Impact of Next Generation Sequencing Techniques in Food Microbiology

Understanding the Maxam-Gilbert and Sanger sequencing as the first generation, in recent years there has been an explosion of newly-developed sequencing strategies, which are usually referred to as next generation sequencing (NGS) techniques. NGS techniques have high-throughputs and produce thousands or even millions of sequences at the same time. These sequences allow for the accurate identification of microbial taxa, including uncultivable organisms and those present in small numbers. In specific applications, NGS provides a complete inventory of all microbial operons and genes present or being expressed under different study conditions. NGS techniques are revolutionizing the field of microbial ecology and have recently been used to examine several food ecosystems. After a short introduction to the most common NGS systems and platforms, this review addresses how NGS techniques have been employed in the study of food microbiota and food fermentations, and discusses their limits and perspectives. The most important findings are reviewed, including those made in the study of the microbiota of milk, fermented dairy products, and plant-, meat- and fish-derived fermented foods. The knowledge that can be gained on microbial diversity, population structure and population dynamics via the use of these technologies could be vital in improving the monitoring and manipulation of foods and fermented food products. They should also improve their safety.     

“Ethno-microbiology” of ethnic Indian fermented foods and alcoholic beverages

The concept of “ethno-microbiology” is to understand the indigenous knowledge of the Indian people for production of culturally and organoleptically acceptable fermented foods by natural fermentation. About 1000 types of common, uncommon, rare, exotic and artisan fermented foods and beverages are prepared and consumed in different geographical regions by multi-ethnic communities in India. Indian fermented foods are mostly acidic and some are alkaline, along with various types of alcoholic beverages. A colossal diversity of microorganisms comprising bacteria mostly belongs to phylum Firmicutes, filamentous moulds and enzyme- and alcohol-producing yeasts under phyla Ascomycota and Mucoromycota, and few bacteriophages and archaea have been reported from Indian fermented foods. Some microorganisms associated with fermented foods have functionalities and health promoting benefits. “Ethno-microbiology” of ethnic Indian people has exhibited the proper utilisation of substrates either singly or in combination such as fermented cereal-legume mixture (idli, dosa and dhokla) in South and West India, sticky fermented soybean food (kinema and related foods), fermented perishable leafy vegetable (gundruk and related foods), fermented bamboo shoots (soibum and related foods) and fermented fish (ngari and others) in North East India, and fermented meat and sausage-like products in the Indian Himalayas, fermented coconut beverage (toddy) in coastal regions, and various types of naturally fermented milk products (dahi and related products) in different regions of India. This review has also highlighted the “ethno-microbiology” knowledge of the people involving the consortia of essential microorganisms in traditionally prepared amylolytic starters for production of cereal-based alcoholic beverages. The novelty of this review is the interpretation of ethno-microbiological knowledge innovated by ethnic Indian people on the use of beneficial microorganisms for food fermentation to obtain the desired fermented food products for consumption.     

Assessment of peanut allergen Ara h1 in processed foods using a SWCNTs-based nanobiosensor

The goals of this research were to develop a rapid single-walled carbon nanotube (SWCNT)-based biosensor and to employ it to commercial food products for Ara h1 detection. The SWCNT-based biosensor was fabricated with SWCNTs immobilized with antibody (pAb) through hybridization of 1-pyrenebutanoic acid succinimidyl ester (1-PBASE) as a linker. The resistance difference (ΔR) was calculated by measuring linear sweep voltammetry (LSV) using a potentiostat. Resistance values increased as the concentration of Ara h1 increased over the range of 1 to 10⁵ ng/L. The specific binding of anti-Ara h1 pAb to antigen including Ara h1 was confirmed by both indirect ELISA kit and biosensor assay. The biosensor was exposed to extracts prepared from commercial processed food containing peanuts, or no peanuts, and could successfully distinguish the peanut containing foods. In addition, the application of present biosensor approach documented the precise detection of Ara h1 concentrations in commercially available peanut containing foods.     

Surface plasmon resonance based biosensor technique: A review

Optical Surface plasmon resonance (SPR) biosensors represent the most advanced and developed optical label‐free biosensor technology. Optical SPR biosensors are a powerful detection and analysis tool that has vast applications in environmental protection, biotechnology, medical diagnostics, drug screening, food safety and security. This article reviews the recent development of SPR biosensor techniques, including bulk SPR and localized SPR (LSPR) biosensors, for detecting interactions between an analyte of interest in solution and a biomolecular recognition. The concepts of bulk and localized SPs and the working principles of both sensing techniques are introduced. Major sensing advances on biorecognition elements, measurement formats, and sensing platforms are presented. Finally, the discussions on both biosensor techniques as well as comparison of both SPR sensing techniques are made. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The Importance of Amine-degrading Enzymes on the Biogenic Amine Degradation in Fermented Foods: A review

Biogenic amines (BAs) are a class of harmful compounds often be found in high protein foods, especially naturally fermented foods. BAs derive from free amino acid decarboxylation through microbial activities and can cause toxic effects (headache, heart palpitations, vomiting) on humans, depending on individual sensitivity. Indigenous amine-degrading strains or strains producing amine-degrading enzymes (ADEs) have drawn great attention since they play an important role in affecting BA accumulation, and enzymes/genes involved in the biosynthetic mechanisms. They also help maintain the sensory quality of the final products. Besides, due to ADEs’ harmless catalytic products, they can be further utilized in fermented foods and beverages to reduce BAs. This review describes in detail the mechanisms of BAs formation, as well as the diversity of ADEs able to degrade BAs in a model or real food systems. A deeper knowledge of this issue is crucial because ADEs’ activities are often associated with strains rather than species or genera. Moreover, this information can help to improve the selection and characterization of strains for further applications as starters or bioprotective cultures, to obtain high-quality foods with reduced BAs contents.     

Evaluation of free D-glutamate in processed foods

Monosodium glutamate (MSG) is added to many processed foods at significant levels for flavor enhancement. It is also naturally occurring at high levels in some foods. The enantiomeric composition of free glutamate in foods was examined and all foods analyzed were found to contain D -glutamate. The relative percent of D -glutamate in the food products studied depended on the origin of the glutamate. Foods to which MSG was added by the manufacturer had a high total level of MSG but a lower relative percentage of the D -enantiomer (usually less than 0.8%). In comparison, fermented foods tend to have high relative levels of D -glutamate but a lower total amount of the amino acid. The relative percent of D -glutamate in nonfermented foods containing no added MSG was also found to be low compared to fermented products. In some cases the percent D -glutamate could be related to the relative amounts of other food ingredients such as cheese. © 1994 Wiley-Liss, Inc.     

食品中农药残留的新型生物检测技术研究进展

食品安全是关系国计民生的重大战略问题。农药残留（简称农残）是造成食品质量与安全问题的重要因素。食品中农药残留不仅发生率高，而且超标严重，涉及范围广，长期接触或食用含有农药残留的食品会危害人体健康。传统的农残分析技术已经很难满足多样化食品安全检测的实际需求，现代新型生物技术因特异性强、灵敏度高、简单快速等优势，在食品的农残检测中得到广泛应用和持续发展。分析了食品中农药残留及检测技术现状，综述了酶联免疫吸附测定法、荧光免疫法和生物条形码免疫法等分析技术，及电化学、光学、压电传感器等生物传感技术在食品农残检测中的应用进展，讨论了各种技术的优势和局限，以期为相关研究提供参考，从而进一步提高我国食品农残的检测效率，保障食品安全，促进我国食品行业的可持续发展。     

Extent of genetic lesions of the arginine and pyrimidine biosynthetic pathways in Lactobacillus plantarum,L. paraplantarum,L. pentosus,and L. casei: prevalence of CO(2)-dependent auxotrophs and characterization of deficient arg genes in L. plantarum

Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO(2). We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum, 32 strains of L. pentosus, 15 strains of L. paraplantarum, and 10 strains of L. casei. The distribution of prototroph and auxotroph phenotypes varied between species. All L. casei strains, no L. paraplantarum strains, two L. pentosus strains, and seven L. plantarum strains required arginine for growth. Arginine auxotrophs were more frequently found in L. plantarum isolated from milk products than in L. plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In L. plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one L. pentosus strain, a high CO(2) requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated.     

Traditional healthful fermented products of Japan

A variety of fermentation products, such as foods containing probiotic bacteria, black rice vinegar (kurosu), soy sauce (shoyu), soybean-barley paste (miso), natto and tempeh, are sold in food stores in Japan. These fermented food products are produced by traditional methods that exploit mixed cultures of various non-toxic microorganisms. These microorganisms include lactic acid bacteria, acetic acid bacteria, sake yeast, koji molds and natto bacteria. Many traditional fermented foods have been studied and their effects on metabolism and/or immune system have been demonstrated in animal and/or human cells. This review summarizes the scientific basis for the effects of these traditional food products, which are currently produced commercially in Japan.     

Koji – where East meets West in fermentation

Almost all biotechnological processes originate from traditional food fermentations, i.e. the many indigenous processes that can be found already in the written history of thousands of years ago. We still consume many of these fermented foods and beverages on a daily basis today. The evolution of these traditional processes, in particular since the 19th century, stimulated and influenced the development of modern biotechnological processes. In return, the development of modern biotechnology and related advanced techniques will no doubt improve the process, the product quality and the safety of our favourite fermented foods and beverages. In this article, we describe the relationship between these traditional food fermentations and modern biotechnology. Using Koji and its derived product soy sauce as examples, we address the mutual influences that will provide us with a better future concerning the quality, safety and nutritional effect of many fermented food products.     

合成生物学细胞传感技术在食品安全快速检测中的应用

合成生物学细胞传感技术为快速、现场检测食品污染物提供了一种新型替代方法。由于细胞内环境相对稳定,合成生物学细胞传感器有较强的抗干扰能力;由于细胞能够通过自我复制而实现增殖,细胞传感器在生产上具有简单、廉价、快速的特点,因此在食品安全快速检测中具有良好的应用前景。本文综述了合成生物学细胞传感器核心元件的组成、构建方法和类型,介绍了多功能细胞传感器的合成生物学基因回路,列举了细胞传感器在食品安全快速检测中的商业化应用前景,并阐述了细胞传感器在食品安全快速检测中的挑战和发展趋势。     

Influence of ethanol,malate and arginine on histamine production of <Emphasis Type="Italic">Lactobacillus hilgardii</Emphasis> isolated from an Italian red wine

Wine, like other fermented foods, may contain biogenic amines produced by lactic acid bacteria via amino acids decarboxylation. The most relevant amines from the toxicological standpoint are histamine and tyramine. The complexity of fermented substrates makes it difficult to suggest a priori how variables can modulate amine production. Lactobacillus hilgardii ISE 5211 was isolated from an Italian red wine. Besides producing lactate from malate, this strain is also able to convert arginine to ornithine and histidine to histamine. In the present investigation we studied the influence of malate, arginine and ethanol on histamine accumulation by L. hilgardii ISE 5211. Ethanol concentrations above 13% inhibit both histamine accumulation and bacterial growth; concentrations below 9% affect neither growth nor histamine production. However, an ethanol concentration of 11% allows a low but continuous accumulation of histamine to occur. Arginine also delays histamine accumulation, while malate appears to have no effect on histidine-histamine conversion.     

Importance of lactic acid bacteria in Asian fermented foods

Lactic acid bacteria play important roles in various fermented foods in Asia. Besides being the main component in kimchi and other fermented foods, they are used to preserve edible food materials through fermentation of other raw-materials such as rice wine/beer, rice cakes, and fish by producing organic acids to control putrefactive microorganisms and pathogens. These bacteria also provide a selective environment favoring fermentative microorganisms and produce desirable flavors in various fermented foods. This paper discusses the role of lactic acid bacteria in various non-dairy fermented food products in Asia and their nutritional and physiological functions in the Asian diet.     

Enterococci from foods

Enterococci have recently emerged as nosocomial pathogens. Their ubiquitous nature determines their frequent finding in foods as contaminants. In addition, the notable resistance of enterococci to adverse environmental conditions explains their ability to colonise different ecological niches and their spreading within the food chain through contaminated animals and foods. Enterococci can also contaminate finished products, such as fermented foods and, for this reason, their presence in many foods (such as cheeses and fermented sausages) can only be limited but not completely eliminated using traditional processing technologies. Enterococci are low grade pathogens but their intrinsic resistance to many antibiotics and their acquisition of resistance to the few antibiotics available for treatment in clinical therapy, such as the glycopeptides, have led to difficulties and a search for new drugs and therapeutic options. Enterococci can cause food intoxication through production of biogenic amines and can be a reservoir for worrisome opportunistic infections and for virulence traits. Clearly, there is no consensus on the acceptance of their presence in foodstuffs and their role as primary pathogens is still a question mark. In this review, the following topics will be covered: (i) emergence of the enterococci as human pathogens due to the presence of virulence factors such as the production of adhesins and aggregation substances, or the production of biogenic amines in fermented foods; (ii) their presence in foods; (iii) their involvement in food-borne illnesses; (iv) the presence, selection and spreading of antibiotic-resistant enterococci as opportunistic pathogens in foods, with particular emphasis on vancomycin-resistant enterococci.     

A review of studies and measures to improve the mycotoxicological safety of traditional Japanese mold-fermented foods

Miso (fermented soybean paste), shoyu (soy sauce) and sake (rice wine) are traditional moldfermented foods in Japan and have been consumed throughout much of its history. These have long been considered safe foods. In this contribution we review and summarize long-term studies to investigate potential problems with mycotoxin contamination of these products. The fungal cultures used for fermentation of these products are called “koji-molds” and mainly consist of strains ofAspergillus oryzae. A. oryzae belongs to theA. flavus group taxonomically, which is generally known to be a main producer of aflatoxins. Therefore, we studied the productivity of aflatoxins by various koji-molds, as well as the possibility of aflatoxin contamination of rice (which is used in the production of fermented foods), miso, shoyu and sake. Rice was found to be free from aflatoxins. Furthermore, none of the tested koji-molds produced any detectable levels of aflatoxins, consequently no aflatoxins were found in miso, shoyu, or sake. However, some koji-molds are known to produce cyclopiazonic acid (CPA) and kojic acid (KA). We studied the production of CPA and KA by various commercial koji-molds and identified some strains that produce relatively high amounts of CPA or KA. Consequently, we advised food industry not to use these strains. Although mycotoxin contamination of these products is therefore presently very low, further attempts should be made to completely eliminate CPA and KA from fermented foods.     

Antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food

Aim: To develop antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food products. Methods and Results: Aptamer, a single‐stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer‐A8, specific for internalin A, an invasin protein of L. monocytogenes, was used in the fibre‐optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti‐Listeria antibody, P66, was immobilized on streptavidin‐coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647‐conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 10³ CFU ml^?1. This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10² CFU 25 g^?1) ready‐to‐eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. Conclusion: Based on the data presented in this study, the antibody–aptamer functionalized fibre‐optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. Significance and Impact of the Study: The study demonstrates feasibility and novel application of aptamer on fibre‐optic biosensor platform for the sensitive detection of L. monocytogenes from food products.     

A rapid chemical spot test for the detection of lactic acid as an indicator of microbial spoilage in preserved foods

A ckland , M.R. & R eeder , J.E. 1984. A rapid chemical spot test for the detection of lactic acid as an indicator of microbial spoilage in preserved foods. Journal of Applied Bacteriology 56 , 415–419.
A rapid method for the detection of lactic acid in preserved foods has been developed, based upon a chemical spot test which does not require solvent extraction or derivatisation of the lactic acid in the sample. The test can be completed within 5 min and was shown to confirm microbial spoilage detected by cultural techniques in a range of preserved foods. In addition the test was able to indicate microbial spoilage in samples where cultural techniques failed because the spoilage organisms were dead. The test may not be appropriate for products which have a naturally high lactic acid content.     

A high density microelectrode array biosensor for detection of E. coli O157:H7

A high density microelectrode array biosensor was developed for the detection of Escherichia coli O157:H7. The biosensor was fabricated from (100) silicon with a 2 microm layer of thermal oxide as an insulating layer, an active area of 9.6 mm2 and consists of an interdigitated gold electrode array. The sensor surface was functionalised for bacterial detection using heterobifunctional crosslinkers and immobilised polyclonal antibodies to create a biological sensing surface. Bacteria suspended in solution became attached to the immobilised antibodies when the biosensor was tested in liquid samples. The change in impedance caused by the bacteria was measured over a frequency range of 100 Hz-10 M Hz. The biosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The biosensor was able to discriminate between cellular concentrations of 10(4)-10(7)CFU/mL and has applications in detecting pathogens in food samples.     

Extent of Genetic Lesions of the Arginine and Pyrimidine Biosynthetic Pathways in Lactobacillus plantarum, L. paraplantarum, L. pentosus, and L. casei: Prevalence of CO2-Dependent Auxotrophs and Characterization of Deficient arg Genes in L. plantarum

Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO₂. We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum, 32 strains of L. pentosus, 15 strains of L. paraplantarum, and 10 strains of L. casei. The distribution of prototroph and auxotroph phenotypes varied between species. All L. casei strains, no L. paraplantarum strains, two L. pentosus strains, and seven L. plantarum strains required arginine for growth. Arginine auxotrophs were more frequently found in L. plantarum isolated from milk products than in L. plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In L. plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one L. pentosus strain, a high CO₂ requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated.