In Japan, the institutionalisation of HACCP (Hazard Analysis and Critical Control Point) came into full effect in June 2021. Globally, HACCP-based approaches have widely adopted preconditioning programmes (PRPs) as preventive control systems. At present, microbiological testing is often integrated into the preventive control system. In this context, microbiological testing is expected to play an important role in monitoring, validating and verifying the programme. Appropriate microbiological testing is also essential to verify the effectiveness of the HACCP system. In addition, microbiological testing is required to demonstrate compliance with food microbiological standards (e.g. standards, guidelines, specifications). In the EU, microbiological testing is also used to investigate suspected deviations in process control, particularly for hygiene indicator bacteria. In Japan, the introduction of HACCP is expected to lead to a shift towards microbiological testing of process controls in food manufacturing plants. Furthermore, in the food industry, microbiological challenge testing, storage testing and growth modelling are essential tasks in quality design to estimate and test what can happen to food during processing, distribution and subsequent handling. Microbiological testing also plays an essential role as a tool for this work. Thus, with the further development of food safety management, microbiological testing is expected to continue to play an impor tant role in assessing whether food safety objectives are being met. On the other hand, traditional microbiological detection and identification methods for foodborne pathogens are well known to be time-consuming and laborious, and are increasingly recognised as insufficient to meet the demands of rapid food testing. Recently, rapid detection, identification and monitoring methods for various types of foodborne pathogens have been developed, including nucleic acid-based methods, immunological methods, and MALDI-TOF MS-based methods, in addition to conventional culture methods. In view of the growing interest in rapid methods for microbiological testing of foods, the aim of this special issue is to describe the current developments, research needs and future trends in this field.　There is a wide range of rapid methods for food microbiological testing, and it is not possible to cover them all in this special issue. In this article, five experts in the field of microbiological food testing will introduce the rapid techniques that are expected to develop in the future. We also discuss the concept of introducing methods necessary for the introduction of HACCP and the microbiological testing methods suitable for the transition to process control.

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The importance of the validated microbiological test method and how to select the test method based on the fitness for purpose will be explained. Based on this, I will show how to select a microbial test method after the transition to process control such as the HACCP (Hazard Analysis and Critical Control Point), and how to utilize microbial test for risk management.

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The HACCP (Hazard Analysis and Critical Control Point) system was fully enforced for all food business operators in July 2021 as a response to the globalization of food distribution. Additionally, considerations on food microbiological testing have significantly changed. During the self-inspection process of food companies, there is an increasing demand for a shift from the conventional method of collecting one sample of the final product and then using the official method to a quick and direct method that is useful for managing the manufacturing process. With the development of science, a method has been developed for differentiating the colonies with a color- developing enzyme-substrate medium targeting an enzyme produced by bacteria. A ready-to-use simple medium, that can overcome the drawbacks of agar media that must be prepared in-house, has been developed and is extensively used during the inspection of many food companies. The simple media require validation by an international validation agency. Simple media are effectively used in cases such as the processing of multiple samples under the same conditions, responding to urgent test requests, and reducing the time required for tests to prioritize the original quality control work.

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In recent years, the HACCP (Hazard Analysis and Critical Control Point) system was introduced to food processing industries in Japan. Consequently, the rapid and sensitive detection method of foodborne pathogens is now focusing on ensuring the safety of food in the processing line. PCR (polymerase chain reaction) is expected to be a rapid, high specificity and highthroughput detection method for foodborne pathogens versus the conventional culture method. Moreover, realtime PCR technology has the potential to quantify the concentration of target pathogens in food material. Predictive microbiological analysis for pathogenic bacteria is a new approach for ensuring the safety of food. Accordingly, some analytical models are being developed to predict the growth of target pathogens in specific food material. The analysis for injured bacteria as a new risk potential in causing microbial food poisoning is also one of the focuses in food safety research. Therefore, a high-resolution analysis method to determine the level of injured bacteria is required to estimate bacterial killing efficiency in the food processing line. This article introduces a trial of real-time PCR application for rapid detection of bacteria, construction of predictive bacterial growth models, and determination of bacterial injury levels in food materials. These real-time PCR applications may not only provide for the detection of foodborne pathogens but may also support the formulation of new food composition and optimize bacterial killing conditions in the processing line.

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In recent years, the identification of microbial species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has gathered attention because of its convenience, rapidness, and low operating cost. MALDI-TOF MS obtains spectrum data from ribosomal proteins with molecular weight ranging from m/z 2,000 to 20,000. There are two methods for analyzing the spectrum data and for identifying microbial species. One method analyzes genetic data, assigning the peaks to the several ribosomal proteins as biomarkers, and analyzes the patterns of the differences of molecular weight. This method is complex to perform and is thus unsuitable for the rapid identification of species. However, it does provide the possibility of achieving identification of strains at high resolution. The other method is a finger-printing method designed to analyze the main peaks of the spectrum data without identifying specific proteins. The finger-printing method is convenient and rapid and thus is frequently applied clinically. The finger-printing method is being introduced in the food industry; however, data on food-related bacteria are insufficient and sometimes do not completely identify microbial species. In order to adequately utilize the finger-printing method in the food industry, it is necessary to construct a comprehensive database system. We have established a consortium composed mainly of food companies and worked for expansion of a comprehensive database by sharing of food company in-house databases.

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It has been more than 15 years since the advent of Next Generation Sequencing (NGS), which has made it possible to read DNA sequences much faster and at a lower cost than the Sanger method. A few years ago, Third Generation Sequencing (TGS) was also introduced to replace the short reads (only single fragments of 300 bp can be read) of Next Generation Sequencing with long reads (>100 Kbp can be sequenced at once). The pace of innovation in this field has accelerated rapidly over the last few years. This paper provides an overview of the applications of next generation sequencing and third generation sequencing in food microbiology testing as of 2021. It also provides a forecast of the impact of these new technologies on food microbiology testing in the near future. Due to space constraints, the third generation sequencers are discussed with particular emphasis on the molecular epidemiological analysis of foodborne illness caused by foodborne pathogens carried out by public authorities. In particular, third generation sequencing, especially of long reads, is expected to revolutionise the detection of foodborne pathogens, which has so far relied on culture and PCR methods. This will be of great help in determining the causes illness caused by foodborne pathogens. The analysis of microorganisms in food is currently undergoing rapid changes with NGS and TGS.

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The intake of fruit has been reported to decrease risk of diseases such as cancer, cardiac infarct, and apoplexia cerebri, by research in nutritional epidemiology. Citrus fruits contain several functional substances, such as flavonoids, coumarins, and phenylpropanoids. These substances are known for antioxidative activity, anticancer activity, and hypotension effects. The content of functional substances (flavonoids, coumarins, and phenylpropanoids) in local special citrus fruits was investigated. The content of functional substances in lemon (produced in the Seto Inland Sea area), Meyer lemon (produced in Kihou of Mie and others), niihime (produced in Kumano of Mie), su-daidai (produced in Suou-Oshima of Yamaguchi), hetsuka-daidai (produced in Osumi island of Kagoshima), sanboukan (produced in Wakayama), new-amanatsu (produced in Wakayama and others), suruga-elegant (produced in Suruga of Shizuoka), genko (produced in Madara island of Saga), yuuko (produced in Doinokubi of Nagasaki), kabosu (produced in Oita), sudachi (produced in Tokushima), yuko (produced in Tokushima), yuzu (produced in Kochi and elsewhere), nagato-yuzukichi (produced in Hagi of Yamaguchi), and hebesu (produced in Hyuga of Miyazaki) were local special citrus fruits that were analyzed by HPLC, and the quantitative values were subjected to a multivariate statistics principal component analysis. Lemon contained abundant eriocitrin and hesperidin. Eriocitrin, 6,8-C -diglucosylapigenin, 6,8-C -diglucosyldi osmet in, 5-geranyl oxy-7- methoxycoumarin, 5-geranyloxypsolaren, and 8-geranyloxypsolaren were characteristically contained in lemon. Meyer lemon contained abundant narirutin, hesperidin, and 6,8-C -diglucosyldiosmetin. The flavonoid composition in Meyer lemon was different from standard lemon. Meyerin, a novel antioxidant, was isolated from Meyer lemon peel. Citropten, 7-methoxy-5- prenyloxycoumarin, and meyerin were characteristically contained in Meyer lemon. Niihime contained abundant eriocitrin, hesperidin, and nobiletin. Saccharic acid 1,4-lactone 3,5-di-O-ferulat, was a novel substance isolated from niihime peel. Eriocitrin, nobiletin, and saccharic acid 1,4-lactone 3,5-di-O -ferulat were characteristically present in niihime. Su-daidai contained abundant neoeriocitrin, naringin, and neohesperidin. Neoeriocitrin and citropten were characteristically contained in su-daidai. Hetsuka-daidai contained abundant naringin and neohesperidin. Neoeriocitrin in hetsuka-daidai was present in a small amount. Sanboukan contained abundant narirutin, hesperidin, and 6,8-C -diglucosylapigenin. Er ioci tr in and 6,8-C -diglucosylapigenin were characteristically contained in sanboukan. New-amanatsu and surugaelegant contained abundant naringin, neohesperidin, and auraptene. Auraptene was characteristically present in the citrus fruits. Genkou and yuuko were abundantly present in narirutin and hesperidin. Kabosu, sudachi, yuko, yuzu, nagato-yuzukichi and hebesu contained abundant narirutin, naringin, hesperidin, and neohesperidin. Eriocitrin, neoeriocitrin, and sudachitin were characteristically present in sudachi. Eriocitrin and neoeriocitrin was characteristically found in yuko. The functional substances in citrus fruits of local special products are expected to make them recognized health foods in the future.

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I introduce the efforts of the Department of Life Science and Health, Kobe Institute of Health for the past 20 years to ensure food safety in the field of chemical inspection. Most of the inspection work is focused on systematic inspections at the Sanitation Inspection Office, Food Safety Inspection Office, and Meat Safety Inspection Office, which are subordinate organizations of the Public Health Management Center. During the 20 years from 2000 to 2019, a total of 448,972 items were inspected, including 19,837 items for food additive inspection, 396,492 items for residual pesticide inspection, 17,534 items for veterinary drug inspection, 1,560 items for specific raw material (allergic substance) inspection, 1,747 items for genetically modified food inspection, 4,880 items for inspection of standards and food ingredients, 4,105 items for inspection of utensils, containers and packaging, 787 items for inspection of food radioactive substances, and 2,030 items for inspection of household products. Violations of the standards were found in food additives, pesticide residues, veterinary drugs, specified raw materials (allergens), household products, etc. As withdrawal tests, 668 samples related to chemical food poisoning and symptomatic complaints were tested during the 20 years from 2000 to 2019. In the last 20 years, by utilizing the latest equipment, we have been able to analyze offflavors using GC-MS-SPME, natural poisons using LC-MS/MS, and contaminants using FTIR and fluorescent X-ray. As the scientific and technical core of hygiene administration in Kobe City, we will continue to respond to social conditions, citizens' needs, etc., and strive to ensure food safety in cooperation with government agencies.

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During a meal, we taste food by perceiving not only the taste, but also shape, color, aroma, texture, sound, and other elements. In recent years, with the rapid evolution of virtual reality and augmented reality technologies, more and more research has been conducted to reproduce or extend the food experience, which used to be complex and difficult to realize. In this paper, the latest studies on food VR/AR are introduced for vision, audition, haptics, olfaction, and taste. In addition, a detailed description of the author's own research, a system that produces a virtual drinking sensation, is presented.

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Phytochemicals are attracting attention, and interest is increasing in the functionality of plant-derived food additives such as rosemary. By clarifying the functionality of plants that are rarely used in Japan, we thought that we could broaden the range of cooking options. In this study, we evaluated the antioxidant activity and melanin synthesis inhibitory effect of eight kinds of plants used as herbs and food additives in the Kingdom of Thailand. In addition, as a new attempt, we investigated the activation of the YAP/TAZ transcription factor, which is the mechanism that induces immortalization of ES cells, and the downstream expression of the CCN gene group. As a result, high antioxidant activity was confirmed in 7 types of plants, and a strong melanin synthesis inhibitory effect was confirmed in 7 types of plants. The expression of CCN1 was significantly increased by a 70% ethanol extract of Curcuma aromatica Salisb. Decreased CCN2 expression was confirmed in water extracts of Stevia rebaudiana L., Pandanus amaryllifolius Roxb. and Illicium verum Hook.fil. Plants with high antioxidant activity and melanin synthesis inhibitory effects can be expected to reduce cancer risk and have cosmetic effects. The increase in CCN1 limits the synthesis and deposition of an extracellular matrix by myofibroblasts and reduces the risk of fibrosis during wound healing, so that these plants can be expected to be used for medical treatment and cosmetics. The decrease in CCN2 is expected to be useful for breast cancer treatment.

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