Safety assessment of dairy microorganisms: the Enterococcus genus

Unlike most of lactic acid bacteria, the Enterococcus genus is not considered "generally recognized as safe" (GRAS). Safety assessment for enterococci remains controversial. While enterococci are considered "positive" or useful in cheese technology, isolates of this genus have emerged as opportunistic pathogens for humans. Thus these bacteria have the paradoxical position of being useful in dairy fermentations, but also potentially dangerous. The aim of this review is to summarize both the positive and negative traits of enterococci that illustrate the controversial nature of this bacterial genus. According to food safety assessment guidelines, we propose a case-by-case evaluation of each potential technological strain and suggest several lines of research before using enterococci in fermented food products.     

Safety assessment of dairy microorganisms: the Lactococcus genus

The Lactococcus genus includes 5 species. Lactococcus lactis subsp. lactis is the most common in dairy product but L. garviae has been also isolated. Their biotope is animal skin and plants. Owing to its biochemical characteristics, strains of L. lactis are widely used in dairy fermented products processing. Cases of human infections due to lactococci are very seldom reported even if Lactococcus garviae can be involved in fish diseases. Then L. lactis can be considered as safe and it is most commonly considered as Generally Recognized as Safe.     

Safety assessment of dairy microorganisms: the Lactobacillus genus

Lactobacilli are Gram positive rods belonging to the Lactic Acid Bacteria (LAB) group. Their phenotypic traits, such as each species' obligate/facultative, homo/heterofermentation abilities play a crucial role in souring raw milk and in the production of fermented dairy products such as cheese, yoghurt and fermented milk (including probiotics). An up to date safety analysis of these lactobacilli is needed to ensure consumer safety. Lactobacillus genus is a heterogeneous microbial group containing some 135 species and 27 subspecies, whose classification is constantly being reshuffled. With the recent use of advanced molecular methods it has been suggested that the extreme diversity of the Lactobacillus genomes would justify recognition of new subgeneric divisions. A combination of genotypic and phenotypic tests, for example DNA-based techniques and conventional carbohydrate tests, is required to determine species. Pulsed-Field gel Electrophoresis (PFGE) has been successfully applied to strains of dairy origin and is the most discriminatory and reproducible method for differentiating Lactobacillus strains. The bibliographical data support the hypothesis that the ingestion of Lactobacillus is not at all hazardous since lactobacillemia induced by food, particularly fermented dairy products, is extremely rare and only occurs in predisposed patients. Some metabolic features such as the possible production of biogenic amines in fermented products could generate undesirable adverse effects. A minority of starter and adjunct cultures and probiotic Lactobacillus strains may exceptionally show transferable antibiotic resistance. However, this may be underestimated as transferability studies are not systematic. We consider that transferable antibiotic resistance is the only relevant cause for caution and justifies performing antibiotic-susceptibility assays as these strains have the potential to serve as hosts of antibiotic-resistance genes, with the risk of transferring these genes to other bacteria. However, as a general rule, lactobacilli have a high natural resistance to many antibiotics, especially vancomycin, that is not transferable. Safety assessment requirements for Lactobacillus strains of technological interest should be limited to an antibiotic profile and a study to determine whether any antibiotic resistance(s) of medical interest detected is (or are) transferable. This agrees with the recent EFSA proposal suggesting attribution of a QPS status for 32 selected species of lactobacilli.     

Safety assessment of dairy microorganisms: the hemiascomycetous yeasts

Hemiascomycetous yeasts constitute a class of unicellular fungi often associated with the food and drink processing industries. A number of species including Kluveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica, play a key role in the cheese-making process by providing aroma, affecting texture and/or permitting the growth of other microorgansisms. The large majority of yeast infections are due to a few opportunistic species presently classified within the genus Candida, and occur in immunocompromised patients. Recent advances in taxonomy have provided evidence for the incorrect classification of a number of yeasts and suggest that their association with the genus Candida should be reconsidered. Indeed, none of the most common pathogenic Candida species are found in cheese. Improved techniques, combined with more advanced analytical methods have brought to light several emerging pathogens, some of which are involved in cheese-making, for example D. hansenii and Y. lipolytica. Other emerging pathogens may also be found as rare occurrences in cheese. Problems in designation of these isolates are due in part to the still limited range of specific methods of identification and are exacerbated by lack of consensus concerning yeast taxonomy. These organisms cause rare infections in immunocompromised and hospitalized patients, which are generally mild and either self-limiting or easily treated. From studies with Saccharomyces cerevisiae, it seems that it is more the exposure to high doses of yeast than the identity of the species or strain that is associated with infection. As such yeasts in cheese cannot be considered to constitute a risk for healthy individuals.     

Safety assessment of dairy microorganisms: Streptococcus thermophilus

Streptococcus thermophilus is a major dairy starter used in yogurt and cheese production. In Streptococcus genus, S. thermophilus is the only one food species among commensal and opportunistic pathogen species. Comparative genomics suggest that this species recently emerged and evolved by combination of loss-of-function and horizontal gene transfer events. These gene transfer events detected in S. thermophilus have originated from other dairy species and might contribute to its adaptation to the milk environment.     

Safety assessment of dairy microorganisms: Geotrichum candidum

Geotrichum candidum is a ubiquitous filamentous yeast-like fungus commonly isolated from soil, air, water, milk, silage, plant tissues, digestive tract in humans and other mammals. This species is widely used as adjunct culture in the maturation of cheese. The genus Geotrichum is composed of 18 species. A recent taxonomic revision concluded that the old Galactomyces geotrichum/G. candidum complex contained four separate species of which Galactomyces candidus sp. nov./G. candidum. M13 primer can be used for identifying species of the Geotrichum genus. Used in combination, RAPD-PCR and RAM-PCR permit strains to be differentiated. The species can be unambiguous differentiated from the two species most frequently described in human pathology: Geotrichum clavatum (reclassified Saprochaete clavata) and Geotrichum capitatum (reclassified Magnusiomyces capitatus/Saprochaete capitata). Sources of exposure are food ingestion--cheese consumption playing a major role--inhalation and contact. A bibliographic survey was conducted to assess corresponding hazards and risks. G. candidum infections (mainly pulmonary or bronchopulmonary, but also cutaneous, oral, disseminates) are very rare: fewer than 100 cases reported between 1842 and 2006. Moreover, cases were not all confirmed by repeated isolations and demonstration of the fungus' presence in tissues, a prerequisite to establish a true diagnosis of geotrichosis. Immunocompromised population was recently shown as a target for opportunistic infection. The most effective treatments include either azole drogs as ketonazole, iconazole and clotrimazole, or polyene antibiotics as amphotericin B, nystatin and pimaricin, or voriconazole-amphotericin B association. Less than 1 case/year of disease was possibly caused by G. candidum and it never included dairy products or foodborne infection. The risk of developing an infection due to G. candidum in connection with its technological use and consumption of dairy products is virtually nil. For these reasons, G. candidum should be proposed for QPS status.     

Safety assessment of dairy microorganisms: bacterial taxonomy

Safety assessment requires uniform and reproducible nomenclature schemes for all the micro-organisms deliberately added in fermented food products. The QPS approach described by EFSA is based on the identity of an isolate at the highest taxonomic unit that is appropriate for the purpose for which the evaluation is intended. This depends upon the body of knowledge available for the micro-organism to be assessed and upon the nature of the micro-organism being assessed. Species identification is the fundamental unit of biological classification and is critical for describing, understanding and comparing biological diversities at different levels among ecological niches. Accurate taxonomic identification of a micro-organism is essential for assessing safety assessment, and this section describes current and advanced knowledge on bacterial taxonomy.     

Safety assessment of dairy microorganisms: coagulase-negative staphylococci

The genus Staphylococcus is made up of 36 validated species which contain strains that are pathogenic, saprophytic, or used as starter cultures for the food industry. Staphylococci species used in cheese-making are novobiocin-resistant, coagulase-negative and are not usually identified at species level by routine laboratories. A bibliographic survey was conducted to assess safety status of CNS used in fermented dairy foods. Commercial kits based on phenotypic discrimination can't reliably identify these species because of the variable expression of some phenotypic traits. Several molecular targets, including 16S rRNA, hsp60, tuf, SodA and rpoB genes can be used for identifying species of the Staphylococcus genus. No coagulase-negative staphylococci isolated from milk or dairy products has ever been involved in a case of food poisoning or human pathology following ingestion of dairy products. Nevertheless, a few case of nosocomial infection caused by some species (S. caprae, S. capitis, S. sciuri) have been reported in patients with depressed immune systems or who have undergone long severe hospital treatments, or in the presence of an indwelling catheter or foreign materials such as cardiac prostheses. They may therefore be regarded as exceptional opportunistic pathogens in certain clinical situations.     

Safety assessment of dairy microorganisms: Propionibacterium and Bifidobacterium

Streptococcus suis present in raw pork meats sold in local retail markets was enumerated by Most Probable Number (MPN)-PCR method. This method combined the conventional MPN technique with a specifically designed PCR assay based on the amplification of a 294-bp S. suis species-specific 16S rRNA gene sequence. A total of 78 raw pork lean meat samples purchased at two different supermarkets (Site A and B) and a wet market (Site C) were tested. Results indicated that S. suis could be detected from the enriched MPN tubes of all, except one, sample homogenates. The concentration of S. suis ranged from <3 to 4600 MPN/g of pork meat, with a total bacterial count (TBC) varying from 3.6 log to 7.4 log CFU/g. Statistical analyses indicated that pork meats purchased from the supermarket at Site B in summer contained significantly higher concentration of S. suis organisms than those from other retailers in any season. A significant correlation existed between log S. suis concentration and log TBC of the samples. This study revealed that raw pork meats available in local supermarkets or wet markets could contain S. suis at concentrations that were usually difficult to detect with traditional culture method. Field application of this method may contribute to a measurable evaluation, and thus the effective control, of human S. suis infection due to raw pork or pig carcass handling.     

Safety assessment of dairy microorganisms: aerobic coryneform bacteria isolated from the surface of smear-ripened cheeses

The group of "coryneform bacteria" belongs to the class of Actinobacteria including a diverse and heterogeneous collection of bacteria of various genera. Most of them are known as environmental residents and/or commensal flora of humans and they are isolated frequently in clinical studies. Actinobacteria include also several aerobic species, present at the surface of smear-ripened cheeses for decades and used as ripening culture in the dairy industry. Their clinical significance is controversial because an easy combination of phenotypic and molecular methods to characterize Actinobacteria at the species level is still lacking. A bibliographical survey was conducted to assess the safety status of Actinobacteria species used as starter culture in fermented dairy foods, according to their technological interest. Aerobic coryneform bacteria isolated from smear-ripened cheeses are most commonly recovered from soil, the environment or food. To date, no clinical infection or food toxi-infection related to smear cheese coryneform bacteria ingestion has been reported. From a taxonomic viewpoint, dairy species are distant from the reference species associated with known pathologies. From a physiological viewpoint, cheese smear coryneform bacteria appear to be related to particular ecological niches: they are all oxidative species, and most are psychrotrophic and unable to grow at 37 degrees C whereas medically relevant coryneform bacteria are facultative anaerobes and grow at 35-37 degrees C. Consequently, technological strains must be selected according to taxonomic criteria (nonpathogenic species) and ecological criteria.     

乳制品中明串珠菌的分离鉴定及其生物学特性研究

对内蒙古自治区呼伦贝尔市牧区98个乳样中的明串珠菌进行了分离及生物学特性研究。共分离到明串珠菌3株，分别属于乳明串珠菌(EW21)，肠膜明串珠菌葡聚糖亚种(EW24—2)和肠膜明串珠菌肠膜亚种(CB8—1)。它们均可在pH=4．8的环境中生长，且菌株EW24—2和CB8—1可利用蔗糖生成葡聚糖。     

Optical instrument for the rapid detection of microorganisms in dairy products

MicroFoss, an optical instrument capable of detecting metabolic changes due to microbial activity, was tested for the detection of various groups of microorganisms in dairy products. Optical changes in the growth medium are monitored in a semi-fluid zone that separates the liquid medium containing the sample. Raw and pasteurised milks were evaluated for total viable counts (TVC), coliform and Enterobacteriaceae. Yoghurt was evaluated for coliform, Enterobacteriaceae and yeast.The MicroFoss TVC method for both pasteurised and raw milk utilised pre-filled vials containing 8 mL of Nutrient Broth with Bromcresol purple with 2 mL of milk sample. In a comparison of the instrument TVC methodology to the standard plate count methodology, correlation coefficients R=−0.93 and R=−0.92 were obtained for raw and pasteurised milk, respectively. An advantage of using the MicroFoss method was that results are obtained in 2–11 h rather than 24–48 h, with samples with bacterial counts above the acceptable levels detecting in most cases within an 8 h shift.The MicroFoss methodology for the estimation of numbers of coliform and Enterobacteriaceae utilises pre-filled vials containing 5 mL of broth. In a comparison between the MicroFoss coliform methodology and the corresponding plate count methodology utilising Violet Red Bile Agar (VRBA), a correlation coefficient of R=−0.95 was obtained. An identical correlation coefficient of R=−0.95 was obtained for the comparison of the MicroFoss Enterobacteriaceae methodology and the corresponding plate count methodology utilising VRBA+1% glucose. In 60 pasteurised milk samples tested, only 2 samples were positive by the MicroFoss method, one of which had 1 cfu mL⁻¹ by the plate method while the other had <1 cfu mL⁻¹. Coliform or Enterobacteriaceae were isolated from the detecting vials demonstrating the higher sensitivity of MicroFoss.The MicroFoss method for the detection of the presence of yeast in yoghurt required a 1:10 dilution of the product followed by a pH adjustment. Presence of yeast in yoghurt and soft cheeses was always detected without any false positive results. Results were available in less than 40 h.     

Authenticity assessment of dairy products

The authenticity of dairy products has become a focal point, attracting the attention of scientists, producers, consumers, and policymakers. Among many others, some of the practices not allowed in milk and milk products are the substitution of part of the fat or proteins, admixtures of milk of different species, additions of low-cost dairy products (mainly whey derivatives), or mislabeling of products protected by denomination of origin. A range of analytical methods to detect frauds have been developed, modified, and continually reassessed to be one step ahead of manufacturers who pursue these illegal activities. Traditional procedures to assess the authenticity of dairy products include chromatographic, electrophoretic, and immunoenzymatic methods. New approaches such as capillary electrophoresis, polymerase chain reaction, and isotope ratio mass spectrometry have also emerged alongside the latest developments in the former procedures. This work intends to provide an updated and extensive overview since 1991 on the principal applications of all these techniques together with their advantages and disadvantages for detecting the authenticity of dairy products. The scope and limits of different tools are also discussed.     

Ingredient selection criteria for probiotic microorganisms in functional dairy foods

The selection of potentially probiotic microorganisms for use in functional dairy foods is reviewed. Two groups of selection criteria are distinguished encompassing technological and medico-scientific properties. The former ensure provision of viable cultures of the desired cell density that are robust to the intended product application, while the latter ensure that the culture provided possesses activity in terms of some specified nutritional/health benefit at a specified site in the gastrointestinal tract. It is concluded that an understanding of the scientific principles and mechanisms involved in probiotic activities in the human host is continuing to emerge. Further advances will require a concerted collaborative effort between medical, pharmacological, nutritional, microbiological and technological experts. These advances will improve the basis upon which potentially probiotic microorganisms are selected and delivered by functional foods in the diet for specified human nutritional/health benefits .     

Genetic diversity of thermoduric spoilage microorganisms of milk from Brazilian dairy farms

When correctly pasteurized, packaged, and stored, milk with low total bacterial counts (TBC) has a longer shelf life. Therefore, microorganisms that resist heat treatments are especially important in the deterioration of pasteurized milk and in its shelf life. The aim of this work was to quantify the thermoduric microorganisms after the pasteurization of refrigerated raw milk samples with low TBC and to identify the diversity of these isolates with proteolytic or lipolytic potential by RFLP analysis. Twenty samples of raw milk were collected in bulk milk tanks shortly after milking in different Brazilian dairy farms and pasteurized. The mean thermoduric count was 3.2 (±4.7) × 10² cfu/mL (2.1% of the TBC). Of the 310 colonies obtained, 44.2% showed milk spoilage potential, 32.6% were proteolytic and lipolytic simultaneously, 31% were exclusively proteolytic, and 48 (36.4%) were only lipolytic. Regarding the diversity, 8 genera were observed (Bacillus, Brachybacterium, Enterococcus, Streptococcus, Micrococcus, Kocuria, Paenibacillus, and Macrococcus); there was a predominance of endospore-forming bacteria (50%), and Bacillus licheniformis was the most common (34.1%) species. Considering the RFLP types, it was observed that the possible clonal populations make up the microbiota of different milk samples, but the same milk samples contain microorganisms of a single species with different RFLP types. Thus, even in milk with a high microbiological quality, it is necessary to control the potential milk-deteriorating thermoduric microorganisms to avoid the risk of compromising the shelf life and technological potential of pasteurized milk.     

基于ATP再生体系快速检测乳品中微生物

随着新型食品安全检测技术的快速发展,食源性致病菌快速检测技术改善了传统检测方法周期长、灵敏度低等缺陷,对于保障民众生命健康和经济社会发展起到重要作用,而标准化则是推动快速检测技术应用推广的关键和前提。本文系统介绍了生理生化检测技术、免疫学检测技术、分子检测技术等目前使用较多的食源性致病菌快速检测方法,总结了各类不同方法的技术原理、研究进展及优缺点,并从标准化角度进一步介绍了国内外快速检测技术的标准现状以及应用实践情况。新型快速检测技术具备灵敏、快速、特异性强的优势,但也存在一定的缺陷,如免疫检测技术抗体前处理较为麻烦,生理生化检测技术有污染菌混淆问题,分子检测技术有一定假阳性等。此外,大部分检测方法仍属于非法定方法,缺乏统一的判定标准,国内外相关标准体系仍不完善,制约了快速检测技术的标准化推广应用。基于此,本文从标准化角度提出了食源性致病菌快速检测技术未来发展的建议,以期为后续食品安全相关技术的研究与标准化建设提供参考。

     

Inactivation of microorganisms using pulsed electric fields: the influence of process parameters on Escherichia coli,Listeria innocua,Leuconostoc mesenteroides and Saccharomyces cerevisiae

This study examines the killing effect of pulsed electric fields (PEF) on four organisms suspended in a model medium. Escherichia coli, Listeria innocua, Leuconostoc mesenteroides and Saccharomyces cerevisiae differ in size, shape and cell wall construction. The electric field strength, pulse duration and number of pulses were varied in the ranges of 25–35 kV/cm, 2–4 μs and 20–40 pulses, respectively. The results showed that S. cerevisiae was the most sensitive organism with a 6-log reduction, followed by E. coli with a 5.4-log reduction, when they were exposed to 30 kV/cm, and 20 pulses with 4 μs duration. The most resistant organisms were L. innocua and L. mesenteroides with only a 3-log reduction, however, by increasing the parameters to 35 kV/cm and 40 pulses with 4 μs pulse duration; marked viability reductions of 8 and 7 log, respectively, were observed. Heat, which is generated during the process, has limited killing effect on the cells, hence the observed reduction can be ascribed to the PEF treatment. Although transmission electron microscopy of PEF treated cells did not confirm membrane damage, observations suggest that PEF treatments have profound direct or indirect effects on the intracellular organisation of microorganisms.     

Symposium review: Scientific assessment of affective states in dairy cattle

Affective states, which refer to feelings or emotions, are a key component of animal welfare, but these are also difficult to assess. Drawing upon a body of theoretical and applied work, we critically review the scientific literature on the assessment of affective states in animals, drawing examples where possible from research on dairy cattle, and highlighting the strengths and weaknesses of scientific methods used to assess affective states in animals. We adopt the “valence/arousal” framework, describing affect as a 2-dimensional space (with valence referring to whether an experience is positive or negative, and arousal referring to the intensity of the experience). We conclude that spontaneous physiological and behavioral responses typically reflect arousal, whereas learned responses can be valuable when investigating valence. We also conclude that the assessment of affective states can be furthered using mood assessments and that the use of drug treatments with known emotional effects in humans can be helpful in the assessment of specific affective states in animals.     

乳制品中有害微生物检测新技术研究进展

乳及乳制品因营养丰富而成为沙门氏菌(Salmonella)、金黄色葡萄球菌(Staphylococcus aureus)、单核细胞增生李斯特菌(Listeria monocytogenes)、克罗诺杆菌(Cronobacter)和蜡样芽孢杆菌(Bacillus cereus)等多种有害微生物的天然培养基,近年来,乳制品安全事件屡见不鲜,已成为食品安全研究热点。传统检测技术因检测周期长且准确率低,无法满足乳制品安全检测高通量、时间短的需求。随着分子生物学和免疫学的发展,重组酶介导等温核酸扩增(recombinase aided amplification,RAA)技术、环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术、纳米探针技术和三磷酸腺苷(adenosine triphosphate,ATP)发光技术等因灵敏度高、检测时间短被逐渐应用于乳品安全检测。鉴于此,本文结合国内外相关研究,从乳制品安全角度出发,重点介绍了上述技术在乳制品安全检测中的研究现状,并进行了分析讨论,以期对未来乳制品有害微生物快速检测技术的发展提供思路。     

乳品安全风险评估的研究进展

对乳品污染物进行风险评估是保障乳品安全的重要手段之一。简单总结了风险评估的基本概念和研究方法,分别从化学性危害、生物性危害、物理性危害和乳品加工技术(如转基因技术和纳米技术)等方面,综述了近十年来国内外乳品安全风险评估的研究进展,小结了乳品安全风险评估研究中的难点和薄弱环节,并展望了未来的发展方向和潜在的研究热点。