠,向42家实验室随机发放样品,在规定时间内回收检测数据。对检测结果进行稳健统计分析,以Z值评价各个实验室的检测能力,并对非满意结果的实验室通过查阅原始记录进行技术分析。结果表明：制备的能力验证样品均匀、稳定,满足能力验证计划要求;42家实验室总体结果满意率为81.0%,表明实验室对肉制品中亚硝酸盐含量的检测能力较好;部分实验室发现检测过程中所存在的问题,并加以改正,使自身检测能力进一步提高。     

Application of polynomial models to predict growth of mixed cultures of Pseudomonas spp. and Listeria in meat

Three models for one rapid and one slow growing strain of Pseudomonas fragi and one slow growing strain of P. fluorescens were developed in a meat broth; they were designed to take account of variations in growth and to provide a growth response interval. These models, and another for Listeria monocytogenes (Lm14 model), were used to predict the growth of spoilage Pseudomonas spp. and pathogenic Listeria in meat products. The Pseudomonas and Listeria models provided satisfactory predictions concerning inoculated strains grown in decontaminated beef meat. It was also possible to use the Pseudomonas models to predict the growth of the natural flora (mainly Pseudomonas spp.) of refrigerated meat stored under aerobic conditions. In experiments with mixed populations, three situations were observed: (1) in decontaminated meat, L. monocytogenes inoculated alone grew well at 6 degrees C, and this result was correctly predicted by the model; (2) in decontaminated meat inoculated with Listeria and Pseudomonas strains, L. innocua grew well and was not affected by the presence of Pseudomonas, and the growth of both organisms was correctly predicted by the models; (3) in naturally contaminated meat inoculated with Listeria, the strain did not grow until Pseudomonas had reached the stationary phase. The models satisfactorily predicted the growth of Pseudomonas spp. but not that of Listeria. In conclusion, the Lm14 model cannot be used for refrigerated meat stored aerobically as the results suggest a 'fail-safe' level which may be too high: meat had already reached a spoilage state even though no increase in the level of Listeria was observed. The Pseudomonas models accurately predicted the growth of naturally occurring Pseudomonas spp.     

PCR-DGGE法结合传统技术研究4℃冷链贮运条件下三文鱼菌相变化

为研究三文鱼在冷链贮运4 ℃条件下的细菌腐败机制,运用聚合酶链式反应-变性梯度凝胶电泳（polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE）技术、传统鉴定技术以及PCR技术分析了4 ℃冷链贮运条件下的三文鱼中腐败菌菌相变化规律,并通过产量因子测得各优势菌株致腐能力,从而确定特定腐败菌。DGGE指纹图谱显示,贮藏期间微生物多样性降低,假单胞菌属和希瓦氏菌属条带亮度却逐渐提高。这表明这两个属的微生物在三文鱼冷藏期间逐渐成为优势菌。通过分离和鉴定贮藏末期腐败三文鱼的优势腐败菌,本实验得到5 株优势腐败菌,分别为麦芽糖肉食杆菌（Carnobacterium maltaromaticum LMA28）、丁香假单胞菌（Pseudomonas syringae pv. syringae B728a）、荧光假单胞菌（Pseudomonas fluorescens SBW25）、肉食杆菌（Carnobacterium sp. WN1359）和波罗的海希瓦氏菌（Shewanella baltica OS678）。将这5 株纯培养的腐败菌分别接种到无菌三文鱼中并冷藏一定时间后,各腐败菌的挥发性盐基氮（total volatile basic nitrogen,TVB-N）产量因子分别为1.26×10－7、1.25×10－7、1.36×10－7、1.08×10－7 mg TVB-N/CFU和1.03×10－7 mg TVB-N/CFU。这5 株腐败菌对三文鱼致腐败能力的顺序依次为荧光假单胞菌SBW25＞麦芽糖肉食杆菌LMA28＞丁香假单胞菌B728a＞肉食杆菌WN1359＞波罗的海希瓦氏菌OS678。     

Oxygen tension measurement as a means of detecting incipient spoilage of raw milk by psychrotrophic bacteria?

A large fall (89% to 10% saturation) in the oxygen tension of raw milk incubated with constant aeration and agitation at 7°C in a fermenter was observed to occur prior to the onset of extracellular protease and lipase production by microorganisms. This same pattern with respect to oxygen tension and lipase production was also observed when raw milk samples from creamery silos were incubated at 7°C in an orbital incubator. The results are in agreement with earlier fermenter studies in which strains of Pseudomonas fluorescens were grown in a simulated milk medium at 7°C. Measurement of oxygen tension may be able to be used to detect incipient enzyme-mediated spoilage of raw milk by psychrotrophs.     

Antihypertensive activity of casein-enriched milk fermented by Lactobacillus helveticus

Milk proteins contain peptidic angiotensin I-converting enzyme (ACE) inhibitors, which can be released by proteolysis during milk fermentation by some strains of Lactobacillus helveticus. Reconstituted milk media containing skim milk powder (12%), skim milk powder (10%) with added sodium caseinate (5%) or whey protein isolate (5%) were fermented by L. helveticus strains R211 and R389, and further tested for bacterial growth, proteolysis (free NH₃ groups) and ACE-inhibitory activity. The antihypertensive activity in spontaneously hypertensive rats (SHR) was also investigated with caseinate-enriched milk unfermented (UFM) and fermented by the two strains of L. helveticus. Caseinate-enriched milk fermented by both strains showed higher proteolysis and ACE-inhibitory activity, indicating that ACE-inhibitory peptides are probably released from caseins during milk fermentation. Significant decreases in mean arterial blood pressure in SHR rats were measured following oral administration of UFM milk at doses of 1.0 and 2.5 g kg⁻¹ of body weight, and milk fermented by R211 or R389 strains at doses of 0.5, 1.0 and 2.5 g kg⁻¹ of body weight. The antihypertensive activity of UFM could be explained by the release of ACE-inhibitory peptides from caseins during the digestion process.     

Growth kinetics and hydrolytic enzyme production of Pseudomonas spp. isolated from pasteurized milk

Psychrotrophs, particularly Pseudomonas spp. are known to be the main determinants of the shelf-life of pasteurized milk and refrigerated raw milk. It is presumed that they mainly cause spoilage through the elaboration of proteinase and lipase enzymes. At the time of this research, under the relevant European Directive, one of the means of determining the quality of pasteurized milk was the pre-incubated count, which involves incubating the milk sample for 5 d at 6 degrees C followed by a plate count. Examination of numerous pre-incubated counts revealed a bimodal rather than a normal distribution indicating that the types of contaminants in pasteurized milk may be as important as their initial concentration. Pseudomonads that gave particularly high (> 5 x 10(6) cfu/ml) and low (< 10(3) cfu/ml) pre-incubated counts were isolated (high and low count isolates respectively). After the organisms had been subjected to a cold shock no consistent trend between the groups of isolates was detected with respect to lag phase duration. However, the high count isolates consistently had a faster exponential growth rate. Unexpectedly, with the exception of one isolate, the low count isolates produced detectable proteinase and lipase earlier. In addition, with one exception, maximal proteinase and lipase production was observed with the low count isolates. These findings indicate that there is no causal relationship between selective growth advantage and ability to produce proteinase and lipase. It also indicates that the spoilage of pasteurized milk is a complex phenomenon and is worthy of further research.     

Effect of various dairy packaging materials on the shelf life and flavor of pasteurized milk

Milk from three different dairies (each a separate trial: 1, 2, and 3) was standardized to 2% fat and pasteurized at 92.2, 84.0, and 76.4 degrees C (temperatures 1, 2, and 3, respectively) for 25 s and packaged into six different packaging boards, [standard (A) milk boards with standard seam; juice boards with standard (B) and J-bottom (D) seams; barrier boards with standard (C) and J-bottom (E) seams; and foil (F) boards with J-bottom seam], resulting in 18 different treatments. Standard plate count (SPC) was used to test for microbial quality, and taste a panel was employed for flavor acceptability and difference on the milk stored at 6.7 degrees C at 1, 2, 3, and 4 wk. Statistical analysis of taste panel data showed that the flavor of milk samples A2, B2, and D2 deteriorated faster than the blind control (freshly high temperature, short time pasteurized low fat milk processed at 80.6 degrees C for 25 s). The flavor of milk packaged in standard (A) and juice (B and D) boards deteriorated at a faster rate than milk packaged in barrier (C and E) and foil (F) boards. Microbial counts showed that milk samples stored at 6.7 degrees C in trials 2 and 3 produced high SPC at wk 3 (ranges of bacteria in cfu/ml for trial 2: 9.9 x 10(1)-1.8 x 10(6) and trial 3: 2.5 x 10(5)-5.5 x 10(8)). In trial 1, high SPC began at wk 4 (9.9 x 10(1)-5.5 x 10(5) cfu/ml). Milk processed at 76.4 degrees C had the lowest bacterial growth rate, and milk processed at 84.0 degrees C had the highest bacterial growth rate. Different boards had no effects (P > 0.05) on the bacterial growth rates. It appeared that the lower the SPC of the raw milk, the slower the bacterial growth rate after 2 wk of storage. Milk samples stored at 1.7 degrees C maintained low SPC at wk 4, with counts of 0 to 40 cfu/ml for trial 2 and 0 to 200 cfu/ml for trial 3.     

Monitoring quality loss of pasteurized skim milk using visible and short wavelength near-infrared spectroscopy and multivariate analysis

Visible and short wavelength near-infrared diffuse reflectance spectroscopy (600 to 1,100 nm) was evaluated as a technique for detecting and monitoring spoilage of pasteurized skim milk at 3 storage temperatures (6, 21, and 37°C) over 3 to 30 h (control, t = 0 h; n = 3). Spectra, total aerobic plate count, and pH were obtained, with a total of 60 spectra acquired per sample. Multivariate statistical procedures, including principal component analysis, soft independent modeling of class analogy, and partial least squares calibration models were developed for predicting the degree of milk spoilage. Principal component analysis showed apparent clustering and segregation of milk samples that were stored at different time intervals. Milk samples that were stored for 30 h or less at different temperatures were noticeably separated from control and distinctly clustered. Soft independent modeling of class analogy analysis could correctly classify 88 to 93% of spectra of incubated samples from control at 30 h. A partial least squares model with 5 latent variables correlating spectral features with bacterial counts and pH yielded a correlation coefficient (R = 0.99 and 0.99) and a standard error of prediction (0.34 log₁₀ cfu/mL and 0.031 pH unit), respectively. It may be feasible to use short wavelength near-infrared spectroscopy to detect and monitor milk spoilage rapidly and noninvasively by correlating changes in spectral features with the level of bacterial proliferation and milk spoilage.     

基于下一代测序技术分析巴氏杀菌乳中残留细菌在贮藏期间的动态变化

采用高通量测序技术,分析巴氏杀菌乳在常见贮藏温度0、4、10 ℃条件下分别贮藏0、3、6、9、12、15 d内细菌16S rRNA的V3-V4区基因序列,进而比较不同贮藏条件下巴氏杀菌乳中微生物的群落组成及动态变化。结果共获得1 887 个可操作分类单元,共检测到支原菌属（Mycoplasma）、草酸杆菌属（Oxalobacteraceae）、假单胞菌属（Pseudomonas）、不动细菌属（Actinetobacter）、链球菌（Streptococcus）等34 类细菌菌属。多样性分析表明,不同贮藏温度条件下巴氏杀菌乳中微生物的组成及多样性在门、属水平上有明显差异。在0 ℃贮藏15 d内的巴氏杀菌乳微生物多样性保持最完整,并且0 ℃其营养品质也保持较好;而在4、10 ℃贮藏期间巴氏杀菌乳的微生物多样性及营养品质都有较大影响,菌群构成及优势菌群都发生了变化,主要菌群也随贮藏温度的升高变为假单胞菌属、气单胞菌属等。研究表明,在4 ℃贮藏3 d和9 d、10 ℃贮藏6 d作为巴氏杀菌乳品质腐败的关键时间点,都出现了之前未被检测出的类芽孢菌属（Paenibacillus）、沙雷氏菌属（新Serratia）,初步推测此两种菌属是导致巴氏杀菌乳品质腐败的关键决定因素。