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1、1Food safety aspects of cell-based foodFOOD SAFETY ASPECTS OFCELL-BASED FOODFOOD SAFETY ASPECTS OF CELL-BASED FOOD2Food and Agriculture Organization of the United NationsWorld Health OrganizationRome,2023Required citation:FAO&WHO.2023.Food safety aspects of cell-based food.Rome.https:/doi.org/10.406

2、0/cc4855en The designations employed and the presentation of material in this information product do not imply the expression of any opinion whatsoever on the part of the Food and Agriculture Organization of the United Nations(FAO)or the World Health Organization(WHO)concerning the legal or developm

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4、AO or WHO in preference to others of a similar nature that are not mentioned.The views expressed in this information product are those of the author(s)and do not necessarily reflect the views or policies of FAO or WHO.ISBN(FAO)978-92-5-137723-9(print)ISBN(WHO)978-92-4-007094-3(electronic version)ISB

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8、ted Nations(FAO)or the World Health Organization(WHO).Neither FAO nor WHO is responsible for the content or accuracy of this translation.The original English edition shall be the authoritative edition.Disputes arising under the licence that cannot be settled amicably will be resolved by mediation an

9、d arbitration as described in Article 8 of the licence except as otherwise provided herein.The applicable mediation rules will be the mediation rules of the World Intellectual Property Organization http:/www.wipo.int/amc/en/mediation/rules and any arbitration will be conducted in accordance with the

10、 Arbitration Rules of the United Nations Commission on International Trade Law(UNCITRAL).Third-party materials.Users wishing to reuse material from this work that is attributed to a third party,such as tables,figures or images,are responsible for determining whether permission is needed for that reu

11、se and for obtaining permission from the copyright holder.The risk of claims resulting from infringement of any third-party-owned component in the work rests solely with the user.Sales,rights and licensing.FAO information products are available on the FAO website(www.fao.org/publications)and can be

12、purchased through publications-salesfao.org.Requests for commercial use should be submitted via:www.fao.org/contact-us/licence-request.Queries regarding rights and licensing should be submitted to:copyrightfao.org.viiviiiix113333444591718181828292930384040404042474851CONTENTSACKNOWLEDGEMENTSABBREVIA

13、TIONS AND ACRONYMSEXECUTIVE SUMMARYA.INTRODUCTION 1.Background 2.Working terminologies 3.Objectives and target audience 4.Scope of this document 5.Document compositionB.TECHNICAL BACKGROUND ISSUES 1.Terminologies 1.1.Introduction 1.2.Literature synthesis results 1.3.Impact of the terminologies 1.4.D

14、iscussion 2.Generic understanding of production processes 2.1.Introduction 2.2.Literature synthesis results 2.3.Discussion 3.Regulatory frameworks 3.1.Introduction 3.2.Literature synthesis results 3.3.DiscussionC.COUNTRY CASE STUDIES 1.Israel country contexts 1.1.Terminology 1.2.Current status 1.3.R

15、egulatory framework 1.4.Food safety assessment 1.5.Other key considerations outside food safety 1.6.Discussion 2.Qatar country context 2.1.Terminology 2.2.Current status 2.3.Regulatory framework 2.4.Food safety assessment 2.5.Other key considerations outside food safety 2.6.Discussion 3.Singapore co

16、untry context 3.1.Terminology 3.2.Current status 3.3.Regulatory framework 3.4.Food safety assessment 3.5.Other key considerations outside food safety 3.6.DiscussionD.FOOD SAFETY HAZARD IDENTIFICATION 1.Overview of the Expert Consultation 2.Technical Panel experts and resource people 3.Expert Consult

17、ation methodologies 3.1.Approach for the hazard identification 3.2.Approach for developing practical guide for relevant communication 4.Results of the Expert Consultation 4.1.Overview 4.2.Hazard tables by four production stages 4.3.Explanations about the identified hazards 4.4.Concerns not included

18、in the scope of hazard identification 4.5.Food safety communication and building consumer trusts 4.6.Special considerations on terminologies E.CONCLUSIONS AND A WAY FORWARDREFERENCES525252525860616262626267707172727374747576767699110112 116118120BOXES,FIGURES AND TABLESBoxesBox 1.A generic overview

19、of the cell-based food production processBox 2.Cell sourcing and isolation of chicken and bovine muscle satellite cellsBox 3.Cell sourcing and fluorescence-activated cell sorting to enrich bovine muscle satellite cellsBox 4.Cell harvesting scenarios in cell-based food productionFiguresFigure 1.Relat

20、ive share of the synonyms of“cell-based”meat modifiersFigure 2a.The number of mentions of various terms for the period 20102021Figure 2b.Relative share of the various synonymsFigure 3.Product packaging shown to participants in the studyFigure 4.Flow diagram for the sourcing and isolation of cultured

21、 muscle satellite cellsFigure 5.Examples of potential food safety hazards and concerns at different phases of cell-based food productionFigure 6.Global investments in cell-based food products in 2021Figure 7.Regulatory agencies and ministerial offices that may be involved in various stages of a cell

22、-based food start-up company up to and including pre-market approval and issuing the required licences for commercial manufacturingFigure 8.Approval process for novel foods in IsraelFigure 9.Summary of the food safety regulatory competent authority agencies in QatarFigure 10.Process of seeking the S

23、ingapore Food Agencys pre-market approval for cultured meatFigure 11.Generic components of food safety risk analysis paradigmFigure 12.Four stages of the cell-based food productionTablesTable 1.Synonyms of modifier terms for animal“cell-based”food products and their common use in professional sector

24、sTable 2.Studies on modifier terminologies for cell-based food products,their preferred use and associated attributesTable 3.A generic map of potential hazards/concerns in production processesTable 4.Developments in different countries relevant for cell-based food products and their safetyTable 5.Ha

25、zards identified by the Technical Panel for the cell sourcing stageTable 6.Hazards identified by the Technical Panel for the production stageTable 7.Hazards identified by the Technical Panel for the harvesting stageTable 8.Hazards identified by the Technical Panel for the processing stage 1920212478

26、811202841424553657476514253177859195viACKNOWLEDGEMENTSThe Food and Agriculture Organization of the United Nations(FAO)and the World Health Organization(WHO)would like to express their appreciation to the many people who provided advice and guidance during the preparation of this document.It was prep

27、ared for FAO and WHO,and the development process of the document was coordinated by Masami Takeuchi(FAO)in collaboration with FAO and WHO colleagues under the overall guidance of Markus Lipp(FAO).This publication has been published as a joint publication of FAO and WHO,while the earlier chapters wer

28、e initially drafted by various experts under the oversight of FAO.Technical contributions provided by Juliana De Oliveira Mota(WHO)and Moez Sanaa(WHO)throughout the process are herewith acknowledged.Section B was authored by Mark Sturme and Gijs Kleter,Wageningen Food Safety Research,the Netherlands

29、.Technical reviews were conducted by various international experts,namely Ousama Abubaker Abushahma,Joshua Ayers,Laura Braden,Stan Chan Siew Herng,Kern Rei Chng,Jonatan Darr,Breanna Duffy,Jeremiah Fasano,Antonio Fernandez,William Hallman,Ziva Hamama,Melissa Hammar,Natsuo Komoto,Teng Yong Low,Paul Mo

30、zdziak,Rick Mumford,Glen Neal,Kimberly Ong,Atiq Rehman,Yadira Tejeda Saldana,Jo Anne Shatkin,Elliot Swartz,Mehdi Triki,Hanna Tuomisto,Ruth Willis and Johnny Yeung Chun Yin.Technical and editorial inputs were provided by various FAO colleagues and technical editing was provided by Jennifer Parkinson.

31、For the Section C,various government officials and experts have contributed to the country case studies and the following individuals have authored each chapter(alphabetical order).C-1(Israel):Jonatan Darr,Ziva Hamama,Joseph Haskin,Yogev Magen and Shay Reicher C-2(Qatar):Ousama Abubaker Abushahma,Ir

32、shad Ahmed Abdul Samad,Hend Ali Al-Tamimi and Mehdi Triki C-3(Singapore):Joanne Chan Sheot Harn,Angela Li,Teng Yong Low,Kern Rei Chng,Johnny Yeung Chun Yin,Stan Chan Siew Herng,and How Chee OngTechnical reviews for the country case studies were provided by various international experts who have part

33、icipated in the relevant FAO initiative in the Technical Working Group,namely:Amie Adkin,Joshua Ayers,Darren Cutts,Jeremiah Fasano,Melissa Hammar,Rick Mumford,Glen Neal,Matthew OMullane and Atiq Rehman.The authors of each country case have also provided technical reviews on cases from other countrie

34、s.Technical and editorial inputs on the country case studies were provided by various FAO colleagues including Shan Chen and Markus Lipp,and technical editing was provided by Jeannie Marshall.Section D was developed based on the results of the Expert Consultation and all Technical Panel members have

35、 made substantial technical contributions to draft and review each section.Various FAO colleagues,including Maura DiMartino,Vittorio Fattori,and Keya Mukherjee,several Technical Working Group members and FAO experts have provided both technical and editorial contributions.In particular,significant c

36、ontributions to the final text of the documents provided by Jeffrey Farber are gratefully acknowledged.viiviiiAMPS Association for Meat,Poultry and Seafood InnovationANPR Advance Notice of Proposed RulemakingCEPA Canadian Environmental Protection ActEFSA European Food Safety AuthorityFAO Food and Ag

37、riculture Organization of the United NationsFBS fetal bovine serumFDA Food and Drug AdministrationFRESH Future Ready Food Safety HubFSANZ Food Standards Australia New Zealand FSEH Department of Food Safety and Environmental HealthFSIS Food Safety and Inspection Service of the United States GAP good

38、agricultural practicesGCC Gulf Cooperation CouncilGCCP good cell culture practicesGHP good hygiene practicesGLP good laboratory practicesGM genetically modifiedGMO genetically modified organismGMP good manufacturing practicesGRAS generally recognized as safeGSO Gulf Standardization OrganizationHACCP

39、 hazard analysis and critical control pointsIEC International Electrotechnical CommissionISO International Organization for StandardizationJECFA Joint FAO/WHO Expert Committee on Food AdditivesMME Ministry of Municipality and EnvironmentMOCI Ministry of Commerce and IndustryMOH Ministry of HealthMOP

40、H Ministry of Public HealthNFS National Food ServiceNSNR New Substances Notification RegulationsOECD Organisation for Economic Co-operation and DevelopmentQS Qatar General Organization for Standards and MetrologyR&D research and development SD standard deviationSFA Singapore Food AgencyUSDA United S

41、tates Department of AgricultureUSDA-FSIS United States Department of Agricultures Food Safety and Inspection ServiceUS FDA United States Food and Drug AdministrationWHO World Health OrganizationABBREVIATIONS AND ACRONYMSixEXECUTIVE SUMMARYAnimal-based meat production has evolved over thousands of ye

42、ars to meet the demand for safe and affordable sources of protein.Cell-based food production,which is the field of growing animal agricultural products directly from cell cultures,has been explored as an alleged sustainable alternative to the conventional livestock agricultural system.As commercial

43、cell-based food production continues to expand,the urgency increases to address one of the most important questions of consumers,the question of food safety.Thus,the Food and Agriculture Organization of the United Nations(FAO),in collaboration with the World Health Organization(WHO),has developed th

44、e present document to engage with respective Members and relevant stakeholders by proactively sharing the current knowledge to identify concrete ways to inform consumers and all other stakeholders about the food safety considerations for cell-based food products.This document includes a literature s

45、ynthesis of relevant terminology issues,principles of cell-based food production processes and the global landscape of regulatory frameworks for cell-based food production.Case studies from Israel,Qatar and Singapore have been included to highlight different scopes,structures and contexts surroundin

46、g their regulatory frameworks for cell-based food.The results of the FAO-led Expert Consultation,where comprehensive food safety hazard identification was conducted,form the core of the document and the identified hazards are summarized with causal-chain examples.Hazard identification is the first s

47、tep of the formal risk assessment process.During the Expert Consultation,all potential hazards were discussed in the four stages of the cell-based food production,namely:1)cell-sourcing;2)cell growth and production;3)cell harvesting;and 4)food processing.Experts agreed that while many hazards are al

48、ready well known and existing equally as well in conventionally produced food,the focus may need to be put on the specific materials,inputs,ingredients(including potential allergens),and equipment that are more unique to cell-based food production.While the list of hazards identified forms a strong

49、basis for the next steps,more data generation and sharing at the global level are essential to create an atmosphere of openness and trust that will enable the positive engagement of all stakeholders.International collaborative efforts would benefit various food safety competent authorities,particula

50、rly those in low-and middle-income countries,to employ an evidence-based approach to prepare any necessary regulatory actions.The way forward will consist of continuing to invest in research and development in order to understand whether the alleged benefits in increased sustainability can be realiz

51、ed.In this regard,it will be important to closely observe as to what extent,if any,cell-based foods result in differences from conventionally produced foods.Keywords:food safety,cell-based food,cell culturing,cultured meat,cultivated meat,terminology,nomenclature,production process,regulatory framew

52、ork,risk analysis,hazard identification,risk assessment,expert consultation,food standards,Codex AlimentariusFood safety aspects of cell-based foodFAOA.Introduction11.BackgroundThe world is facing tremendous food challenges as estimates are that our growing world population will reach 911 billion by

53、 2050.In concert with this,as the global demand for proteins grows and because of potential health and environmental concerns,more consumers are looking to reduce their consumption of animal origin products.The increasing recognition of the challenges related to feeding a growing global population,w

54、hile at the same time producing food more sustainably,is spurring food system innovations which are shaping our future agrifood landscape.For example,many in the food sector are looking for opportunities to expand the sources of alternative proteins that can be both environmentally sustainable and n

55、utritionally sound.In terms of traditional meat/protein production,there are also other mitigating factors such as there being a limited amount of global arable land as well as real and unknown threats due to climate change.Cell-based food production,or cellular agriculture,which is the field of gro

56、wing animal agricultural products directly from cell cultures instead of using livestock,and which has been referred to as cell-based foods,cell-cultured foods and cultivated meat,has been explored as a potentially sustainable option to complement the conventional livestock agricultural system.Some

57、of the cell-based food products are already under various stages of development across the world,making it critical to objectively assess the benefits they might bring,as well as any risks associated with them-including food safety and quality concerns.Since the initial studies in the early 2000s,me

58、thodologies for cell-based food production,have been well characterized and have moved from laboratories to production facilities.In 2013,the first beef burger produced through this technology was presented to the world.In December 2020,the first cell-based chicken nuggets were approved in Singapore

59、.In November 2022,the United States Food and Drug Administration(US FDA)completed its first pre-market consultation for human food(chicken)made using animal cell culture technology.The voluntary pre-market consultation is not an approval process;however,it means that after analyzing the data submitt

60、ed by the company,the US FDA states that it has no further questions at this time about the safety conclusions.Currently,there are more than 100 start-ups developing various cell-based food products around the world.This commercial landscape is expanding very quickly,with many different types of pro

61、ducts and commodities such as various meats,poultry,fish,aquatic products,dairy and eggs in the pipeline for future commercialization.One of the most important questions consumers would raise is food safety.In addition to safety,there are several other legitimate issues that are important to conside

62、r,such as ethical issues,environmental considerations,animal welfare,consumer preference/acceptance,production cost,prices of the end products,as well as regulatory requirements such as approval mechanisms and labelling rules.As cell-based food production may involve a set of relatively new technolo

63、gies,techniques and/or production steps,it is likely that many countries are currently thinking about and would consider implementing a regulatory process that addresses all the relevant issues,before such products become available in the marketplace.The Food and Agriculture Organization of the Unit

64、ed Nations(FAO)and the World Health Organization(WHO)consider that the time is ripe to initiate the discussion on the potential benefits and drawbacks of cell-based food production.It is important for FAO/WHO as well as the respective Members,to engage in the proactive sharing of relevant knowledge

65、and information among various stakeholders to identify concrete ways to assure the safety of cell-based food products for consumers.The intention of the present FAO/WHO work on cell-based food is to capture the key food safety issues in a timely manner,before the products can become widely available

66、 in the global market,so that competent authorities,A.INTRODUCTION2Food safety aspects of cell-based foodparticularly those in low-and middle-income countries,will be equipped with up-to-date information and scientific knowledge related to cell-based food production,to consider potentially important

67、 regulatory actions.This document,extensively studying food safety aspects of cell-based food,does not have an intention to endorse the technology.It is not FAO/WHOs role to promote any types of food products or production methodologies,however,it is not FAO/WHOs intention to block any relevant tech

68、nological developments and innovations.FAO and WHO support their Members to assure any food,no matter how it is produced,is safe for the consumer.2.Working terminologiesThroughout this document,the terms“cell-based food”,“cell-based food products”and“cell-based food production”are used as a set of w

69、orking terminologies to indicate the products or production processes involving culturing cells isolated from animals.A literature synthesis was conducted on various relevant terminologies(see Section B-1)and the results showed that while some different preferences exist among different sectors,the

70、term“cell-based food”was found to be less confusing,conveniently overarching and generally well-accepted by consumers.However,there is no term that is 100 percent scientifically correct.In theory,any organism made of cells can be described as“cell-based”,therefore,it does not automatically distingui

71、sh the technology to grow edible tissues from“cells”.Also,the term“cell-based”has never been used for food,therefore some food business operators may prefer not to use the term.The terms“cultured”and“cultivated”can be confusing as they are often used in the aquaculture sector to indicate farmed fish

72、 and fisheries products.The term“cellular agriculture”can be considered too general as it may include the topic of plant cell culturing or fermentation,which can use a wide variety of methodologies and techniques.There has also been a challenge identified on the use of commodity names such as“meat”,

73、“chicken”or“fish”together within the terminologies(see Section D-4.6.3),thus the consistent use of“food”and“food products”has been maintained in the document.Nomenclature can have a significant impact on consumer perception,marketing efforts and relevant regulatory actions such as labelling.While th

74、e present document uses the term“cell-based food”,experts(see Section D-2 for more details about the Technical Panel experts)have suggested to have good studies before considering international harmonization of the terminology.While it is ideal to have an internationally harmonized set of terminolog

75、ies,experts have indicated that it may be more important to start with recommending a set of key elements for the food safety competent authorities to consider and use within their cultural and geographical contexts as well as their languages.The experts also suggested not to use a direct translatio

76、n of the English terms,without considering the impact of such terms in the local language.3.Objectives and target audienceThe primary objective of this document is to provide readers with up-to-date technical knowledge on the multidisciplinary topic of cell-based food production,with a focus on the

77、food safety aspects,through the process of literature synthesis and expert elicitation.Overarching specific objectives include:1.to summarize the relevant technical matters for national food safety competent authorities,particularly those in low-and middle-income countries,to consider their potentia

78、lly required actions;2.to share information on technical knowledge and good practices among competent authorities on various regulatory frameworks on cell-based foods to learn from each other;3.to summarize the results of the Expert Consultation where food safety hazard identification for cell-based

79、 food products has been conducted;and 4.to identify the needs for possible follow-up actions by international organizations like FAO/WHO.This will facilitate global discussions and action planning with the partner agencies and stakeholders at the global level.3A.IntroductionMore specific objectives

80、include reviewing and describing information so that national food safety competent authorities can:1.understand how various countries and organizations are describing and using terminology related to cell-based foods,so that this information can serve as a basis to support stakeholders worldwide in

81、 making informed decisions on selecting those cell-based food terminologies that could be used in communications or accepted in legislation on cell-based food products;2.understand the various technologies that are currently being used to produce cell-based foods and the potential hazards that have

82、been identified;3.understand the results of food safety hazard identification conducted by the Technical Panel and initiate generation of relevant data for the next steps of risk assessment;and 4.learn about the current regulatory thinking and developments that currently exist for cell-based foods i

83、n different countries and jurisdictions.While the primary target audience of this document was set for national food safety competent authorities,the global community of scientists,developers,the cell-based food industry as well as academics doing research in the area of cell-based food production m

84、ay benefit from reading this document.4.Scope of this documentThe scope of this document is strictly technical and prioritizes any potential food safety issues associated with cell-based food products.The scope includes examining what are the existing terminologies that are now being used in the fie

85、ld,providing an overview of the scientific literature on the cell-based food production technologies currently being developed along with any potential hazards that have been identified,as well as discussing the current regulatory developments that apply for cell-based foods in different countries.I

86、t is recognized that there are several other issues with respect to cell-based foods that are important to consider,including ethical issues,environmental considerations,animal welfare,consumer preference/acceptance,nutrition aspects,production costs,prices of the end products,and regulatory require

87、ments such as approval mechanisms and labelling rules.Although these issues are critical in moving the whole area of cell-based foods forward,they are not within the scope of this document,however,they may be the subject of future FAO and/or WHO consultations.In addition,besides cell-based foods,the

88、re are several other alternative protein sources that fall under the area of“new foods and production systems”,a field that is growing fast and it is very likely to grow even more so over time.Some of the more prominent topics covered under this category include seaweed,microalgae,edible insects,pla

89、nt-based protein alternatives and 3-D printed foods.However,these latter potential alternative food protein sources are also not included within the scope of this document.5.Document compositionThis document has 5 sections from A to E.Section A is the present introductory chapter,and Section B consi

90、sts of three technical background issues namely 1)terminologies,2)production process and 3)regulatory frameworks.Section C has three country case studies from Israel,Qatar and Singapore.Section D summarizes the results of an Expert Consultation meeting where potential food safety hazards for cell-ba

91、sed food production have been identified by the Technical Panel experts and resource people.Section E concludes the document with a way forward.4Food safety aspects of cell-based foodB.TECHNICALBACKGROUND ISSUES1.Terminologies1.1.IntroductionThe increasing global demand for animal-sourced protein ad

92、ds to the existing pressure on ecosystems and biodiversity(FAO,2018).Intensifying animal production may also threaten broader sustainability objectives,such as climate change and public health,resulting in trade-offs in various aspects of environmental protection,food security and animal welfare(FAO

93、,2019,Henchion et al.,2021,OECD,2021).These factors have triggered research efforts for developing more sustainable ways of producing animal meat as well as a research focus on a“protein transition”wherein consumption of animal protein will be at least partially replaced by alternative protein sourc

94、es,such as from plants and microorganisms but also in vitro produced animal protein(Aiking and de Boer,2020),in order to accommodate the increased demand for protein and assure global food security.One of the technological developments that could produce analogues of animal proteins without slaughte

95、ring animals is via in vitro cultivation of animal cells on a large scale,which could then be processed into products that are substantially equivalent to conventional meat.Such products are often called“cell-based”,“cultured”or“cultivated”meat,and currently there are several terms in use to define

96、this type of products around the globe.While research in this area has been ongoing since the early 2000s,the development of the products was presented to the general public in 2013,when researchers from the Netherlands demonstrated the first product describing it as a“lab-grown”beef burger at a pre

97、ss conference in London(BBC News,2013).In December 2020,so-called“cultured”chicken nuggets became the first commercialized product of its kind,after market approval in Singapore;these particular nuggets are a blend of cultured chicken and plant-based ingredients(Carrington,2020).On a broader scale,t

98、he production of analogues of animal products,such as meat,poultry,seafood,dairy,and eggs produced through cell-based culture techniques has been advancing quickly in the past few years and at least 76 companies have been developing similar products in 22 different countries since 2013(Byrne,2021).B

99、ecause of the novelty of the cell-based food production process and products,assurance of food safety is one of the main concerns of nutritionists,food technologists,the competent authorities and consumers.In addition,the national competent authorities will have to consider various socioeconomic iss

100、ues relating to these products,including consumer preference,acceptance,ethical issues,production costs,trade issues and market prices.When there is a need for clear labelling of such products and/or special authorization processes are to be conducted by competent authorities,then appropriate regula

101、tory frameworks need to be adjusted or newly employed,as these products may enter their jurisdictions or appear at the border at any time,via e-commerce for example.In order to discuss the relevant technical issues about cell-based food production,it is important to use clear and consistent terminol

102、ogies that can be accepted by all the stakeholders.Terminologies and labels are also an important and direct means of communicating information to consumers(FAO,2021).However,currently many different terms and labels exist for these types of products in both the scientific literature and public comm

103、unications,thereby potentially creating confusion.It is therefore important to make an inventory of these terms and their current usage,framing and legal consequences,in order to achieve a consensus on the terminology to use at the global level.This will also contribute to a better understanding of

104、the topic as well as encourage further discussions on cell-based food products in different parts of the world.5B.Technical background issuesTo aid the scientific advice activities provided by the Food and Agriculture Organization of the United Nations(FAO),it is essential to use clear terminologies

105、 to describe the relevant processes,associated technologies,techniques and products in animal cell-based food production.The chapter focuses on the terminologies used in different sectors and describes the associated issues,by making a systematic inventory of the available scientific literature as w

106、ell as non-scientific reports and public communications.This overview employed the systematic-review methodology,and it does not include any political nor opinion-based views.The aim of the chapter is not to define the relevant terminologies but to simply collect the existing ones with the attribute

107、d analyses,so that subject-matter experts and/or policymakers at the national level can use this overview as a reference to make informed decisions.1.2.Literature synthesis results1.2.1.Modifiers found in various literatureA list of the synonyms used for cell-based food products,such as cell-based m

108、eat and seafood products,and their use by different professional sectors is provided in Table 1 based on the outcomes of several consumer and industry studies on the perception,acceptance and preference for terminologies for the modifier part(e.g.“cultured”)of the terminologies.Table 1.Synonyms of m

109、odifier terms for animal“cell-based”food products and their common use in professional sectorsSectorModifier termaAuthoritiesIndustry and developersAcademiaMediaanimal-freeXXartificialXXcell-basedXXXXcell-cultivatedbXcell-culturedXXXXcellularXXcleanXXcruelty-free XcultivatedXXXXculturedXXXXfakeXXFra

110、nkenmeatXhealthyXXimitationXin vitroXXlab-grownXXmadeXMeat 2.0cXShmeatXslaughter-freeXsyntheticXXtest tubeXvat-grownXNotes:a)Based on scientific articles collected from the literature search,grey literature and media;b)Hallman,W.K.,Hallman,W.K.II,&Hallman E.E.(2021 Cell-Based,Cell-Cultured,Cell-Cult

111、ivated,Cultured,or Cultivated.What is the best name for meat,poultry,and seafood made from the cells of animals?https:/www.biorxiv.org;c)Meat 2.0 is a term that is used to cover“cell-based”meat,but also plant-based and microbe-based meat replacers.Source:Authors own elaboration.6Food safety aspects

112、of cell-based food1.2.2.Modifier terminologies used by authoritiesThe use of terminologies by authorities such as governmental institutions and regulatory bodies is often expected to be guided by legally accepted terms.Besides,for example,Singapore and the European Union,regulatory bodies in most co

113、untries have not yet ruled as to what existing legislation cell-based food products fall under,or which specific terms for labelling of cell-based food products are to be used.As of February 2022,the Singapore Food Agency(SFA)is the only regulatory body that has implemented a specific section for ce

114、ll-based food products in their“Requirements for the Safety Assessment of Novel Foods”document(SFA,2021a).This document uses the term“cultured”meat,but this is not the only term allowed,as the SFA has indicated that product package labelling will require qualifying terms that clearly communicate the

115、 nature of“cultured”meat food products to consumers so that they can make informed choices.These terms may also include,for example,“cultured”,“cultivated”and“cell-based”(SFA,2021b).Singapore has also published general food labelling guidelines that advise against the use of claims that would cast d

116、oubt on the safety of other foods or imply that a particular food is safer than other similar food,and these would also apply to cell-based food(SFA,2021a).In the United States,the Food Safety and Inspection Service(FSIS)of the United States Department of Agriculture(USDA)published in September 2021

117、 an Advance Notice of Proposed Rulemaking(ANPR)in which it requests comments for“the labelling of meat and poultry products comprised of or containing cultured cells derived from animals”(USDA-FSIS,2021).Similarly,the United States Food and Drug Administration(US FDA),which has labelling authority f

118、or cultured fish and seafood cell products,published in October 2020 a“Request for Information”in which it calls for comments for“the labelling of foods comprised of or containing cultured seafood cells.”(FDA,2020).The FDA intends to use the information and data resulting from this notice to determi

119、ne what type(s)of actions,if any,the agency should take to ensure that these foods are properly labelled.The FSIS and the FDA have agreed to develop joint principles for product labelling and claims to ensure that products are labelled consistently and transparently.Although the FSISs ANPR makes use

120、 of the term“cultured”meat,the US authorities are still in the process of defining the actual food labels that will be allowed in the future,which will impact the terms to be used by these authorities in the future.It is also worth considering that the authorities labelling regulations may have pref

121、erence for terms that describe the process the food has undergone.1.2.3.Modifier terminologies used by industry and developersIn September 2021,a focus group surveyed the Chief Executive Officers(CEOs)of 44 cell-based food companies globally about their preferred nomenclature for their products.Seve

122、nty five percent of the companies were found to use the modifier“cultivated”,20 percent the concept“cultured”meat,and one company(2 percent)“cell-based”.Several quotes from the interviewed CEOs appear to point to a shared view that the use of“cultivated”allows us to differentiate from other products

123、 and at the same time appeal to consumers and be amenable to consumer education.The use of“cultivated”might therefore align the industry viewpoint for the modifier term(Byrne,2021).This survey indicates an increase in adoption of the term“cultivated”since a study in 2020,where this term was found to

124、 be used in 45 percent of relevant websites and promotional material from the cell-based food industry.This is partially in line with the recommendation from the American“cultured”meat industry trade group Association for Meat,Poultry and Seafood Innovation(AMPS)to use either“cultured”,“cultivated”o

125、r“cell-based”and in line with the recommendation by the cell-based meat industries based on the outcomes of the consumer study by Szejda et al.(2019).Following various post-hoc stakeholder meetings,the study executor and stakeholders chose the term“cultivated”meat to go forward with.Towards this end

126、,a communication strategy was devised,where an analogy was drawn between cultivating meat and growing plants in a greenhouse.In addition,the use of cultivation-related language,such as“cultivator”for the reaction vessel in which cells are grown,was considered to expand the narrative to engage people

127、 with the concept of meat cultivation(Szejda et al.,2019).It is important to note that the terminologies used or preferred by industry are subject to change and indicates the need to harmonize terminologies in the industrial sector,which might come from legal approval of specific terms by the author

128、ities.7B.Technical background issues1.2.4.Modifier terminologies used in academic researchThe scientific community uses a wide variety of terminologies(Table 1).However,no studies have been performed to analyze the preferred modifier terminologies among scientists and,therefore,a consensus on accept

129、ed terminologies does not exist.Based on the scientific articles(N1=144)collected from the literature search on this topic for the period 20132022,the most used terms are“cultured”(N=43)and“cell-based”(N=27),followed by“in vitro”(N=17),“artificial”(N=11)and“cellular”(N=10),while other modifier terms

130、 appear to be less commonly used(Figure 1).Figure 1.Relative share of the synonyms of“cell-based”meat modifiers1.2.5.Modifier terminologies used by the media and othersUsing the News on the Web corpus(Davies,2016)via the website English-Corpora.org,a large collection of texts was searched through to

131、 verify the frequency that“cell-based”meat terms were mentioned in the media between 2010 and 2021(Figure 2).This showed that media coverage of“cell-based”meat developments has markedly increased in the last 10 years(Figure 2a)and uses a wide variety of synonyms(Figure 2b and Table 1).The most frequ

132、ently used terms since 2010 were,among others,“cultured”(30 percent),“lab-grown”(19 percent)and“fake”(14 percent)and“clean”(9 percent).It has to be noted that the preferentially used terms in the media have shifted in the last years:while in the initial years,terms such as“in vitro”,“cultured”or“cle

133、an”meat were often used alongside“cultured”meat,currently other terms are more frequently encountered,such as“cultivated”or“cell-based”meat(Southey,2021).Note:Terms used in the titles of scientific articles collected from the literature search for the years 20132021(as mentioned in Table 1).Source:A

134、uthors own elaboration.ArtificialCellularCultivatedCleanCell-culturedLab-grownAnimal-freeSlaughter-freeSyntheticIn vitroCell-basedCultured1 N=144 means that the number(N)of scientific articles was 144.8Food safety aspects of cell-based foodFigure 2a.The number of mentions of various terms for the pe

135、riod 20102021Figure 2b.Relative share of the various synonyms12001400160010008006004002000201020112012201320142015201620172018201920202021Cultured meatCultivated meatLab-grown meatFake meatCell-based meatSynthetic meatClean meatArtificial meatHealthy meatImitation meatIn-vitro meatOthers(1%)Note:“fa

136、ke meat”and“imitation meat”were also used for other meat analogue types;“healthy meat”occurred in many unrelated contextsSource:Davies,M.2016.Corpus of News on the Web(NOW).https:/www.english-corpora.org/nowClean meat,9%Cultivated meat,7%Cell-based meat,4%Synthetic meat,4%Artificial meat,3%Healthy m

137、eat,3%Imitation meat,2%Others(34 percent)of interviewed Brazilian respondents were willing to consume cell-based meat(Bryant and Krelling,2021;Forte Maiolino Molento et al.,2021).There is variability,though,between interviewees of different age groups and from different urban areas of Brazil when as

138、ked if they would consume“meat from cellular agriculture”10Food safety aspects of cell-based food(Forte Maiolino Molento et al.,2021).After having been presented texts with one out of four different names for cell-based meat,subjects in another study found“clean meat”to be less descriptive and less

139、distinguishable from conventional meat and plant-based alternatives than“cultivated meat”,“cell-based meat”,and“slaughter-free meat”(Bryant and Krelling,2021).It should be noted that both these studies were performed using the Portuguese equivalents of the English modifier terms.Bryant et al.(2019)d

140、id a pre-test among Chinese consumers to rank various potential names for cell-based meat in Mandarin for appeal and descriptiveness.Based on the outcomes,these authors selected the term“purity meat”(similar to“clean meat”),for use in a survey to further study consumer perception.1.3.2.Language barr

141、iers and translation issuesLanguage-specific perception barriers may also exist for the use of certain terminologies.Direct translation from English may not always be straightforward or might be problematic due to non-familiarity or negative connotations of the translated terms.For example,several r

142、espondents to a consumer survey in Japan expressed their dislike of the translation of“cultured”meat into Japanese(Baiyo-niku)(CAIC,2021).Among ten cell-based meat-related terms submitted to a cross-section of German society in a study survey,“direct meat”(Direktfleisch in German)attained the highes

143、t scores for appeal,accuracy,and clear differentiation.This term was nonetheless excluded from further study due to its dissimilarity to the English synonyms currently used and the low acceptability among industrial stakeholders(Janat et al.,2020).Similar issues in perception of specific terms might

144、 also exist in other languages and should be evaluated before using terms.Bryant et al.(2019)employed back-translation of the related terms and a study questionnaire from English into Mandarin to achieve equivalent meaning.Back-translation entails the translation of a questionnaire into a target lan

145、guage by a bilingual person as a first step.This translated text is subsequently translated back into the source language by another bilingual person who is unaware of the original text.The original text and the second translation can then be compared.Any ambiguities and discrepancies can then be re

146、solved,and the text revised and refined accordingly(Jones,1998).1.3.3.Modifier terminologies that are fit for purposeHallman and Hallman(2020)extended on the findings by Bryant and Barnett(2019)in their study on possible names for“cultured”seafood products.They noted that past consumer studies had f

147、ocused on meat,yet that the category of“cultured”seafood products was also at an advanced stage of development.Moreover,previous studies had not addressed the distinguishability between“cultured”and conventional products.In the case of seafood,there is already a need to distinguish products of farme

148、d and wild-caught seafood,and this now needs to be further clarified for the term“cultured”seafood as well.The authors formulated three additional requirements for a designation for cell-based food products,namely that they 1)are appropriate from the consumers point of view;2)do not disparage one or

149、 any other category of foods;and 3)do not raise a response inconsistent with the idea that“cultured”seafood is safe,healthy and nutritious.The term chosen should be able to modify not only seafood but also poultry and meat.Three additional phrases were used for the investigation,including“produced u

150、sing cellular aquaculture”,“cultivated from the cells of.”,and“grown directly from the cells of.”(Hallman and Hallman,2020).All the concepts using the term“cell”were most accurately identified as being neither farm-raised nor caught in the wild,and also scored significantly lower in consumer accepta

151、nce than the conventional products(Hallman and Hallman,2020).All concepts used were equally well identified as products not to be consumed by people who are allergic to seafood.The phrases“cultivated from the cells of.”and“grown directly from the cells of.”were most accurately identified as not bein

152、g“ocean-caught”or“farm-raised”.They were also somewhat less appetizing(1718 percent versus 26 percent)than the other concepts and evoked the least positive initial responses.With several others,participants imagined products labelled with these two phrases to be less tasty and less safe to eat as we

153、ll.They also thought products labelled with the concepts“cell-cultured”and“cultivated from the cells of.”to be less nutritious than conventionally farmed and wild-caught seafood(Hallman and Hallman,2020).The authors abandoned“cultivated”,“cultured”,and“produced using cellular aquaculture”due to an a

154、pparent misidentification 11B.Technical background issuesSource:Hallman,W.K.&Hallman,W.K.,II.2021.A comparison of cell-based and cell-cultured as appropriate common or usual names to label products made from the cells of fish.Journal of Food Science,86(9):3798-3809.dx.doi.org/10.1111/1750-3841.15860

155、.as being from conventional aquafarming,widely known as aquaculture.They also abandoned the descriptors“cultivated from the cells of.”and“grown directly from the cells of.”given the negative responses to these concepts and the association with genetic modification.Survey participants expressed posit

156、ive initial responses to the two remaining concepts of“cell-based”and“cell-cultured”.While both these concepts performed well on many counts,“cell-based”outperformed“cell-cultured”in terms of perceived nutritional value and taste of the product,purchasing intention,and consumption advice to children

157、.The authors concluded that“cell-based”met all criteria and was an appropriate name for product description(Hallman and Hallman,2020).In a follow-up study,the authors compared the two selected terms“cell-based”and“cell-cultured”in a more focused way using a group of American consumers as respondents

158、(Hallman and Hallman,2021).Participants(N=1200)were shown two pictures of imaginary pouches containing salmon substitute products.The front of the pouch featured a picture of a salmon fillet(suggested serving),the name“Atlantic salmon fillets”in large font with a smaller subscript“cell-based”seafood

159、 on the left and“cell-cultured”seafood on the right,on top of a nutritional fact table plus storage advice and product weight(Figure 3).Figure 3.Product packaging shown to participants in the study12Food safety aspects of cell-based foodThe outcomes confirmed those of the previous study in that many

160、 participants correctly identified both products as not being derived from farm-raised or wild-caught fish,and that they should not be consumed by persons with allergies.For the remaining incorrect identifications,“cell-cultured”was more often associated with farm-raised products than“cell-based”,wh

161、ich was also the case for ocean-caught fish.Moreover,many participants correctly assumed that both products were derived from salmon cells.Initial,subsequent,and overall reactions to“cell-based”were more positive than to“cell-cultured”.Products with both concepts performed equally positive in some r

162、espects:consumers considered both somewhat-to-moderately safe to eat,moderately nutritious,slightly good-tasting,and neither natural nor unnatural.“Cell-cultured”was associated more with genetic modification than“cell-based”,while purchasing and tasting intentions were slightly greater for“cell-base

163、d”than for“cell-cultured”products(Hallman and Hallman,2021).Ong et al.(2020)also studied the term“cell-based”meats,reviewing the evolving production and regulatory landscapes for these products.As regards nomenclature,they considered the possibility of adding additional terms implying edibility,heal

164、thiness,sustainability and no involvement of animals.While for edibility,the ingredients and production processes used should be proven to be safe,various claims and labelling rules and guidelines may apply to claims of healthiness,sustainability and absence of cruelty to animals.As regards healthin

165、ess,depending on the regulatory frameworks,certain claims may be permitted provided that evidence can be provided in support of these claims.The authors considered that reference to“animal-free”might still be controversial as cells from animals will be used as donors in the initial stage,although th

166、e use of lines of immortalized cells could further decrease dependency on animals,as does the avoidance of the use of animal-derived additives to the production media(Ong et al.,2020).Szejda et al.(2019),in collaboration with several cell-based food companies,carried out a study in which focus group

167、s(N=27)discussed a narrative for the“cultured”meat presented to them,followed by another study with segmented consumer groups(enthusiasts,sceptics,opponents).They concluded that,for example,the concepts“cultivated”meat and“cultured”meat had the most appeal and were moderately descriptive.“Cell-based

168、”and“cell-cultured”were only somewhat appealing yet scored better on the descriptiveness scale as being moderately to very descriptive.The modifiers“cultivated”,“cultured”,and“cell-based”differentiated moderately and moderately to very much from conventional meat.It was argued that“cultivated”evoked

169、 positive responses,considering appeal,neutrality,and descriptiveness criteria,for many of the participants.1.3.4.Other considerations for terminologiesAllergen labellingThe product noun,such as“salmon”in the collocation“cultured salmon”might impart important information to allergy patients who are

170、allergic to the traditional form of the product from the same animal species(salmon in this example).It is important to ensure that the modifiers do not conceal this,such as in the example“cell-based artificial salmon product”(Lamb,2018).In addition,it is also important to consider proper allergen l

171、abelling,as cell-based food products can have the same level of risks for allergic reactions as conventional counterparts(Hallman and Hallman,2020).This will entail the declaration of ingredients(listed on the product label)that may cause hypersensitivities,such as egg,crustaceans,fish,and milk(Code

172、x Alimentarius,2018).These may then have to be highlighted in bold font,for example,so as to stand out for consumers reading the product label.Commodity terminologies in the regulatory frameworkWhile no internationally harmonized definition of the term exists and nothing indicates restrictions on th

173、e use of any terms,there are potential and significant restrictions in many countries on using commodity terms such as“meat”,“chicken”,“fish”,“milk”and so forth.Cell-based food can be considered as“novel food”in certain jurisdictions(e.g.in the European Union),which may place additional requirements

174、 on the terms used and provides an opportunity to define terms,as certain regulatory requirements of“meat”may not apply to this type of product(Seehafer and Bartels,2019).In the United States of America,new agency regulations for labelling of meat and poultry products derived from animal cells is un

175、der consultation in a so-called“advance notice of proposed regulation(ANPR)”(USDA,2021).13B.Technical background issuesWhile the ANPR touches upon issues of regulation and safety,it is notable that it also addresses the various aspects identified by the scientific investigations into the impact of n

176、aming of these products on acceptance and interpretation accuracy.The term“cellular agriculture”As of February 2022,several terms are in use in science,industry and the media,such as“cellular agriculture”,“cellular food technologies”,“cell-based techniques”and“cell-based food production.The use of t

177、hese terms is currently dictated by the end user,and no studies have been performed on the perception and acceptance of alternative terms by different social or professional groups.The term“cellular agriculture”is used by many stakeholders and it indicates the production method that can be used to m

178、ake acellular or cellular products,where acellular products are made of organic molecules like proteins and fats and contain no cellular or living material in the final product,while cellular products are made of living or once-living cells.For example,acellular animal-sourced foods(like milk protei

179、ns or gelatine)are produced without animals through fermentation using microorganisms like yeast or bacteria(often referred to as precision fermentation).In contrast,cellular products are formed by growing cells from a particular animal species and tissue type in vitro,followed by assembly of cells

180、on a scaffold to form tissue-like structures and further processing into products(Rischer et al.,2020).The use of the term is also documented in various sources(CAIC,2021).However,it should also be noted that for the scientific community,the term“cellular agriculture”encompasses not only the product

181、ion of cell-based food but also the utilization of cell cultures of a whole variety of host organisms(animals,plants,microorganisms)for the production of agricultural food products rather than production from farmed animals or crops(Mattick,2018;Rischer et al.,2020).Table 2 provides a summary of the

182、 various studies analyzed in detail examining the impact of terminology on the perception of cell-based meat products by consumers.The results show that“cultivated”was the preferred modifier in 5 studies,while“cultured”and“cell-based”were preferred twice in separate studies and“clean”in one study.Ce

183、llX/Ning Xiang14Food safety aspects of cell-based foodTable 2.Studies on modifier terminologies for cell-based food products,their preferred use and associated attributesSector/social groupCountryTerm preferencePreference(%)or best perception/acceptanceStudy set-upReferenceConsumersCell-based food i

184、ndustryNon-profit advocatesUnited States of AmericaCultivatedPreference of consumers based on survey,and of relevant compa-nies and associations.Appeal:cultivated and cultured more appealing than cell-based and cell-cultured.Descriptiveness:cell-based and cell-cultured more descriptive than cultivat

185、ed and cultured.Differentiation from conventional meat:cultivated,cell-based,and cultured were moderately and cell-cultured was moderately to very differentiating.all terms were moderately differentiating.Mixed methods consumer survey and focus groups(N=27).University students:participants expressed

186、 a diverse range of political views,skewed toward a younger age(primarily 1821 years),majority female(59%),and the majority were omnivores.(Szejda,2019)Survey reportConsumersUnited States of AmericaClean“Clean meat”showed significantly more positive associations than“animal-free”,“cultured”or“lab-gr

187、own”.“Clean meat”and“animal-free meat”also triggered more positive attitudes-and“clean meat”more positive intentional behaviours-than“lab-grown meat”.Between-subjects design(N=185).Participants perception assessed for 4 product names:(1)“cultured meat”,(2)“clean meat”,(3)“lab-grown meat”,and(4)“anim

188、al-free meat”.Participants were recruited through Amazon MTurk(online platform),and were 57.8%male,42.2%female,aged 2068 years(mean=34.86,standard deviation(SD)=10.38).The country was not recorded,though 75%of MTurk workers are in the United States of America.(Bryant and Barnett,2019)Scientific arti

189、cleCell-based food industry WorldwideCultivatedCultured75%preference.20%preference.Study poll-49 company CEOs consulted.(Friedrich,2021)Poll reportCell-based food industryWorldwideCultivatedCulturedCell-basedCell-cultured37%preference.25%preference.18%preference.7%preference.Analysis of websites,Lin

190、kedIn profiles,and media statements of all known cultivated meat start-ups.(Byrne,2021)Survey reportCell-based food industryUnited States of AmericaCultivatedCell-basedCulturedCell-culturedPreferred terms neutral and scientifically accurate,and clear distinction from“plant-based protein”and“animal-b

191、ased meat”.Statement by AMPS Innovation member companies.(AMPS,2022)Opinion15B.Technical background issuesSector/social groupCountryTerm preferencePreference(%)or best perception/acceptanceStudy set-upReferenceConsumersUnited States of AmericaCell-basedCell-based best term for clarity,perception and

192、 acceptance.Cell-based seafood,cell-cultured seafood,cultivated seafood,and cultured seafood were compared.Between-subjects online experiments(N=3186).Study participants were recruited from a web-based consumer panel with more than 3.2 million active members enrolled in the United States.The experim

193、ent was performed during an 18-day period in 2020.A total of 8 485 randomly selected E-rewards panel members were sent an e-mail invitation to participate in the study.Demographic information(education level,year of birth,ethnicity,race,and gender)was used to produce a sample balanced to 2010 United

194、 States of America census data.(Hallman and Hallman,2020)Scientific articleConsumersUnited States of AmericaCell-basedCell-based versus cell-cultured seafood was compared.Two-group between-subjects design(N=1200).Data were collected in 2020.Study participants consisted of adult American consumers(18

195、 and older)recruited from the YouG web-based consumer panel.A sample of 1 600 participants were selected to produce the final dataset,matching a sampling frame derived from the 2018 AmericanCommunity Survey.Of these 1 600 participants,1 200 were randomly assigned to one of the two experimental condi

196、tions.A total of 591 participants viewed packages displaying the“Cell-Based Seafood,”and 609 viewed packages displaying“Cell-Cultured Seafood”.Median length of the experiment was 11.8 minutes.Consistent with census data,51.3%of the 1 200participants were female.Mean age was 47.41,SD=17.69.(Hallman a

197、nd Hallman,2021)Scientific articleConsumersUnited Kingdom of Great Britain and Northern Ireland,United States of AmericaCultivated CulturedPreferred terms for social context and product packaging,and considered more appealing.Both terms were perceived very similar.Cell-based and Cell-cultured not th

198、e preferred terms,but considered more descriptive.Both terms were also perceived as very similar.Survey and experiments-(N=2 292 for United States of America and N=2 270 for United Kingdom of Great Britain and Northern Ireland).Sampling protocol to match adult population aged 1874 years,by interlock

199、ed sex and age groups to fit within generational groups.Geographical region and race/ethnicity quotas in the United States of America,and region quotas in the United Kingdom of Great Britain and Northern Ireland were accounted for.(Szejda,2021)Scientific article16Food safety aspects of cell-based fo

200、odSector/social groupCountryTerm preferencePreference(%)or best perception/acceptanceStudy set-upReferenceConsumersPortugalN.A.Only the term“lab-grown”was included in comparison between eight different food products:red and white meat,fish and seafood,insects,legumes,tofu,seitan,and lab-grown meat.“

201、Lab-grown”meat was perceived negatively as the least natural and most processed of all meat alternatives,associated with health risks and artificiality and it was seen as the least sustainable and most expensive.Study 1(N=138)-participants 58.1%female,aged 1852 years(Median age=26.77,SD=8.89).More t

202、han half(58.9%)had a higher education degree(BSc,MSc or Doctorate),38.8%had completed secondary education and 2.3%primary education.Most participants included animal products(meat or fish)in their diets(82.8%),3.7%followed a vegetarian diet,and 6%a vegan diet;7.5%reported to have“other”dietary orien

203、tations.Study 2(N=285)-participants(68%female)aged 1866 years(M=30.21,SD=10.19).More than half(56.8%)had a higher education degree(BSc,MSc or Doctorate),41.1%completed secondary education,and 2.1%primary education.Most participants were employed(60.4%)or students(22.1%).Most participants included me

204、at or fish in their diets(59.6%),and 15.1%followed a vegetarian diet,21.1%had a vegan diet,and 4.2%reported“other”dietary orientations.On average,participants lived in predominantly urban areas.(Possidonio et al.,2021)Scientific articleConsumersEuropean Union,United Kingdom of Great Britain and Nort

205、hern Ireland,United States of AmericaCell-basedThe“clean meat”label was evaluated negatively.The authors mention that the term“clean”meat was chosen,as it tends to be associated with more positive evaluations of the product compared with other labels such as“cultured”,“in vitro”,or“lab-grown”meat.Th

206、us one of the more positive labels was used to avoid strong negative effects induced by the label alone.Images of“clean meat”-labelled dishes were more negatively evaluated than images of“regular meat”-labelled dishes by omnivores.“Clean meat”-based dishes were perceived as lower in safety and/or lo

207、wer in naturalness.Experiment 1-participants(N=270)recruited through the crowdsourcing platform Prolific and received financial compensation.Only omnivores were retained.The sample consisted of 54.9%men and 45.1%women,with a mean age of 30.42 years(SD age=10.95).Most participants were from the Europ

208、ean Union(45.3%),United Kingdom of Great Britain and Northern Ireland(27.9%),or the United States of America(11.4%).Experiment 2-participants(N=626)were recruited through opportunity sampling on social media and received no financial compensation.Only omnivores and vegans retained.Sample consisted o

209、f 21.8%men and 78.2%women,with a mean age of 36.41 years(SD age=16.41).Of this sample,455 were omnivores(74.7%women;Median age=37.47 years,SD age=17.07)and 171 were vegan(87.8%women;Median age=33.35 years;SD age=14.45).Participants were not asked for their nationality.Experiment 3-participants(N=273

210、)were recruited through the crowdsourcing platform Prolific and received financial compensation.Only omnivores were retained.The sample consisted of 56.1%men and 43.9%women,with a mean age of 28.19 years(SD age=9.36).Most participants were from the European Union(57.4%),the United Kingdom of Great B

211、ritain and Northern Ireland(18.7%),and the United States of America(6.7%).(Krings,Dhont and Hodson,2022)Scientific articleSource:Authors own elaboration.17B.Technical background issues1.4.DiscussionOverall,through the examination of both scientific and grey literature,“cell-based”,“cultivated”and“cu

212、ltured”are the three major terminologies used or preferred by consumers,industry and the authorities.These terms are also commonly used in scientific publications,but a broader range of terms can also be found in many cases in science,including the terms“in vitro”,“artificial”and“clean”that were use

213、d more frequently in the early days of the technology developments.However,industry prefers to use“cultured”,“cultivated”or cell-based”,while the media use a more diverse array of terms including,but not limited to,“cultured”,“lab-grown”,“fake”,“clean”,“cultivated”,or“cell-based”.As for consumers,on

214、ly a small number of well-designed quantitative studies in a limited number of countries have addressed the appropriateness and relevant consumer perception and acceptance of different terminologies.Moreover,these studies did not always include the same set of terms to be analyzed and compared.Despi

215、te these limitations,consumer studies indicated that the term“cultivated”was often considered the most appealing,and“cultured”,“cell-based”and“clean”to a lesser extent.These studies did not always test whether these four terms were also considered to be the clearest.It is recommended that,from the e

216、arly stages,the national competent authorities establish clear and consistent terminologies that fit in with their national and language contexts so that they can mitigate potential miscommunications on this subject in the future.If English is the language to be used,based on the data currently avai

217、lable and consumer studies,the potential candidates are“cell-based”,“cultivated”or“cultured”,whereby the specific use might be further determined by the target audience or language-specific associations of these terms.It is important to note that“cultured”and“cultivated”may be wrongly interpreted wh

218、en used for cell-based seafood products,as both terms can be perceived as being“farmed fish”(Hallman and Hallman,2020).In addition,United States federal agencies use the term“cultivated”to identify farmed shellfish.To make the terminology non-commodity-specific,“cell-based”may be useful as in cell-b

219、ased food,cell-based food products,or cell-based food production,while“cultivated”and“cultured”most likely need to be followed by a commodity name,such as meat,chicken,fish and so forth.FAO/Oded AntmanCEFET MG/UFMG/Leonardo Dutra Luz18Food safety aspects of cell-based food2.Generic understanding of

220、production processes2.1.IntroductionFood safety is an essential element to achieve food security,and regardless of how food is produced,consumers expect all food products to be safe to eat.While various steps are involved in overall food safety assurance,one of the first practical and important step

221、s to ensure food safety is the identification of potential hazards in food production chains,in order to further assess the risks associated with implementing measures to reduce or mitigate any adverse health impact.Cell-based food production encompasses the in vitro cultivation of animal cells or m

222、icrobial cells for the production of analogues of animal or plant products(e.g.animal tissues or specific animal or plant proteins and fats),with nutritional properties matching those of conventionally produced components.Technologies in this area are rapidly developing and various types of large-sc

223、ale cell-based food production are on the horizon.These technologies could possibly play an important role in supporting the increasing global demand for animal-sourced protein(Henchion et al.,2021)and provide more sustainable ways of producing animal protein in the so-called“protein transition”(Aik

224、ing and de Boer,2020).It is important to apply the same level of food safety assurance of currently commercialized food products to animal cell-based food products as well,thus a basic understanding of the cell-based food production processes is an important preliminary step prior to food safety haz

225、ard identification.To this end,the aim of this chapter is to provide an overview of the available literature for a generic understanding of the relevant technologies and production processes for animal cell-based food and the potential food safety hazards and/or relevant concerns.Animal cell-based f

226、ood production can employ a wide variety of cells to initiate the production process in order to develop cellular products such as proteins,fats or tissues from whole animal cells of poultry,cattle,pork,fish(e.g.salmon and tuna),game animals(e.g.kangaroo and quail),shrimp,crab,and lobster(Hong et al

227、.,2021;Miller,2020).The specific production processes for each cell-based food product may vary considerably.This chapter therefore primarily focuses on the processes that are common for the majority of production chains for animal cell-based food products.Therefore,this chapter can be interpreted a

228、s an overview of the main characteristics of generic production process steps and the relevant potential food safety hazards or concerns.In addition,as FAO aims to provide scientific information to the relevant competent authorities,particularly those in low-and middle-income countries,key considera

229、tions for countries with limited knowledge,resources and capacity are included.2.2.Literature synthesis results2.2.1.General processes for animal cell-based food productionManufacturing processes for animal cell-based food products may significantly vary depending on the type of cell line used(lives

230、tock,poultry,fish or seafood)and the nature of the final product(e.g.a burger,steak or nuggets).Nevertheless,a general process includes four key production stages(i)target tissue or cell selection,isolation,preparation and storage,(ii)cell proliferation and possible cell differentiation during large

231、-scale biomass production(iii)tissue or cell harvesting,and(iv)processing and formulation of food products(Ong et al.,2021).Depending on the commodity and desired final product,each of these stages can have different sub-steps for completing the specific stage.To present a high-level understanding o

232、f the production process,an overview of the common cell-based food production process has been summarized in Box 1.19B.Technical background issues2.2.2.Cell selection sourcing,storage,isolation and preparationCell sourcingProduction of cell-based food starts with the selection of the desired cell so

233、urces(livestock,poultry,game,fish,seafood)and cell types(e.g.non-differentiated stem cells,muscle precursor cells,fibroblasts or adipose-derived cells)to be used for developing the final product.Small tissue samples can be obtained by taking a biopsy from live or slaughtered animals,after which the

234、desired cell type can be isolated or reprogrammed for in vitro cultivation.It is important that,before taking biopsies,the health status of the animal is confirmed.Cells used for cell-based food production can be e.g.embryonic stem cells,which are pluripotent cells that are located within blastocyst

235、s and have an unlimited capacity for self-renewal and the ability to differentiate into any somatic cell type,induced pluripotent stem cells(iPSCs)that are derived from reprogrammed adult somatic cell and have regained their capacity to differentiate into any cell type found in the body,mesenchymal

236、stem cells or adult stem cells such as myosatellite cells(Ben-Arye and Levenberg,2019;Ong et al.,2021;Reiss,Robertson and Suzuki,2021).For some products,primary cell lines that are freshly isolated from specific organ tissues and maintained for growth in vitro might be used,which is the case for mos

237、t fish cell lines,as they are not readily available from cell culture collections(Rubio et al.,2019).Mesenchymal stem cells can readily be obtained from bone marrow or adipose tissue,while muscle precursor cells are sourced from muscle.Cell isolationTissues obtained from biopsies are either explante

238、d(a method whereby a sample adheres to a plate,which encourages cell migration to the culture surface)or further processed through mechanical and enzymatic steps that liberate the cells.One example is the isolation of muscle cells,where enzymatic digestion uses e.g.trypsin or collagenase to release

239、cells from muscle samples(see Figure 4 and Box 2).In general,the use of digestive enzymes enables the isolation of muscle stem cells from a large piece of muscle without damaging the cells,though some digestion of cell surface antigens may occur.As for all isolation methods,it also carries the risk

240、of contamination with other types of cells.Complementary methods are therefore warranted for further purification of muscle stem cells from these initial extracts.Methods that have been successfully used to this end can be cost-demanding(though negligible in the overall costs for the production proc

241、ess)and include selective plating,differential adhesion,cytochasalin-B-based detachment of myogenic cells from myoblast cultures(Choi et al.,2021),cell capture using magnetic beads with cell-specific antibodies or fluorescence-activated cell sorting(FACS)(Post et al.,2020;Rubio et al.,2019),or Perco

242、ll density gradient centrifugation.It may therefore be important to develop alternative pre-plating methods amenable to industrial-scale production(Guan et al.,2021).Figure 4 shows a general scheme for isolation of muscle satellite cells from livestock and poultry for cultured muscle tissue(CMT)prod

243、uction.Specific cell isolation procedures may apply,depending on the desired cell types;thus two examples for livestock and poultry have been provided in Box 2 and Box 3 to illustrate the different cell sourcing and isolation processes.Detailed isolation procedures also exist for other cell types,su

244、ch as adipose-derived stem cells(Lu et al.,2014;Sampaio et al.,2015),mesenchymal stem cells(Feyen et al.,2016;Vassiliev and Nottle,2013)or fibroblasts(Park et al.,2022).For cells derived from fish or seafood,protocols are currently not publicly available.Box 1.A generic overview of the cell-based fo

245、od production processSource:FAO.2022.Thinking about the future of food safety-A foresight report.Rome.www.fao.org/3/cb8667en/cb8667en.pdfHarvestinga.Cell proliferationb.Cell differentiationProductionFood processing and formulationa.Cell sourcingb.Cell isolation c.Cell preparationd.Cell storageCell s

246、election123420Food safety aspects of cell-based foodBox 2.Cell sourcing and isolation of chicken and bovine muscle satellite cellsFigure 4.Flow diagram for the sourcing and isolation of cultured muscle satellite cellsSource:Joo,S.T.,Choi,J.S.,Hur,S.J.,Kim,G.D.,Kim,C.J.,Lee,E.Y.,Bakhsh,A.et al.2022.A

247、 Comparative Study on the Taste Characteristics of Satellite Cell Cultured Meat Derived from Chicken and Cattle Muscles.Food Science of Animal Resources,42(1):175185.10.5851/kosfa.2021.e72Source:Example protocol from Joo Seon-Tea et al.A Comparative Study on the Taste Characteristics of Satellite Ce

248、ll Cultured Meat Derived from Chicken and Cattle Muscles.Food Sci Anim Resour.2022;42(1):175185.https:/doi.org/10.5851/kosfa.2021.e72.The flow diagram for the cultured muscle satellite cell isolation is shown in Figure 4.Cell sourcingSkeletal muscle samples were derived from 4 to 6week-old broiler c

249、hickens or 24 to 27month-old cattle steers.Animals were euthanized following approved human methods.Several small pieces of the pectoralis major muscle from chickens and the biceps femoris muscle from cattle were removed from the carcasses immediately after slaughter.The collected muscle pieces were

250、 sterilized with 70 percent ethanol,placed in Hanks Balanced Salt Solution containing 3 percent antibiotic-antimycotic(containing penicillin,streptomycin,and amphotericin B),and transported to the cell culture laboratory.On a clean bench,muscle pieces were washed once with 70 percent ethanol and pla

251、ced in a Petri dish.Each muscle tissue was rinsed 35 times with a 4-fold volume of cold phosphate-buffered saline(PBS),followed by the removal of visible adipose and connective tissue.Muscle tissue was cut into very small pieces using scissors after spraying with 0.25 percent trypsin-EDTA.Muscle tis

252、sue was minced,and 4 grams of minced muscle were transferred and 5 times the volume of 0.25 percent trypsin-EDTA was added.Muscle tissue was transferred to a tube and incubated in a water bath at 37 C for 30 minutes while gently inverting every 10 minutes.The digested muscle tissue was collected by

253、low-speed centrifugation,and after removing supernatant,10 mL of proliferation medium(PM)was added to the pellet and serially filtered through 100,70,and 40 m strainers.The filtered cell suspension was centrifuged to collect the cell pellet.Isolation of muscle satellite cellsMuscle satellite cells(M

254、SCs)were separated by the pre-plating method utilizing the difference between the cell adhesion rate and the growth rate.The cell pellet obtained after sourcing was re-suspended in PM and plated onto a cell culture dish coated with 0.2 percent gelatine.The cell culture dish was incubated at 37 C in

255、the presence of 5 percent CO2 for 1 hour(pre-plating 1,PP1).Fibroblasts quickly adhered to the bottom of the cell culture flask,while MSCs remained in the supernatant.The supernatant containing MSCs was collected in a centrifuge tube and centrifuged for 10 minutes at 500g.The MSC pellet was re-suspe

256、nded with PM,plated onto a cell culture dish and incubated at 37 C with 5 percent CO2 for 2 hours(pre-plating 2,PP2).The supernatant and non-attached cell suspensions were recovered,centrifuged again,and only the cell pellets were cultured for 24 hours(PP3).This pre-plating process was repeated up t

257、o PP5 to isolate muscle satellite cells that are as pure as possible in the final PP5 fraction.Cells in all steps from PP1 to PP5 were cultured in PM.Muscle BiopsyCMT HarvestCultured Satellite CellsNon adhering Cellsat 37OC,5%Co2incubatorRepeatPre-platingProcessSatellite CellsTrypsinized CellsIsolat

258、ed Muscle CellsPP1PP2PP3PP4PP5Seeding Muscle CellsSCsNonSCsNonSCsNonSCsNonSCsNon21B.Technical background issuesBox 3.Cell sourcing and fluorescence-activated cell sorting to enrich bovine muscle satellite cellsSource:Example protocol from Joo Seon-Tea et al.A Comparative Study on the Taste Character

259、istics of Satellite Cell Cultured Meat Derived from Chicken and Cattle Muscles.Food Sci Anim Resour.2022;42(1):175185.https:/doi.org/10.5851/kosfa.2021.e72.Preparation of robust production cell linesMany cell lines currently used for cell-based food production are not genetically modified(Hadi and B

260、rightwell,2021;Post et al.,2020;Zhang et al.,2020).These cell lines therefore may not necessarily possess the exact physiological or genetic characteristics desired for optimized growth and prolonged cultivation in large-scale bioreactors,such as a limited number of cell divisions,or low resistance

261、to shear stress and sub-optimal oxygenation.Developing so-called immortalized cell lines is one of the approaches that could lead to cells with an extended proliferation capacity.This can be achieved by,for example,targeting the telomerase activity through genetic modification and thereby preventing

262、 senescence(Soice and Johnston,2021),but can also be obtained through non-GM methods whereby primary cells are serially sub-cultured until clonal populations of immortalized cells arise from spontaneous genetic variation over time.Cell storageThe cell type used for cell-based food production has a l

263、arge impact on the parameters used in the production process,as each cell type has its particular requirements that might be beneficial for or detrimental to efficient production.For consistent production of cell-based food products,it is also vital to use stable cell lines that maintain the same ge

264、netic and physiological characteristics and exhibit uniform/consistent production performance over time.This necessitates storage of cells after isolation from animals(primary cells)or storage of cells from specific stages of the production process.To this end,cells are stored as frozen aliquots in

265、master cell banks after the addition of cryopreservation fluid(Ong et al.,2021).Individual vials of the master cell bank can then be used to generate large“working”cell banks from which individual vials are used to initiate cultures for each production run or period of experimentation(Healy et al.,2

266、011)Prior to cryopreservation,cell lines are screened for the presence of microbial contaminants,and may be verified for the species cell line identity to ensure that cell cultures are not contaminated during the seed phase of biomass production(Andriolo et al.,2021).Cell banks for animal cell lines

267、 have not been developed yet for many animal species,in particular for fish,and establishing such cell banks is therefore an important factor for future large-scale cell-based food production(Ramani et al.,2021).Alternative isolation protocol for bovine muscle satellite cells(not related to Figure 4

268、).Cell sourcingBovine satellite cells were derived from fresh(within 30minutes of euthanasia)muscle samples obtained from 1 to 2year-old male cattle.Freshly harvested bovine muscle was immediately transferred to the lab on ice and washed with 75 percent ethanol for 1minute,followed by washing 2 time

269、s in phosphate-buffered saline(PBS).Tissues were mechanically dissected and dissociated with collagenase II(CLS-2,0.2 percent)in Dulbeccos Modified Eagle Medium(DMEM)supplemented with 1 percent penicillin-streptomycin at 37 C for 1.5hours.The mixture was mixed by vortexing or triturated with a pipet

270、te every 10minutes.After digestion,20 percent fetal bovine serum(FBS)in DMEM was added and mixed well with a pipette.Muscle fragments were centrifuged at 80 g for 3minutes and the supernatant was collected as mononuclear cell suspension.The precipitated debris was again triturated with a 20-gauge ne

271、edle in PBS and centrifuged at 80 g for 3minutes.The supernatant was collected and mixed with previous mononuclear cell suspensions.After centrifugation at 1,000 g for 5minutes,the cells were washed twice with PBS followed by DMEM with 20 percent FBS.After that,the cells were filtered through a 100

272、m cell strainer followed by a 40 m cell strainer.The cells were then centrifuged at 1,000g for 5minutes at 4 C and incubated with the erythrocyte lysis buffer(ACK)buffer for 5minutes on ice.The cells were washed twice with PBS and cell pellet was reconstituted with FACS buffer(1 percent bovine serum

273、 albumin BSA in PBS)or frozen in FBS supplement with 10 percent dimethyl sulphoxide(DMSO)until further use.Fluorescence-activated cell sortingFrozen cells were recovered in a 37 C water bath and washed with PBS twice before further processing.The cells were resuspended in FACS buffer and stained wit

274、h selected APC anti-human CD29 Antibody(1:10),PE-CyTM7 anti-human CD56(1:10),FITC anti-sheep CD31(1:10),FITC anti-sheep CD45(1:10)for 3045minutes on ice.After antibody incubation,the cells were washed twice with cold PBS and reconstituted in F-10 with 20 percent FBS.The viable CD31CD45CD56+CD29+cell

275、s(bovine satellite cells)were isolated by cell sorting.Cell sorting was performed with a BD FACSAria cell sorter using 405 nm,488 nm and 640 nm lasers.Unstained cells were routinely used to define FACS gating parameters.22Food safety aspects of cell-based food2.2.3.Production-cell proliferation and

276、differentiation and large-scale productionCell proliferationFor large-scale production,isolated cells need to be proliferated on a large scale and to a high cell density and in many cases differentiated into specific cell types,which will involve several scale-up steps from the seed stage to full sc

277、ale production in large bioreactors(1 00010 000 L volumes or higher).The cell source and type used have an important impact on the proliferation and scale-up requirements.Cell types such as skeletal muscle cells,fibroblasts,satellite cells,and iPSCs are in general being favoured,on their own or in c

278、ombination with adipogenic stem cells,and each have their particular benefits and requirements for proliferation factors such as oxygenation,pH and temperature(Swartz,2021).While most mammalian cells typically need to be proliferated at a narrow range of temperatures from 36.5 to 37.5 C(Choi et al.,

279、2021),fish cells can be grown at much lower temperatures in a wider range between 15 and 30 C.Moreover,fish cells are expected to tolerate lower oxygen levels compared with mammalian cells and to be more adaptable to a wider pH range,based on the physiology of fish and aquatic invertebrates(Fernande

280、z et al.,1993;Rubio et al.,2019).The use of a fresh/non-exhausted medium is also considered important,as it was found that medium exchange was critical in maintaining good cell growth(Hanga et al.,2020).For creating cell-based fat,mesenchymal stem cells isolated from fat or bone marrow may be an opt

281、ion as these multipotent stem cells have the capability to develop into fat cells(adipocytes)(Fish et al.,2020).iPSCs,for example,can still develop into myotubes,a propensity also exploited in research into human tissue engineering for medical purposes.In addition,adipose tissue-derived stem cells(A

282、DSCs)can also be triggered to develop into various types of cells,such as bone-,muscle-,and fat-cells(Balasubramanian et al.,2021).The tendency of the isolated cells to proliferate and differentiate may differ depending on the tissues from which they are sourced,as shown for muscle satellite cells(C

283、hoi et al.,2021).Reiss et al.(2021)point out that pluripotent stem cells may be costlier to obtain and to grow,and that more time may be needed to have them differentiate into cells with the desirable phenotype.They also note that primary adult stem cells,for example,may be easier to obtain from e.g

284、.biopsies of animal muscle tissue.For seafood,the fact that fish muscle consists of three different types of muscle(red,white,pink)opens up possibilities when designing cultivation systems(Rubio et al.,2019).Co-cultures of different types of cells,such as muscle and fat cells,may not only help to mi

285、mic the structure and characteristics of meat,poultry or fish closely(e.g.marbling)but different types of cells may also secrete factors and matrices that induce other cell types to proliferate and differentiate(Balasubramanian et al.,2021).Various authors,for example,used a technique to layer alter

286、nating sheets of muscle and fat cells on top of each other(Pandurangan and Kim,2015;Shahin-Shamsabadi and Selvaganapathy,2021).Co-cultures may also be used for the creation of“self-organizing”methods of cell-based food production,as an alternative to methods employing scaffolds.One challenge in this

287、 regard is the transport of nutrients and oxygen throughout the mixed-cell-type tissues being formed,which may be done with the aid of artificial blood-circulation-imitating concepts(Bhat et al.,2015).Cell differentiationAfter cell proliferation,cells need to be induced to differentiate into cell ty

288、pes with the desired characteristics for the cell-based food product.Cell differentiation can be stimulated e.g.by changing to a culture medium with an altered composition of signalling molecules,environmental conditions or by changing scaffolds.Medium composition for cell differentiation can be ach

289、ieved by addition or removal of growth factors,vitamins,amino acids or trace minerals.The media used are complex,and besides the proper amounts of,e.g.lipids,amino acids,and vitamins,the addition of essential growth factors is also required to stimulate the proliferation and differentiation of those

290、 cell types that do not produce such factors themselves in culture(Arshad et al.,2017).The chemical and biochemical compounds that could act as hormones or growth factors for this purpose range widely,including for example steroids,signalling molecules,insulin and insulin-like growth factors(IGFs),f

291、ibroblast growth factors(FGFs),transforming growth factor beta-2(TGF-s),and interleukins(Choi et al.,2021).As cell differentiation is never 100 percent efficient,further purification might still be required of the target cell type.Whilst plasma and serum from animals,such as foetal calf serum,may be

292、 added for cell proliferation and differentiation(up to 20 percent),23B.Technical background issuesthis might not align with the strategy directed towards animal-slaughter-free production.Alternatives that can be used include recombinant growth factors,the recycling of growth factors used by culture

293、d cells themselves and adapting cell lines to grow in serum-free media or in alternative media containing plant or microbial components(ONeill et al.,2021).Besides or as an alternative to growth factors,mechanical stimulation such as contraction,fluid flow,or magnetic particles may be used to stimul

294、ate muscle cells in particular.Process design for large-scale production Bioreactor configuration and process design takes into account factors such as oxygenation,shear stress,pH through carbon dioxide concentration and temperatures that are optimal for proliferation of the selected mammalian,fish

295、or seafood cell line(Allan et al.,2019;Arshad et al.,2017).Fish and seafood cell lines might be more amenable towards temperature,oxygen and pH ranges than other animal cell lines and might therefore be proliferated using a simpler(and cheaper)reactor design.In contrast,avian cell lines might requir

296、e optimal growth temperatures higher than 37 C.Different types of bioreactors might be used for cell-based food production,such as stirred tank bioreactors and rocking bed bioreactors,but also those using fluidized or packed beds,or hollow fibres(Allen,2013;Choi and Hyun-Jae,2019;Djisalov et al.,202

297、1;Hanga et al.,2020).It is important that the reactor configuration that is used for a specific cell line is scalable without negative effects on the cell proliferation and differentiation capacities that could,for example,be introduced by increased shear stress or reduced oxygenation.The stirred ta

298、nk bioreactor is currently preferred for the large-scale and cost-effective growth of animal cells for food production and elsewhere in the biopharmaceutical sector(Eibl et al.,2021).In all reactor set ups it is important to monitor the process carefully,such as for pH(controlled via carbon dioxide)

299、,dissolved oxygen,temperature,nutrients(e.g.ammonia,glutamate,glucose),biomass,cell density and proliferation,as well as cell image analysis(Djisalov et al.,2021).Medium exchange is expected to be critical to maintaining good cell growth,as was demonstrated in a lab-scale stirred flask model for gro

300、wing fat cells(Hanga et al.,2020),and is therefore a key part of the process design.Cells used for cell-based food production in many cases might need to utilize an adherent surface for proliferation(Ong et al.,2021).These surfaces can be microcarriers(MCs,small beads)or more robust scaffolds that a

301、llow the formation of more complex cell structures such as sheets.MCs are often composed of materials such as gelatine,dextran,collagen or polystyrene(Bodiou et al.,2020).Scaffold materials might include natural components such as polysaccharides(cellulose,alginate,chitosan,decellularized plant mate

302、rials),proteins such as gelatine and collagen(from animal or non-animal sources),textured soy protein or synthetic scaffold materials composed of polymers such as polyethylene glycol(PEG),polylactide(PLA)or polyacrylamide(Ben-Arye and Levenberg,2019;Ng and Kurisawa,2021;Seah et al.,2022).Composites

303、of natural and synthetic materials may also be used.In all cases,microcarrier or scaffold materials are preferably biocompatible,biodegradable,edible and safe to use and,in the case of scaffolds,provide the final product with structure and texture(Bomkamp et al.,2022).Matrices may be structured such

304、 that the cells are stimulated to grow into fibre-like structures.Acevedo and co-workers(Acevedo et al.,2018;Orellana et al.,2020)employed an edible film with laser-cut microchannels and observed that cells after seeding did start to form muscle-forming(myogenic)structures.Eibl et al.(2021)note that

305、 the choice of MCs and media to be used in stirred bioreactors are mutually dependent for optimal results and affect the scalability of the process(Bodiou et al.,2020;Eibl et al.,2021).For example,when using an air-lift reactor design,the air bubble size needs to be chosen carefully as the use of MC

306、s requires a smaller bubble size in order to prevent cells from being dislodged from the carrier and harmed(Li et al.,2020).Biopolymers used as microcarriers or scaffolds can also serve a function as an additional fibrous substance in the final product,or to contain molecules that emulate the action

307、 of hormones(Ng and Kurisawa,2021).Park et al.(2021),for example,describe a porous multilayer film containing different polysaccharides with C-phycocyanin.The latter is an algal protein with proliferation-inducing properties and hence a possible substitute for foetal bovine serum as a media additive

308、.Results showed that muscle cells grown on this substrate displayed increased proliferation(Park et al.,2021).Alternatively,they can also be selected or designed to be biodegradable,with their degradation possibly leading to the release of flavour or nutritional compounds.Edible biopolymers are gene

309、rally not cell-adhesive and modifications may be needed for this purpose(Ng and Kurisawa,2021).24Food safety aspects of cell-based food2.2.4.HarvestingOnce cells have reached their maximum density during proliferation and have differentiated into the desired cell type,they are harvested in a way tha

310、t maintains cell/tissue integrity and avoids microbial contamination.Cells can be collected using sedimentation,centrifugation or filtration techniques,and when cells were grown on scaffolds/MCs that are not edible or biodegradable,they must first be dissociated from the scaffold before further proc

311、essing.Dissociation can be done using enzymatic,chemical or mechanical methods(Allan et al.,2019;Bodiou et al.,2020;Rodrigues et al.,2019).Depending on the production system used,only part of the cells might be harvested,after which fresh(or recycled)media can be added to the remaining cells for fur

312、ther cultivation.The implementation of automated cell harvesting systems instead of manual harvesting could be a development that can greatly reduce the risk of contamination during the harvesting stage(Specht et al.,2018;Tan et al.,2017).The literature review did not find any technical articles des

313、cribing specific harvesting processes for cell-based food products.However,Bodiou et al.(2020)discuss three cell proliferation and harvesting scenarios as described in Box 4.Box 4.Cell harvesting scenarios in cell-based food productionScenario 1:Temporary microcarriers(MCs)for satellite cells prolif

314、erationMCs that are used as temporary substrates for the proliferation of satellite cells(SCs)need to be removed before further processing,which requires(1)a high detachment(dissociation)yield and(2)easy separation from the cells.Dissociation of SCs from MCs can be based on(i)chemical,(ii)mechanical

315、 or(iii)thermal principles to detach cell from MCs while maintaining cell viability,proliferation and differentiation capacity.(i)Chemical detachment consists of the enzymatic and non-enzymatic dissociation of cells.The enzymatic detachment is based on proteases in combination with chelating agents

316、for Ca2+to reduce cell binding.Non-enzymatic dissociation agents,such as dextran-sulphate,N-acetyl-L-cysteine and dithiothreitol,mimic enzyme activity that cleaves or degrades MCs coating;(ii)mechanical methods to detach cells from MCs include pipetting,high agitation and vibration and can be used i

317、n combination with enzymes and chelators like trypsin-EDTA;and(iii)thermal response materials used for cell detachment from MCs can undergo a phase transition and/or morphological modification in response to a variation of temperature,leading to cell detachment.The advantages of mechanical and therm

318、al techniques over chemical techniques are that they do not require the use of dissociation agents and do not have washing steps before and after dissociation which leads to longer processing times and extensive manipulation of the culture.Separation systems for separating detached cells from MCs ar

319、e based on one of the following four principles:filtration,centrifugation,inertia and magnetism.The most common filtration methods use dead-end filtration systems,(alternate)tangential flow filtration or continuous centrifugal separators.Magnetism can be used as a separation method when magnetic par

320、ticles(made from iron,nickel,cobalt or their alloys)are incorporated into the MCs core.After dissociation of the cells from the surface of the MCs,the introduction of a magnetic field separates the MCs from the cells.Scenario 2:Non-edible,degradable microcarriersMCs that serve as a temporary substra

321、te for cell proliferation are separated at the end of the process through MC degradation instead of dissociation in order to obtain the cells.Diverse natural or synthetic degradable materials have been used for MC production,including polystyrene,cellulose,collagen,gelatine,alginate,chitosan,poly(L-

322、lactic-co-glycolic acid)(PLGA),polylactide(PLA),or poly(+-caprolactone)(PCL).These polymers can be degraded according to five principles:thermal,chemical,mechanical,photo and biological degradation.The degradation of MCs needs to be controlled in order to be robust,fast and prevent damage or interac

323、tion of the SCs with the degradation products.In addition,premature degradation of MCs should be prevented during cell proliferation.Up to date,only one MC has been commercialized and developed with the purpose of being totally and rapidly biodegradable for cell harvesting.It is made of cross-linked

324、 polygalacturonic acid(PGA)and can be easily dissolved within 1020 minutes using an EDTA solution in combination with pectinase,which that digests the polymer.Other polymers including dextran,cellulose,collagen,pectin or gelatine could be enzymatically digested in a similar way.Thermal and photo deg

325、radation are likely to be less suited for cell culture,as the high temperatures required to thermally degrade polymers,or the ultraviolet radiation needed to induce photo-degradation are known to cause protein and DNA denaturation and damage.Mechanical forces such as stirring speeds,shaking or fluid

326、ization can also be used in combination with chemical degradation(enzymatic or non-enzymatic)to facilitate/accelerate the degradation process and reduce the concentration of enzymes.Finally,slowly degrading materials compatible with SC culture could also be performed.The use of degradable MCs elimin

327、ates the need for separation,thereby simplifying the process and resulting in increased cell recovery.The resulting cell suspension can be washed and directly used for downstream processing.25B.Technical background issues2.2.5.Food processing and formulationHarvested cells/tissues are further proces

328、sed and formulated into a specific type of cell-based food product for commercialization.In most cases,this involves the addition of other food ingredients for flavour and in some cases,it may involve the addition of preservatives.Different cell types may also be combined(e.g.muscle and fat cells)to

329、 replicate the structure and texture of conventional meat or meat cells/tissue combined with plant-based components to produce blended products.Common techniques to achieve structure and texture in cell-based food products include shear-cell technology,extrusion or 3D-printing,depending on the desir

330、ed final product type(Handral et al.,2020).In addition,biopolymers can be used to impart structure to the cell-based meat structure.Ideally,such biopolymers are already used during the cultivation stage as a cost-effective means for triggering myotube formation,for example,in the final stage of cell

331、 cultivation within a scaffold in a fixed bed reactor,following passages through stirred and suspension reactors for the multiplication of cells.Alginate(besides many other polysaccharides such as carrageenan,pectin,gellan,xanthan,etc.)appears to be an attractive candidate for this purpose as this b

332、iopolymer can accommodate smaller parts of cultured tissue into a kind of reconstituted meat product.Its gelatinization can be induced at low temperatures by the addition or release of calcium ions.2.2.6.Potential food safety hazards and concernsOverview Cell-based food production involves various p

333、rocesses,techniques and steps and in some cases,novel inputs,meaning added steps,materials,technologies or techniques that have not commonly been used in conventional food production(e.g.scaffolds or modified cell properties)can be used.To be able to properly identify potential hazards,a generic mapping of potential hazards and concerns is simplified and presented in Table 3.Scenario 3:Edible micr