SINGLE-CELL PROTEIN Safety for Animal and Human Feeding Proceedings of the Protein-Calorie Advisory Group of the United Nations System Symposium "INVESTIGATIONS ON SINGLE-CELL PROTEIN'7 held at the Istituto di Ricerche Farmacologiche 'Mario Negri' Milan, Italy, March 31 - April 1,1977 Edited by SILVIO GARATTINI Istituto di Ricerche Farmacologiche 'Mario Negri' SILVIO PAGLIALUNGA Istituto di Ricerche Farmacologiche 'Mario Negri' and NEVIN S. SCRIMSHAW Massachusetts Institute of Technology PERGAMON PRESS OXFORD NEW YORK TORONTO SYDNEY PARIS FRANKFURT U K. Pergamon Press Ltd., Headington Hill Hall, Oxford OX3 OBW, England U.S.A. Pergamon Press Inc., Maxwell House, Fairview Park, Elmsford, New York 10523, U.S.A. CANADA Pergamon of Canada, Suite 104, 150 Consumers Road, Willowdale, Ontario M2 J1P9, Canada AUSTRALIA Pergamon Press (Aust.) Pty. Ltd., P.O. Box 544, Potts Point, N.S.W. 2011, Australia FRANCE Pergamon Press SARL, 24 rue des Ecoles, 75240 Paris, Cedex 05, France FEDERAL REPUBLIC Pergamon Press GmbH, 6242 Kronberg-Taunus, OF GERMANY Pferdstrasse 1, Federal Republic of Germany Copyright© 1979 Pergamon Press Ltd. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic tape, mechanical, photocopying, recording or otherwise, without permission in writing from the publishers. First edition 1979 British Library Cataloguing in Publication Data PAG International Symposium on Investigations on Single-Cell Protein, Milan, 1977 Single-cell protein. 1. Proteins in human nutrition - Safety measures - Congresses 2. Proteins in animal nutrition - Safety measures - Congresses I. Title II. Garattini, Silvio III. Paglialunga, Silvio IV. Scrimshaw, Nevin Stewart 641. Γ2 TP453.P7 78-40993 ISBN 0-08-023765-7 ISBN 0-08-023764-9 Pbk In order to make this volume available as economically and as rapidly as possible the authors' typescripts have been reproduced in their original forms. This method un- fortunately has its typographical limitations but it is hoped that they in no way distract the reader. Printed and bound at William Clowes & Sons Limited Beccles and London Foreword The Protein-Calorie Advisory Group (PAG) of the United Nations System was established in 1955 by the World Health Organization at a time when the principal international organizations concerned with nutrition — WHO, FAO, UNICEF — were just beginning to realize the extent and magnitude of the problem of childhood malnutrition in devel- oping countries. In recent years, the PAG has been sponsored by FAO, WHO, UNICEF, the World Bank, and the U.N. itself. The issue of safety and nutritional value of all potential sources of foods has been an important concern of the PAG. The Group, from the very beginning, has played an active role in promoting research and devel- opment of various novel protein sources. As part of this activity, it has been moni- toring research and development on single-cell protein (SCP) products by sponsoring international symposia in collaboration with other interested groups, and through holding a series of Working Group Meetings. The First International Conference on Single-Cell Protein was held in 1967 at the Massachusetts Institute of Technology (1). The first and second PAG Working Group Meetings on this subject were held in 1970 and 1971 in Marseilles and Moscow, res- pectively. The PAG actively collaborated with MIT on a Second International Con- ference on Single-Cell Protein in 1973 (2), and conducted its third Working Group Meeting at MIT as part of the Conference. These Working Group Meetings have helped the PAG to issue several recommendations, statements, and guidelines on different aspects of SCP production, evaluation, safety testing, and nutritive value of SCP products, and to identify additional areas for further study and research. The rapid progress in research and development in this area led the PAG to hold a fourth Working Group Meeting in New York in 1973, and a fifth in Geneva in 1975. At this Meeting, it was recommended that the Group hold an international symposium on SCP for animal feeding, so that new information and experience could serve as a basis for review and revision of the earlier PAG recommendations on the subject. An International Symposium on Single-Cell Proteins for Animal Feeding and the sixth Working Group Meeting were held in Brussels, Belgium in March of 1976. The pro- ceedings of this Symposium have been published in PAG Bulletin Vol. VI, Nos. 2-4, 1976. The report of the various PAG Working Group Meetings on SCP and the PAG statements and guidelines on the subject have been published in the various issues of the PAG Bulletin as well as in two separate PAG documents (parts I and II) entitled "Documents on Single-Cell Proteins Issued by PAG." vii viii Foreword The discussions at the PAG Brussels Symposium brought out the need for a further detailed review of all scientific data from clinical and metabolic studies using SCP grown on alkanes. The present publication represents this effort and is based on the PAG International Symposium on Investigations on Single-Cell Protein held in Milan, Italy from 31 March to 1 April, 1977. The meeting was made possible by the cooperation of the Institute of Pharmacological Research ?Mario Negri1, whose scientists have made important research contributions in this subject area. The PAG was helped in the planning of the Conference by Professor N. S. Scrimshaw, who chaired the seventh Working Group Meeting held immediately after the Conference, and Professor S. Garattini, Director of the Institute. The report of the Working Group Meeting held on 2 April, 1977 is included in Appendix I. The PAG ceased its activities at the end of 1977. Its coordinating functions within the U.N. System have been assumed by a newly established Sub-Committee on Nutrition of the U.N. Agency Committee on Coordination (ACC-SCN), and its advisory functions by an Advisory Group on Nutrition (AGN). Its functions relating to single-cell protein and other new protein sources have been taken up by the World Hunger Programme of the United Nations University (UNU-WHP). P. S. Venkatachalam, M.D. Senior Programme Officer, World Hunger Programme, United Nations University (Former Deputy Director, PAG Secretariat) 1. Mateles, R. I. and Tannenbaum, S. R. (eds.) (1968) Single-Cell Protein, MIT Press, Cambridge, Massachusetts. 2. Tannenbaum, S. R. and Wang, D. I. C. (eds.) (1975) Single-Cell Protein II, MIT Press, Cambridge, Massachusetts. Participants D. G. AHEARN, Department of Biology, Georgia State University, Atlanta, Georgia, U.S.A. L. AJELLO, Center for Disease Control, Department of Health, Education and Welfare, Public Health Service, Atlanta, Georgia, U.S.A. H. E. AMOS, The British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey SM5 4DS, England K. ANANTHARAMAN, Laboratoire Biologique, Produits Nestle S.A., Orge, Switzerland S. ARACHI, Mitsui Italia, Piazza Liberty 2, Milan, Italy W. BALLONI, Istituto di Microbiologia Agraria e Tecnica, Universita, Piazzale delle Cascine 27, Florence, Italy I. BARTOSEK, Istituto di Ricerche Farmacologiche ?Mario Negri1, Via Eritrea 62, Milan, Italy R. BATTINI, Snam Progetti, S. Donato Milanese, Milan, Italy P. BELLEZZA, Italproteine S.p.A., Galleria Passarella 2, Milan, Italy D. BELTRAME, TPL, Via della Magliana 821, Rome, Italy E. BERNASCONI, Montedison, Via dei Gracchi 35, Milan, Italy F. BERTONI, Liquichimica Biosintesi, Via Goldoni 10, Milan, Italy M. BIANCHEDI, Istituto Malattie Infettive Vet., Universita, Bologna, Italy L. BIANCHI, Liquigas, Via Roncaglia 12, Milan, Italy R. D. N. BIRTLEY, ICI Ltd., Alderley Park, Macclesfield, Cheshire SK10 4TJ, England A. BIZZI, Istituto di Ricerche Farmacologiche fMario Negri1, Via Eritrea 62, Milan, Italy H. BLUMENTHAL, Division of Toxicology, Bureau of Foods, Food and Drug Administration, Washington, D.C. 20204, U.S.A. B. BONGIOVANNI, Centro Ricerche Liquichimica, Robassomero, Torino, Italy G. F. B0RIES, Institut National de la Recherche Agronomique, 180 Chemin de Tourne- feuille, 31300 Toulouse, France ix X Participants D. D. BRYSON, ICI Ltd., Agricultural Division, Billingham, Cleveland TS23 ILE, England A. CANALE, Istituto di Zootecnica, Via Nizza 52, Torino, Italy C. CANTARELLI, C.N.R., Istituto Tecnologie Alimentari, Via Celoria 2, Milan, Italy L. CARBONEL, TPL, Caracas, Venezuela G. CAROSIO, CTIP, S.p.A., Piazzale Douhet 31, Rome, Italy J. T. CARTER, BP Protein, Britannic House, Moor Lane, London EC2Y 9BU, England S. CASAGRANDE, Istituto Farmacologia, Universita, Via Banchi di Sotto 55, Siena, Italy V. CAVAZZONI, Cattedra Microbiologia Industriale, Via Celoria 2, Milan, Italy L. CHIERICI, Liquifarm, Via Roncaglia 12, Milan, Italy J.J COONEY, Chesapeake Biological Institute, University of Maryland, Solomons, Maryland 20688, U.S.A. P. CUCCHETTI, Credito Industriale Sardo, Via XX Settembre 27, Milan, Italy G.M. CURTO, Istituto Zootecnica Generale, Universita Studi, Via Celoria 2, Milan, Italy G. D'AGNOLO, Istituto Superiore Sanita, Viale Regina Elena 299, Rome, Italy L. DE CESARI, Liquichimica Biosintesi, Via Goldoni 10, Milan, Italy C. DELISE, Montedison, Via dei Gracchi 35, Milan, Italy 0. DE PELET, Apria, 35 Rue du General Fog, 75008, Paris, France G. DONELLI, Istituto Superiore Sanita, Viale Regina Elena 299, Rome, Italy E. EASTHAM, Pédiatrie Gastrointestinal Unit, Massachusetts General Hospital, Fruit St., Boston, Massachusetts 02114, U.S.A. P. FASELLA, Laboratori Ricerche di Base, Snamprogetti, S.p.A., Monterotondo, Rome, Italy C. FERRARI0, TPL, Via della Magliana 821, Rome, Italy G. FLORENZANO, Istituto di Microbiologia Agraria, Universita di Firenze, P. le Cas- éine 27, Florence, Italy F. FORMENTON, Liquichimica Biosintesi, Via Goldoni 10, Milan, Italy S. GARATTINI, Istituto di Ricerche Farmacologiche 'Mario Negri1, Via Eritrea 62, Milan, Italy G. GARGANI, Istituto di Microbiologia, Universita, Viale Morgani 48, Florence, Italy L. GASPARINI, Italproteine S.p.A., Plant, Sarroch, Cagliari, Italy E. GATUMEL, BP Proteins, Britannic House, Moor Lane, London, EC2, England F. GIORGI, Anic S.p.A., Piazza Boldrini 1, S. Dontao Milanese, Milan, Italy A. GUAITANI, Istituto di Ricerche Farmacologiche 'Mario Negri', Via Eritrea 62, Milan, Italy G. GUIROLA, Department of Chemistry, UMIST, P.O. Box 88, Sackville St., Manchester M60 1QD, England S. GULBRANDSEN, Norsa-Hydro AS, P.O. Box 2594, Solli - Oslo 2, Norway A. HEATH, McGraw-Hill Publishing Co., Via Baracchini 1, Milan, Italy Participants xi R. HODQES, BP Proteins Ltd., Britannic House, Moor Lane, London EC2Y 9BU, England D. HOLZSCHU, Department of Biology, Georgia State University, Atlanta, Georgia 30333, U.S.A. J.C. HOOGERHEIDE, Laan van Clingendael 129, Den Haag, The Netherlands G.H. HUDSON, Commission of the European Communities, Directorate General for Agri- culture, 200 Rue de la Loi, 1049 Brussels, Belgium D.A. JONAS, Ministry of Agriculture, Fisheries, and Food, Great Westminster House, Horseferry Road, London WC1, England K. KATOH, Division of Applied Microbiology, National Food Research Institute, Ministry of Agriculture and Forestry, 4-12 Shiohama, Koto-ku, Tokyo, Japan K. KOMAGATA, Institute of Applied Microbiology, University Bunkyo-ku, Tokyo, Japan M. KR0N, Centro Ricerche Liquichimica, Robassomero, Rome, Italy M. LENER, Laboratori Ricerche di Base Snamprogetti S.p.A., Monterotondo, Rome, Italy A. LEONARDI, Istituto di Ricerche Farmacologiche 'Mario Negri', Via Eritrea 62, Milan, Italy A. LEPIDI, Istituto Microbiologia Agraria, Via del Borghetto 80, Pisa, Italy J.D. LEVI, BP Proteins Ltd., P.O. Box 33, Grangemouth, U.K. J. LIBERATORI, Centro Studio Alimentazione Animale del C.N.R., Istituto Chimico Universita, Corso d'Azeglio 48, Torino, Italy J.-P. LIOT, Delegation Generale a la Recherche Scientifique et Technique, 35 Rue St. Dominique, 75007, Paris, France G. LONGOBARDI, CTIP S.p.A., Piazzale Douhet 31, Rome, Italy A. MACRIf, Istituto Superiore di Sanita, Viale Regina Elena 299, Rome, Italy G. MALACART, Italproteine S.p.A., Galleria Passarella 2, Milan, Italy E.A. MALICK, Provesta Corporation, Subsidiary of Phillips Petroleum Co., Bartlesville, Oklahoma, U.S.A. P.L. MANACHINI, Cattedra Microbiologia Industriale, Universita, Via Celoria 2, Milan, Italy F. MARINI, Istituto Biochimico Italiano, Via Lorenzini 2, Milan, Italy A. MARTINI, Istituto Microbiologia Agraria, Universita di Perugia, Borgo XX Giugno, Perugia, Italy R. MATERASSI, Istituto di Microbiologia Agraria e Tecnica, Universita, Piazzale delle Cascine 27, Florence, Italy V. MAZZARACCHIO, Via di Filomarino 13, Rome, Italy S.A. MEYER, Protistology Department, American Type Culture Collection, Rockville, Maryland (now at Department of Biology, Georgia State University, Atlanta, Georgia 30333, USA) C.K. MILNER, Nutrition Section, Shell Toxicology Laboratory, Sittingbourne Research Center, Sittingbourne, Kent, U.K. H. MOGREN, Research Division of Marbou, Sweden A. MORDENTI, Istituto Zootecnia e Nutrizione Animale, Universita, Via S. Giacomo 11, Bologna, Italy I. NORVO, Mitsui Italia, Piazza Liberty 2, Milan, Italy Xll Participants J.D. ORTHS, Westfalia Separator AC, 474 Oelde, Germany J. OIWA, Roniprot LLC, Romanian-Japanese Joint Venture Co., Curtea de Arges, Bvd., Republicii, 105, Romania S. PAGLIALUNGA, Istituto di Ricerche Farmacologiche 'Mario Negri1, Via Eritrea 62, Milan, Italy G. PALIAVICINI, Istituto Chimica Biol., Universita, Pavia, Italy S. PARRUIANI, Centro Ricerche Liquichimica, Robassomero, Rome, Italy C. PASCUCCI RIGHI, BP Oil Company, c/o Britannica Petroli, Piazza di Spagna 15, Rome, Italy L. PEGEN, Snam Progetti, S. Donato Milanese, Milan, Italy A. PELOSO, Italproteine S.p.A., Galleria Passarella 2, Milan, Italy G.C. PERRI, C.R.F., Via Tito Speri 14, Pomezia, Italy H.J. PHAFF, Department of Food Science and Technology, University of California, Davis, California 95616, U.S.A. B. POULLAIN, Laboratoire de Nutrition et des Maladies Métaboliques, 40 Rue Lionnais, 54.000 Nancy, France L. REY, Nestle Alimentana, Avenue Nestle, 1800 Vevey, Switzerland J. RIVKIN, Avalon S.A., 3 Chemin de Mornex, 1003 Lausanne, Switzerland G. ROSSI, Marxer, Loranze d'Ivrea, Torino, Italy V. RUSSO, Universita di Messina, Via D. Galimberti 3, Parma, Italy E. SALVINI, Liquichimica Biosintesi, Via Goldoni 10, Milan, Italy N.S. SCRIMSHAW, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A. J.C. SENEZ, CNRS, Laboratoire Chimie Bactérienne, 31 Chemin Joseph-Aiguier, 13274 Marseille, Cedex 2, France G. SERLUPI-CRESCENZI, Istituto Superiore di Sanita, Viale Regina Elena 299, Rome, Italy C. SHACKLADY, BP Proteins Ltd., Britannic House, Moor Lane, London EC2Y 9BU, England G.K. SKRIABIN, Institute of Microbes Biochemistry and Physiology, USSR Academy of Sciences, Moscow, USSR V. SILANO, Istituto Superiore di Sanita, Viale Regina Elena 299, Rome, Italy F. SINIGALLIA, Istituto Chimica Biol., Universita, Pavia, Italy P.M. SORGO, R&D Representative, Phillips Petroleum Co., Mariahilferstrasse 77-79, 1060 Wien, Austria G.T. STEEL, Analytical Biochemistry Section, ICI Ltd., Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, England D.A. STRINGER, ICI Ltd., Jealott's Hill Research Station, Bracknell, Berkshire, England M.T. TACCONI, Istituto di Ricerche Farmacologiche fMario NegriT, Via Eritrea 62, Milan, Italy M. TAJIMA, National Food Research Institute, 4-12 Shiohama-1, Koto-ku, Tokyo, Japan N. TANAHASHI, Kanegafuchi Chemical Industry Co., Ltd., 3,3-Chome, Nakanoshima, Kitaku, Osaka, Japan Participants xiii G. TOMASSI, Istituto Nazionale délia Nutrizione, Via Lancisis 27-29, Rome, Italy G. TRIVELLATO, Saras Chimica S.p.A., Piazza Boldrini 1, S. Donato Milanese, Milan, Italy J. TULLIEZ, Institut National de la Recherche Agronomique, 180 Chemin de Tournefeuille, 31300 Toulouse, France F. VALFRE1, Cattedra di Alimentazione e Nutrizione Animale, Facolta di Medicina Veterinaria, Universita, Via S. Costanzo 4, Perugia, Italy E.J. VAN WEERDEN, Centre for Animal Nutrition Research (ILOB), Haarweg 8, The Netherlands I. VELEA, Roniprot LLC, Romanian-Japanese Joint Venture Co., Curtea de Arges, Bvd., Republicii 105, Romania E. VENERONI, Istituto di Ricerche Farmacologiche 'Mario Negri1, Via Eritrea 62, Milan, Italy P.S. VENKATACHALAM, World Hunger Programme, United Nations University, 29th Floor, Toho Seimei Building, 15-1 Shibuya 2-chome, Shibuya-ku, Tokyo 150, Japan D. VENTURINI, Plasmon, Corso Garibaldi 97, Milan, Italy G. VENTURINI, Liquifarm, Via Roncaglia 12, Milan, Italy G. VICARI, Istituto Superiore di Sanita, Viale Regina Elena 299, Rome, Italy C. VILLA, Marxer, Loranze d'lvrea, Torino, Italy M. VINCENZINI, CNR, Piazza délie Cascine 27, Florence, Italy K. YAMADA, Sapporo Breweries ltd., 4-1 Mita 1-chome, Megro-ku, Tokyo, 153, Japan A. YEO, BP Proteins Ltd., Britannic House, Moor Lane, London EC2Y 9BU, England F.A. YEPEZ, Ambasciatore Venezuela in Italia, c/o Ambasciata del Venezuela, Viale Bruno Buozzi 109, Rome, Italy M. Y0SHIDA, Laboratory of Feed Resources, National Institute of Animal Husbandry, Chiba-shi 280, Japan P. ZAFFARONI, Snam Progetti, S. Donato Milanese, Milan, Italy V. ZANGRANDI, Liquichimica Biosintesi, Via Goldoni 10, Milan, Italy C. ZORZUT, Centro Ricerche Liquichimica, Robassomero, Torino, Italy Strengths and Weaknesses of Traditional Criteria in the Systematics of Yeasts as Revealed by Nuclear Genome Comparison H. J.Phaff and C.W. Price Department of Food Science and Technology, University of California, Davis, California 95616, U.S.A. INTRODUCTION The purpose of the present review is to survey briefly the historical aspects of yeast classification, to evaluate the current criteria commonly used for species differentiation, and to consider some aspects of molecular taxonomy, specifically those pertaining to nuclear DNA base composition and to DNA/DNA base complementarity between various strains. A natural system of classification corresponds to a biological order that exists independently of the organismal characters selected by the investigator to deduce that order (1). Ideally, a natural classification is devised from an evolutionary standpoint, resting upon the premise that clusters of organisms sharing certain properties result from natural selection during descent from a common ancestor. Until relatively recently, the apparent paucity of microbial features that reflect underlying filiation had largely frustrated attempts to construct an evolutionary classification system for microorganisms. During the past decade, however, it has become possible to assess the evolutionary relationships of microorganisms by a variety of biochemical and genetical techniques. This molecular approach has seen wide application in prokaryotic systematics. Notable examples include comparison of gene arrangements of the tryptophan operon (2), immunological comparisons of aldolase among lactic acid bacteria (3), and polynucleotide sequence comparisons of both selected regions and entire genomes among the Enterobacteriaceae (4). The rationale of the technique of genome comparison is a restatement, at the molecular level, of the phylogenetic principle that organisms grouped together in a system of classification should be descendents from a common ancestor. If two organisms are phylogenetically related, they must retain in their genomes base sequences that have descended from a common ancestral base sequence; closely related organisms will have retained a greater proportion of base sequences in common than organisms that have diverged widely. The techniques of DNA base composition determination, followed by polynucleotide sequence comparison, should therefore provide a sound theoretical foundation for yeast systematics. This approach should also enable one to evaluate the suitability for an evolutionary classification of the conventional characteristics employed by the taxonomic system most widely accepted (5,6) as well as those suggested in several additional classificatory schemes. Practical diagnostic keys that properly reflect phylogenetic principles may then be devised to employ more convenient and less elaborate procedures for the characterization of an organism. 1