乳及乳制品中嗜冷菌的多样性及其腐败危害研究进展

乳是一种营养丰富的物质,同时也是微生物生长繁殖的理想培养基。生乳的质量是影响乳制品产业链的关键因素,随着低温储存和冷链运输技术的发展,生乳中大部分细菌的生长受到抑制,但嗜冷菌的生长并未受到抑制,并逐步成为生乳中的优势菌。生乳在冷藏运输或储存期间,嗜冷菌依旧可以生长繁殖,其分泌的蛋白酶和脂肪酶可耐高温,经过巴氏杀菌或超高温灭菌处理后依旧保持活性,因此,了解嗜冷菌的多样性及其产生的酶对提高乳及乳制品质量、减少腐败和浪费具有重要作用。本文通过介绍乳及乳制品中嗜冷菌污染现状及腐败危害研究,旨在为乳及乳制品行业的风险评估提供背景信息,从源头控制嗜冷菌对生乳的浪费,保证乳及乳制品的品质。     

A 100-Year Review: Microbiology and safety of milk handling

Microbes that may be present in milk can include pathogens, spoilage organisms, organisms that may be conditionally beneficial (e.g., lactic acid bacteria), and those that have not been linked to either beneficial or detrimental effects on product quality or human health. Although milk can contain a full range of organisms classified as microbes (i.e., bacteria, viruses, fungi, and protozoans), with few exceptions (e.g., phages that affect fermentations, fungal spoilage organisms, and, to a lesser extent, the protozoan pathogens Cryptosporidium and Giardia) dairy microbiology to date has focused predominantly on bacteria. Between 1917 and 2017, our understanding of the microbes present in milk and the tools available for studying those microbes have changed dramatically. Improved microbiological tools have enabled enhanced detection of known microbes in milk and dairy products and have facilitated better identification of pathogens and spoilage organisms that were not known or well recognized in the early 20th century. Starting before 1917, gradual introduction and refinement of pasteurization methods throughout the United States and many other parts of the world have improved the safety and quality of milk and dairy products. In parallel to pasteurization, others strategies for reducing microbial contamination throughout the dairy chain (e.g., improved dairy herd health, raw milk tests, clean-in-place technologies) also played an important role in improving microbial milk quality and safety. Despite tremendous advances in reducing microbial food safety hazards and spoilage issues, the dairy industry still faces important challenges, including but not limited to the need for improved science-based strategies for safety of raw milk cheeses, control of postprocessing contamination, and control of sporeforming pathogens and spoilage organisms.     

A microbiological perspective of raw milk preserved at room temperature using hyperbaric storage compared to refrigerated storage

The effects of hyperbaric storage (HS, 50–100 MPa) at room temperature (RT) on endogenous and inoculated pathogenic surrogate vegetative bacteria (Escherichia coli, Listeria innocua), pathogenic Salmonella enterica and bacterial spores (Bacillus subtilis) were assessed and compared with conventional refrigeration at atmospheric pressure for 60 days. Milk stored at atmospheric pressure and refrigeration quickly surpassed the acceptable microbiological limit within 7 days of storage, regarding endogenous microbiota, yet 50 MPa/RT slowed down microbial growth, resulting in raw milk spoilage after 28 days, while a significant microbial inactivation occurred under 75–100 MPa (around 4 log units), to counts below 1 log CFU/mL throughout storage, similar to what was observed for B. subtilis endospores. While inoculated microorganisms had a gradually counts reduction in all HS conditions. Results indicate that HS can not only result in the extension of milk shelf-life but is also able to enhance its safety and subsequent quality.Industrial relevanceThis new preservation methodology could be implemented in the dairy farm storage tanks, or during milk transportation for further processing, allowing a better microbial control, than refrigeration. This methodology is very promising, and can improve food products shelf-life with a considerable lower carbon foot-print than refrigeration.     

Biofilm Formation in Milk Production and Processing Environments; Influence on Milk Quality and Safety

Abstract: Bacteria in milk have the ability to adhere and aggregate on stainless steel surfaces, resulting in biofilm formation in milk storage tanks and milk process lines. Growth of biofilms in milk processing environments leads to increased opportunity for microbial contamination of the processed dairy products. These biofilms may contain spoilage and pathogenic microorganisms. Bacteria within biofilms are protected from sanitizers due to multispecies cooperation and the presence of extracellular polymeric substances, by which their survival and subsequent contamination of processed milk products is promoted. This paper reviews the most critical factors in biofilm formation, with special attention to pseudomonads, the predominant spoilage bacteria originating from raw milk. Biofilm interactions between pseudomonads and milk pathogens are also addressed, as emerging risks and future research perspectives, specifically related to the milk processing environment.     

Effect of nisin and high temperature pasteurization on the shelf-life of whole milk

Two experiments were conducted to assess the effectiveness of nisin on the keeping quality of pasteurized whole milk. After pasteurization, milk samples were stored at 10°C and samples were analysed at intervals for total plate count (TPC), Lactobacillus count, calcium ion concentration, pH and total acidity (TA). In the first experiment nisin was added to milk samples in the range 0 to 50 IU ml^-1 prior to pasteurization at 72°C for 15 s. AH concentrations of nisin used were effective in controlling microbial growth. Milk containing 40 and 50 IU ml^-1 nisin had not spoiled after 41 days' storage compared to spoilage time of 14 days for the control milk. In the second experiment 40 IU ml^-1 of nisin was combined with three 'pasteurization' processes: 72°C for 15 s, 90°C for 15 s and 115°C for 2 s. The milk processed at 72°C for 15 s with nisin showed an increased keeping quality of about 7 days compared with the control and showed a significantly lower count for Lactobacillus. In contrast, nisin had a much greater effect on TPC counts in the milk pasteurized at 90°C for 15 s, and after 28 days' storage at 10°C the milk was still acceptable. Milks treated both with and without nisin at 115°C for 2 s were microbiologically acceptable after 28 days, with counts less than 10 ml^-1. However, the milk with nisin was superior in flavour, as no noticeable off-flavours were apparent after 32 days. All these results are consistent, as shown by the microbiological and chemical analyses. Addition of nisin to milk prior to pasteurization provides an opportunity to achieve extended shelf-life in regions with poor refrigeration.     

Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model

Psychrotolerant spore-forming bacteria represent a major challenge regarding microbial spoilage of fluid milk. These organisms can survive most conventional pasteurization regimens and subsequently germinate and grow to spoilage levels during refrigerated storage. To improve predictions of fluid milk shelf life and assess different approaches to control psychrotolerant spore-forming bacteria in the fluid milk production and processing continuum, we developed a predictive model of spoilage of fluid milk due to germination and growth of psychrotolerant spore-forming bacteria. We characterized 14 psychrotolerant spore-formers, representing the most common Bacillales subtypes isolated from raw and pasteurized milk, for ability to germinate from spores and grow in skim milk broth at 6°C. Complete growth curves were obtained by determining total bacterial count and spore count every 24 h for 30 d. Based on growth curves at 6°C, probability distributions of initial spore counts in bulk tank raw milk, and subtype frequency in bulk tank raw milk, a Monte Carlo simulation model was created to predict spoilage patterns in high temperature, short time-pasteurized fluid milk. Monte Carlo simulations predicted that 66% of half-gallons (1,900 mL) of high temperature, short time fluid milk would reach a cell density greater than 20,000 cfu/mL after 21 d of storage at 6°C, consistent with current spoilage patterns observed in commercial products. Our model also predicted that an intervention that reduces initial spore loads by 2.2 Log₁₀ most probable number/mL (e.g., microfiltration) can extend fluid milk shelf life by 4 d (end of shelf life was defined here as the first day when the mean total bacterial count exceeded 20,000 cfu/mL). This study not only provides a baseline understanding of the growth rates of psychrotolerant spore-formers in fluid milk, it also provides a stochastic model of spoilage by these organisms over the shelf life of fluid milk, which will ultimately allow for the assessment of different approaches to reduce fluid milk spoilage.     

Microbial community analysis of food-spoilage bacteria in commercial custard creams using culture-dependent and independent methods

Custard cream is made from highly nutritive raw materials such as milk and sugar and is easily spoiled by the multiplication of specific microbial contaminants or residents. However, this spoilage microbial community has not been studied. We determined the spoilage microbiota in commercial custard creams using culture-dependent and independent methods. Using the culture-dependent analysis with various agar media, 185 bacterial colonies and 43 eukaryal colonies were isolated from 7 commercial custard cream products. All bacterial isolates were morphologically, physiologically, and genetically identified as bacilli, staphylococci, lactic acid bacteria, and psychrotrophic gram-negative rods. Using culture-independent molecular analysis, the PCR-denaturing gradient gel electrophoresis technique, spoilage of the commercial custard creams was found to be caused by bacilli, staphylococci, lactic acid bacteria, psychrotrophic gram-negative rods, Anoxybacillus sp., Caurobacter sp., and Streptococcus sp. bacteria. The detected spoilage bacteria were the same species as previously detected in spoiled milk products and shown in other reports, suggesting that spoilage bacteria in a raw material easily grow in processed foods made from milk. We determined the spoilage microbial communities in commercial custard creams, and these are the first data concerning spoilage microbiota in nonfermented processed foods using a culture-independent analysis. Our study will be useful for the manufacture and safe preservation of dairy products because the first step toward safe food preservation by food manufacturers is to understand the spoilage microbiota in a target food to select optimal preservatives and to reduce the use of food additives.     

Pseudomonas spp. and Serratia liquefaciens as Predominant Spoilers in Cold Raw Milk

The storage of fresh raw milk at low temperature does not prevent proliferation of psychrotrophic bacteria that can produce heat‐resistant proteolytic enzymes contributing to the reduced shelf life of dairy products. This study aimed to identify the dominant psychrotrophic proteolytic enzyme‐producing population of raw milk from Brazil. Raw milk samples collected in 3 different cooling tanks in Brazil were stored at optimal (45 h at 4 °C followed by 3 h at 7 °C) and suboptimal (45 h at 7 °C followed by 3 h at 10 °C) conditions to simulate farm storage and transportation allowed by Brazilian laws. The highly proteolytic enzyme‐producing strains isolated from stored cold raw milk were characterized by repetitive sequence‐based Polymerase Chain Reaction (PCR) analysis. This clustering resulted in 8 different clusters and 4 solitary fingerprints. The most proteolytic isolates from each rep‐cluster were selected for identification using miniaturized kit, 16S rDNA and rpoB gene sequencing. Serratia liquefaciens (73.9%) and Pseudomonas spp. (26.1%) were identified as the dominant psychrotrophic microorganisms with high spoilage potential. The knowledge of milk spoilage microbiota will contribute to improved quality of milk and dairy products.     

Evaluation of calf milk pasteurization systems on 6 Pennsylvania dairy farms

Waste milk has been fed to calves for many years, but concerns with bacterial contamination as well as possible transmission of diseases have discouraged widespread use of this feed. Pasteurization of waste milk is one option to reduce management risk while utilizing a valuable, low-cost, liquid feed source for calves. However, many farms currently pasteurizing waste milk lack a system to adequately monitor the efficiency of the process. A study was carried out to evaluate 6 on-farm pasteurization systems, including high-temperature, short-time pasteurizers and low-temperature, batch pasteurizers. Milk samples were taken pre- and postpasteurization as well as from the calf buckets and immediately frozen for later bacterial culture. Samples were collected twice daily for 15 d. Milk samples were examined for standard plate count (SPC), coagulase-negative staphylococci count, environmental streptococci count, coliform count, gram-negative noncoliform count, Streptococcus agalactiae count, and Staphylococcus aureus count. Before pasteurization, 68% of the samples had SPC <20,000 cfu/mL, and 39% of samples contained <100 cfu/mL of coliform bacteria. After pasteurization, 96% of samples had SPC <20,000 cfu/mL, and 92% had coliform counts <100 cfu/mL. Bacteria counts were significantly reduced by pasteurization, and pasteurized milk contained acceptable numbers of bacteria in >90% of samples. These results indicate that pasteurization can be very effective in lowering bacterial contamination of milk. However, bacteria numbers significantly increased after pasteurization and, in some cases, bacteria counts in milk fed to calves were similar to prepasteurization levels. Milk handling after pasteurization was identified as an important issue on the farms studied.     

原料乳中腐败微生物对液态乳品质的影响及防控

乳因其营养丰富备受关注,被称为人体的“白色血液”,同时乳也是微生物良好的培养基,因此原料乳品质和液态乳加工过程都会受到严格把控,但目前液态乳在储藏期、流水线或货架期内发生腐败变质现象仍屡屡发生,引起行业对乳及乳制品安全的高度重视。原料乳作为液态乳生产的原料,是其品质保障的基础,原料乳中腐败微生物随着储藏时间的延长而大量繁殖,经过热处理后未被完全杀死的腐败菌及其产生的耐热酶仍会继续影响液态乳品质,此外生物膜的形成也为腐败菌在管道内残留提供了机会,加大了腐败微生物防控的难度。因此本文将从原料乳中腐败微生物多样性进行阐述并探究腐败微生物对于液态乳的不良影响,挖掘腐败微生物破坏液态乳可能存在的作用机制,总结现阶段国内外对腐败微生物的防控手段并探讨其商业应用价值,旨在为今后液态乳质量安全保障及延长货架期提供理论指导。     

Microwave heat treatment application to pasteurization of human milk

A prototype of microwave pasteurizer has been proposed as an alternative for holder pasteurization (HP) routinely used in Human Milk Bank (HMB), ensuring microbiological safety of human milk (HM). It was shown that the time of heat generation was about 15–16 min shorter by applying the microwave than in HP. Total inactivation of heat-sensitive bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis, suspended in milk, occurred in the temperature 62–72 °C in HP. In the case of heat-resistant enterococci the level of inactivation depended on the conditions of the process and the properties of the strains. The application of microwave heating allows to obtain lower D-value than those achieved during HP. The using of microwave heating at 62.5 or 66 °C for 5 or 3 min, respectively, allows to inactivation of HM microbiota. Appropriate microbiological quality of milk is critical for the effectiveness of the pasteurization process.Industrial relevanceLooking for new methods of donor human milk (HM) preservation is dictated by the necessity of providing microbiological safety and, at the same time, maintain its high nutritional and biological value. The holder pasteurization used in the Human Milk Banks (HMB) (heating at 62 °C for 30 min) leads to inactivation of all vegetative forms of microorganisms. Unfortunately, this method causes significant reduction of health benefitting properties of HM. The paper demonstrates the possibility of using the new microwave pasteurizer for preservation of HM, allowing for quick heating of milk to the appropriate temperature and maintaining it in these conditions for a required time. It was shown that the decimal reduction times (D) for strains inoculated to UHT or human milk are several times shorter by using microwave heating than in the commercial pasteurization method. The total inactivation of HM microbiota is obtained after heating at 62.5 and 66 °C for 5 and 3 min, respectively.     

High temperature, short time pasteurization temperatures inversely affect bacterial numbers during refrigerated storage of pasteurized fluid milk

The grade A Pasteurized Milk Ordinance specifies minimum processing conditions of 72°C for at least 15 s for high temperature, short time (HTST) pasteurized milk products. Currently, many US milk-processing plants exceed these minimum requirements for fluid milk products. To test the effect of pasteurization temperatures on bacterial numbers in HTST pasteurized milk, 2% fat raw milk was heated to 60°C, homogenized, and treated for 25 s at 1 of 4 different temperatures (72.9, 77.2, 79.9, or 85.2°C) and then held at 6°C for 21 d. Aerobic plate counts were monitored in pasteurized milk samples at d 1, 7, 14, and 21 postprocessing. Bacterial numbers in milk processed at 72.9°C were lower than in milk processed at 85.2°C on each sampling day, indicating that HTST fluid milk-processing temperatures significantly affected bacterial numbers in fluid milk. To assess the microbial ecology of the different milk samples during refrigerated storage, a total of 490 psychrotolerant endospore-forming bacteria were identified using DNA sequence-based subtyping methods. Regardless of processing temperature, >85% of the isolates characterized at d 0, 1, and 7 postprocessing were of the genus Bacillus, whereas more than 92% of isolates characterized at d 14 and 21 postprocessing were of the genus Paenibacillus, indicating that the predominant genera present in HTST-processed milk shifted from Bacillus spp. to Paenibacillus spp. during refrigerated storage. In summary, 1) HTST processing temperatures affected bacterial numbers in refrigerated milk, with higher bacterial numbers in milk processed at higher temperatures; 2) no significant association was observed between genus isolated and pasteurization temperature, suggesting that the genera were not differentially affected by the different processing temperatures; and 3) although typically present at low numbers in raw milk, Paenibacillus spp. are capable of growing to numbers that can exceed Pasteurized Milk Ordinance limits in pasteurized, refrigerated milk.     

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.     

Conventional and ohmic heating pasteurization of fresh and thawed sheep milk: Energy consumption and assessment of bacterial microbiota during refrigerated storage

This study aimed to assess the energy consumption of ohmic heating (OH) and conventional heating (CH) for pasteurization of fresh and thawed sheep milk and their impact on bacterial microbiota throughout refrigerated shelf-life (4 °C ± 1, 15 days). OH pasteurization using 8.33 and 5.83 V/cm electric field strength spent 72–73% less energy than CH pasteurization (515 KJ). The cultivation-dependent approach showed that at least 4.2 log cycle reductions were achieved in sheep milk submitted to CH and OH pasteurization, regardless sheep milk was fresh or thawed. Data from amplicon sequencing indicated that Staphylococcus was the most prevalent genus in raw samples at day 1 (F1_D1: 58.35%; T1_D1: 69.50%), while Pseudomonas became the most abundant after 15 days under cold storage (F1_D15: 50.15% and T1_D15: 54.50%). The relative abundance of all bacterial genera assessed remained similar on pasteurized samples by CH and OH throughout refrigerated storage.Industrial relevanceOhmic heating (OH) presents as a critical advantage rapid and uniform heating of fluid food material. No studies assessed the use of OH for pasteurization of sheep milk and to evaluate the impact of this technology on sheep milk bacterial microbiota during refrigerated storage. The findings of this study prove the feasibility of sheep milk pasteurization using OH_{8.33 V/cm}. OH also ensured the bacteriological stability of sheep milk during 2 weeks of storage under refrigeration conditions prior to dairy products manufacturing. This approach comprises a cheaper and easier way to store milk when compared to frozen storage, with potential benefits to small farmers and dairy industries.     

16S rDNA high-throughput sequencing and MALDI-TOF MS are complementary when studying psychrotrophic bacterial diversity of raw cows' milk

Refrigeration of raw milk favours the growth of psychrotrophic bacteria (psychrotrophs) that can produce spoilage enzymes, which may deteriorate final dairy products. The psychrotrophs from raw milk across four seasons were identified using culture-dependent and culture-independent approaches. Both 16S rDNA high-throughput sequencing (HTS) and MALDI-TOF MS showed the Pseudomonas genus to predominate; however, predominant species differed between the two identification methods. There was no geographical trend in microbiota, but a seasonal variation was evident. The use of HTS and MALDI-TOF MS was complementary in describing the psychrotrophic bacterial diversity of raw milk and provides an understanding of the raw milk microflora that may influence milk quality. This is the first report to compare data obtained from 16S rDNA high-throughput sequencing and MALDI-TOF analysis to assess the psychrotrophic microbial quality of refrigerated raw milk in New Zealand.     

Effect of heat treatment of milk on activation of Bacillus spores

The quality and shelf life of fluid milk products are dependent on the amount and type of microorganisms present following pasteurization. This study evaluated the effects of different pasteurization processes on the microbial populations in fluid milk. The objective was to determine whether certain pasteurization processes lead to an increase in the amount of bacteria present in pasteurized milk by activating Bacillus spores. Samples of raw milk were collected on the day of arrival at the dairy plant. The samples were pasteurized at 63 degrees C for 30 min (low temperature, long time), 72 degrees C for 15 s (high temperature, short time), 76 degrees C for 15 s, and 82 degrees C for 30 min. The pasteurized samples were then stored at 6 and 10 degrees C for 14 days. The samples were analyzed for standard plate count and Bacillus count immediately after pasteurization and after 14 days of storage. Pasteurization of milk at 72 and 76 degrees C significantly (P < 0.05) increased the amount of Bacillus spore activation over that of 63 degrees C. There was no detection of Bacillus in initial samples pasteurized at 82 degrees C for 30 min, but Bacillus was present in samples after storage for 14 days, indicating that injury and recovery time preceded growth. The majority of isolates were characterized as Bacillus mycoides and not Bacillus cereus, suggesting that this organism might be more a cause of sweet curdling of fluid milk than previously reported.     

Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product

Control of psychrotolerant endospore-forming spoilage bacteria, particularly Bacillus and Paenibacillus spp., is economically important to the dairy industry. These microbes form endospores that can survive high-temperature, short-time pasteurization; hence, their presence in raw milk represents a major potential cause of milk spoilage. A previously developed culture-dependent selection strategy and an rpoB sequence-based subtyping method were applied to bacterial isolates obtained from environmental samples collected on a New York State dairy farm. A total of 54 different rpoB allelic types putatively identified as Bacillus (75% of isolates), Paenibacillus (24%), and Sporosarcina spp. (1%) were identified among 93 isolates. Assembly of a broader data set, including 93 dairy farm isolates, 57 raw milk tank truck isolates, 138 dairy plant storage silo isolates, and 336 pasteurized milk isolates, identified a total of 154 rpoB allelic types, representing an extensive diversity of Bacillus and Paenibacillus spp. Our molecular subtype data clearly showed that certain endospore-forming bacterial subtypes are present in the dairy farm environment as well as in the processing plant. The potential for entry of these ubiquitous heat-resistant spoilage organisms into milk production and processing systems, from the dairy farm to the processing plant, represents a considerable challenge that will require a comprehensive farm-to-table approach to fluid milk quality.     

The microbial safety of fish and fish products: Recent advances in understanding its significance,contamination sources,and control strategies

Microorganisms play a crucial and unique role in fish and fish product safety. The presence of human pathogens and the formation of histamine caused by spoilage bacteria make the control of both pathogenic and spoilage microorganisms critical for fish product safety. To provide a comprehensive and updated overview of the involvement of microorganisms in fish and fish product safety, this paper reviewed outbreak and recall surveillance data obtained from government agencies from 1998 to 2018 and identified major safety concerns associated with both domestic and imported fish products. The review also summarized all available literature about the prevalence of major and emerging microbial safety concerns, including Salmonella spp., Listeria monocytogenes, and Aeromonas hydrophila, in different fish and fish products and the survival of these pathogens under different storage conditions. The prevalence of antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs), two emerging food safety concerns, is also reviewed. Pathogenic and spoilage microorganisms as well as ARB and ARGs can be introduced into fish and fish products in both preharvest and postharvest stages. Many novel intervention strategies have been proposed and tested for the control of different microorganisms on fish and fish products. One key question that needs to be considered when developing and implementing novel control measures is how to ensure that the measures are cost and environment friendly as well as sustainable. Over the years, regulations have been established to provide guidance documents for good farming and processing practices. To be more prepared for the globalization of the food chain, harmonization of regulations is still needed.     

Spoilage microbiota associated to the storage of raw meat in different conditions

The spoilage of raw meat is mainly due to undesired microbial development in meat during storage. The type of bacteria and their loads depend on the initial meat contamination and on the specific storage conditions that can influence the development of different spoilage-related microbial populations thus affecting the type and rate of the spoilage process. This review focuses on the composition of raw meat spoilage microbiota and the influence of storage conditions such as temperature, packaging atmosphere and use of different preservatives on the bacterial diversity developing in raw meat. In addition, the most recent tools used for the detection and identification of meat microbiota are also reviewed.