PRODUCTION OF SINGLE CELL PROTEIN
BY CULTIVATION OF YEASTS
o o o o o >0 Vf) BIBLIOTHEEK TU Delft P 1819 4231
PRODUCTION OF SINGLE CELL PROTEIN
BY CULTIVATION OF YEASTS
ON FATS AND FAT PRODUCTS
PROEFSCHRIFT
TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE TECHNISCHE WETENSCHAPPEN AAN DE TECHNI-SCHE HOGESCHOOL DELFT, OP GEZAG VAN DE REC-TOR MAGNIFICUS IR. H.B. BOEREMA, HOOGLERAAR INDE AFDELING DER ELECTROTECHNIEK, VOOR EEN COMMISSIE AANGEWEZEN DOOR HET COLLEGE VAN DEKANENTE VERDEDIGEN OP WOENSDAG 20
NOVEM-BER 1974 TE 16.00 UUR DOOR
JAN VAN DER VEEN /O/O ^2b/
CHEMISCH DOCTORANDUS GEBOREN TE ROTTERDAM
.<:'^;f^-DIT PROEFSCHRIFT IS GOEDGEKEURD DOOR DE PROMOTOR PROFESSOR DR. T.O. WIKEN
DIT PROEFSCHRIFT WERD BEWERKT OP HET CENTRAAL INSTITUUT VOOR VOEDINGSONDERZOEK
TO
THE FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO)
CONTENTS
PREFACE 10
LIST OF ABBREVIATI ONS 11
Chapter I INTRODUCTION AND SCOPE 13 1. World protein shortage 13 2. General aspects of single cell protein (SCP) production 19
3. Specific problems of fat fermentation 22 4. Availability of fats and fat products 23
Chapter II VARIOUS ANALYTICAL METHODS 26
1. Fat analysis 26 1.1. Moisture, impurities and unsaponifiable matter (MIU) 26
1.2. Saponification value (SV) 26 1.3. Fatty acid content and fat content 26
1.4. Fatty acid profile of fat products 27 1.5. Fat content of emulsions 27 1.6. Emulsifier in fat products 28 1.7. Pesticides in fat products 28
2. Yeast analysis 28 2.1. Yeast concentration in culture liquid 28
2.2. Moisture content and ash content - 30 2.3. Crude protein content and lipid content 30 2.4. Polysaccharide content and crude fibre content 30
2.5. Amounts of emulsifier and pesticides 31 2.6. Elementary analysis and gross formula 31 2.7. Amino acid spectrum and fatty acid profile 31
2.8. Vitamin composition 31 2.9. Elements in ash 33 2.10. Content of ribo-nucleic acid (RNA) and purine base content 34
3. Sanitary analysis of yeast 34
3.1. Introduction 34 3.2. Total counts of aerobic bacteria and spores 35
3.3. Enterobacteriaceae count 35 3.4. Escherichia coli present/absent test 35
3.6. Counts of Clostridium group and Clostridium perfringens 36
3.7. Lancefield group D Streptococci count 36 3.8. Staphylococcus aureus present/absent test 36
3.9. Moulds . 37 4. Nutritional testing of yeast 37 4.1. Protein calories per cent (PCP) 37 4.2. Chemical score and essential amino acid index (EAA-lndex) 37
4.3. Protein efficiency ratio (PER) 40 4.4. Net protein utilization (NPU), true digestibility (TD) and 41
biological value (BV)
5. Safety evaluation of yeast 43
5.1. Introduction 43 5.2. Screening test 44 5.3. Sub~chronic study 44 5.4. Chronic feeding study 44 5.5. Multigeneration study 45 5.6. Teratogenicity studies 45 5.7. Mutagenicity studies 46 5.8. Studies with farm animals 46
5.9. Derived tests 46
Chapter III STANDARD PROCEDURE FOR FAT FERMENTATION 48
1. Fermentation steps 48 1.1. Preparation of the slant cultures 48
1.2. Procedure for the shake flask cultures 50 1.3. Apparatus for 1 liter fermentation 54 1.4. Apparatus for 20 liter fermentation 61 1.5. Harvesting of the yeast cells 64 2. Selection of yeast strain, composition of the media etc. 65
2.1. Selection of the strain 65 2.2. Concentration of the fat products 70
2.3. Fermentation temperature 71
2.4. Fat emulsification 72 2.5. Composition of the salt mixtures 75
2.6. Suppletion of vitamins 81
Chapter IV APPLICATION OF STANDARD PROCEDURE 85 1. Batch fermentations of grease emulsions 85 2. Batch fermentations of fatty acids 90 3. Continuous fermentation of fatty acids 95 4. Batch-to-batch fermentations of fatty acids 98 5. Fermentations of tallow emulsions 102 6. Fermentations of technical fat • 105
Chapter V PROPERTIES OF PRODUCED YEAST 108 5.1. Organoleptic examination 108 5.2. Chemical composition 108 5.3. Biological characteristics 111 5.4. Bacteriological examination , 117
5.5. Toxicological indications 117
Chapter VI DISCUSSION AND CONCLUSIONS 121 1. Discussion of the results 121 2. Economic considerations 121 3. Final conchision 123 LIST OF REFERENCES 125 SUMMARIES, 130 English 130 Dutch 133 Russian ^ 136 CURRICULUM VITAE 143
PREFACE
Ever since the production of single cell protein (SCP) on mineral oil products was started in the early sixties, the Central Institute for Nutrition and Food Research (CIVO—TNO) has been involved in its research and development; especially as far as related biological and toxicological aspects are concerned.
Besides, in 1969, the governmental supervision of the refining and distribution of imported inedible fats was assigned to the CIVO-TNO. The director of this Institute, Dr. CG.J.M. Engel, delegated this task to the author of the present thesis. Small wonder that, with these two streams coming together in one Institute, the idea of using the fats as substrates for the production of SCP gained momentum, particularly since in previous years the idea had been suggested by some CIVO-TNO research workers.
Research was started in the latter part of 1970, and from the very beginning there has been close cooperation with Professor Dr. T.O. Wiken, head of the Laboratory of Microbiology at the University of Technology, Delft. The many inspiring discussions we had over the years have contributed considerably to the quality and the presentation of the thesis; I have very much appreciated them.
As will be clear from the respective Chapters, several departments of CIVO—TNO have contributed to the work. An appreciable number of co-workers have devoted time and keen interest to this research; I herewith thank each of them whole-heartedly.
Finally, I would never have been able to carry out this research, beside my regular work, without the continuous support of my wife. The frequent discussions with her and our three children, about the social relevancy of the subject have been a real stimulus to me. It is with their full consent that this thesis is dedicated to the
Food and Agriculture Organization of the United Nations (FAO).
We all share in the hope that the FAO, being the main platform on which world food supplies are scrutinized, will be able to make the national governments aware of the major interest of food research programs so that, in not too distant future, there may be enough to meet the minimum requirements of food for every human being.
LIST OF ABBREVIATIONS
AV Acid value
atm atmosphere BV Biological value
BBL Baltimore Biological Laboratory CBS Centraalbureau voor Schimmelcultures °C Degree Centigrade
CIVO Central Institute for Nutrition and Food Research cm centimeter
DDT Dichlorodiphenyl Trichloroethane DNA Desoxyribonucleic Acid
EAA—Index Essential Amino Acid Index FAA—slants Fatty Acid Agar slants FA—slants Fat Agar slants
FAD Flavin Adenine Dinucleotide
FAO Food and Agriculture Organization of the United Nations FID Flame lonisation Detection
FMN Flavin Mononucleotide G g g/c/d GYA-slants GRA-slants h HCB H/L HLB lUPAC Gravitative acceleration gram
gram per capita per day
Glucose-Yeast-extract-Agar slants Glucose—Renex—Agar slants hour
Hexachlorobenzene
Hydrophile to Lipophile ratio Hydrophile Lipophile Balance
International Union of Pure and Applied Chemistry Joule
kg kJ
kilo gram kilo Joule
m meter meg micro—gram mg milligram min minute
MIU Moisture, Impurities and Unsaponifiable Matter MJ Mega Joule
ml milliliter mm milUmeter m/m mass to mass ratio
mMol milH—grammolecule Mol grammolecule mV milli-Volt i ^ 10-' g N Nitrogen n normality or normal
NAD Nicotinamide Adenine Dinucleotide
NADP Nicotinamide Adenine Dinucleotide Phosphate NPU Net Protein Utilization
PAG Protein Advisory Group of the United Nations System PEGA Polyethylene Glycol Adipate
PER Protein Efficiency Ratio p.m. pro memory
ppm parts per million RA—slants
RNA rpm
Renex Agar slants Ribonucleic Acid rotations per miijute SCP
SV
Single Cell Protein Saponification value TD True Digestibility t/y tons per year
TNO Netherlands Organization for AppUed Scientific Research vol volume
v/v volume to volume ratio
v/v/h volume (air) per volume (medium) per hour v/v/min volume (air) per volume (medium) per minute
CHAPTER I
INTRODUCTION AND SCOPE
1.1. World protein shortage
Tlie world protein problem has been discussed in so many pubUcations that it would hardly be expected to find anyone who could add some truly new points of view.
Therefore, it is rather amazing that from a number of recent publications the impression emerges that there is no protein shortage at all or that, at least statistically, the amoun of protein yearly available would seem to be more than the minimum level for survival of man. In Table 11-1 this statement is uUustrated by figures, collected by Autret (1969), Engel (1971) and Abbot (1967) about the protein supply per capita per day.
When these figures are related to egg protein by multiplying the vegetable protein figure by the factor of 0.55, and the animal protein figure by the factor of 0.8, the amount available is found to be to 41-43 g/c/d. In FAO Report no. 52 (1973), a theoretical safety level of intake of egg or milk protein calculated for human populations gives an amount of 29 g/c/d. This means that the amount of protein available would be 45% greater than the so-called 'safe level of intake'. The reason for the fact that, in spite of this, a large part of the world population suffers from undernourishment as regards protein is not only a wrong distribution system but has also to do with the circumstance that, in some parts of the world, the protein intake per capita is much higher than the safe level, leaving for people in other regions far less than the required minimum. To illustrate this statement, the protein consumption in India may be compared with that of the United States of North America. As will be seen in Table I 1-2, the average daily intake of both areas together is 47.5 g/c/d egg protein. However the consumption of the Indian people is at the level of 29 g/c/d, whereas the inhabitant in the USA consumes on an average 66.3 g/c/d.
Again the point must be stressed that the fact that, statistically, the amount of protein available for each inhabitant of India is just at the minimum level does not mean that every inhabitant really gets the amount concerned. In the first place, this is due to difficulties of transportation, distribution, etc. Secondly, the same finding that is true for the world as a whole is true also for the area mentioned. The rich people in India are able to buy more than the minimum amount, and will definitely do so, leaving for the poor people an amount far less than the 'safe level' of 29 g/c/d. The figures in Table I 1—2, related to egg protein, were calculated on the
Table 1 I 1 World supply of protein in grams per capita per day, related to egg protein based on figures collected by Autret (1969), Engel (1971) and Abbot (1967) NPU% Cereals 55 Autret Egg protein Engel Egg protein Abbot Egg protein 31 8 1 7 5 33 7 18 5 33 4 1 8 4 Starchy roots 62 2 7 1 7 2 7 1 7 3 2 2 0 Pulses, oil-seeds, and nuts 44 8 0 3 5 9 0 4 0 9 0 4 0 Vege-tables and fruit 80 2 6 2 1 2 7 2 2 2 4 1 9 Total vege table protein 55 45 1 24 8 48 1 26 4 48 0 26 3 Meat and- poul-try 70 9 8 6 9 8 4 5 9 8 8 6 2 Eggs 100 1 4 1 4 1 3 1 3 1 2 1 2 Fish 83 2 6 2 2 4 9 4 1 2 3 1 9 Milk and milk products Total animal pro-tein Total protein g/c/d 85 80 7 2 6 1 6 4 5 4 7 7 6 5 2 1 0 1 6 6 21 0 1 6 7 20 0 1 5 8 66 1 41 4 69 1 43 1 68 0 42 1
Vegetable, animal and total protein, related to egg protein, expressed in grams per capita per day and in percentages
Vegetable protein Animal protein Total protein Egg protein Total protein g/c/d % g/c/d % g/c/d Autret Egg protein Engel Egg protein 45 1 24 8 48 1 26 4 68 2 60 0 69 6 61 2 21 0 1 6 6 21 0 1 6 7 31 8 40 0 30 4 38 8 66 1 41 4 69 1 43 1 100 0 100 0 100 0 100 0 62 6% 62 4%
Table I 1 —2 Average protein supply, related to egg protein, in grams per capita per day, in India and the United States of North America over the period 1960-62 Cereals Starchy roots Pulses, nuts oilseeds etc. Fruit and vegetables Total vege-table protein Meat Egg Fish Milk and milk products Total animal protein Total protein Food protein 31.0 0.9 13.2 0.4 45.5 0.6 0.1 0.5 India Egg protein 17.1 0.6 5.8 0.3 23.8 0.4 0.1 0.4 United States of North America Food protein 15.6 2.2 4.1 5.0 26.9 32.4 5.7 2.5 Egg protein 8.6 1.4 1.8 4.1 15.8 22.7 5.7 2.1 4.8 6.0 51.5 4.1 5.0 28.8 23.5 64.1 91.0 20.0 50.5 66.3 Protein calories Total calories 10.2% 11.7%
assumption that the various proteins serve as the only protein source available. With mixed proteins, other conversion factors have to be applied and this might result in higher values for the net protein utilization.
In this connection it must be emphasized that the net protein utilization of a foodstuff can give a true picture of the nutritional situation only when enough calories are suppHed by the diet concerned. In a well-balanced diet, the contribution of the protein energy to the total energy available should lie between 10% and 12%. As is seen in Table I 1-2, it is 11.7% for North America and 10.2% for India. But if the world population is considered, one finds that the calorie level of 2380 cal/day needs a protein calorie contribution of 238 to 286 calories, which
means a protein intake of 60-72 g/c/d. As will be seen in tabel I 1-1, this value is 66-69 g/c/d for the world population. In this respect, however, there is undoubtedly a protein shortage in India (Table I 1-2). To express this specifically, the term protein calorie malnutrition is used. Abbot (1967) has given figures for particular areas which show that for the Near East, Africa, the Far East and Latin America, (except Argentine, Uruguay and Paraguay), the protein supply, expressed as egg protein, is 33.4 g/c/d and the protein calorie percentage 9.8%. The total population living in these areas is about two thirds of the world population. This means that although the total protein production could cover the minimum requirements, two thirds of the world population are undernourished in so far as they suffer from protein malnutrition.
On this point, a more detailed and exact picture is obtained when vegetable and animal proteins are considered separately. According to Abbot (1967), the production of animal foodstuff has increased in the developed countries since World War II. In the developing areas, however, the total per capita protein supply shows a decrease of about 6% in the same period, accompanied by an increased dependence on cereals. From the data given in Table I 1-2, it is evident that the conversion factor for vegetable protein to egg protein is 0.55 on an average, whereas for animal protein this factor is 0.8. For the 'safe level' intake of 29 g/c/d, the following equation can be given: 29 = V x 0.55 + A x 0.8, in which A denotes animal protein and V vegetable protein. Theoretically this means that, if no animal protein is available, 52.7 g/c/d of vegetable protein is the minimum level. For exclusively animal protein this level is 36.3 g/c/d (V = 0).
Figure I 1 — 1 gives the said relation graphically:
36 3
^ \ Figure I 1-1 Graph of the equation: \ . 29 = 0.55xV + 0.8xA
\^^ 29 = minimum protein level in g/c/d '" \ ^ V = vegetable protein intake in g/c/d
\ , A = animal protein intake in g/c/d
25 \ ,
20 ^ v
10
From Figure I 1 — 1 it will be seen that, if the contribution of animal protein is 15 g/c/d, the amount of vegetable protein required to reach the minimum level is 31 g/c/d. It should be realized that the animal and vegetable proteins are not consumed as such but incorporated in a foodstuff; the other components of this foodstuff contribute to the t(jtal nutritional situation too. Moreover, in discussing the difference in contribution of animal and vegetable proteins for covering the total requirements it is essential to realize that, for the production of a definite amount of animal protein, a far greater quantity of vegetable protein must be available as fodder protein. To illustrate this aspect, the conversion of total feed to edible carcass and of protein ingested to edible protein is given in Table I 1 —3 for various animals (Wilcke, 1973). In addition, the relation of protein consumed to edible protein yield is listed (Wilson, 1971).
It should be taken into account that the fodder protein at least partly consists of
Table I 1—3 Conversion of total feed to edible carcass and of protein consumed to protein formed for a number of animals according to Wilcke (1973).
Class Edible carcass Total feed xl00% Edible protein Protein ingested xl00% Beef Swine Broiler Hen 2.0 17.0 18.3 26.7 4.7 12.0 17.0 23.0 Protein efficiency according to Wilson (1971) Class Broilers Porker Lamb Steer Production level 6 crops at 4 Ib/wt 21/2 litters of 12 at 90 lb carcass 2 litters of 3 at 35 lb carcass 650 lb carcass Annual crude protein yield (lb) 2.5 270 24 75 Annual crude protein consumed (lb) 8 1850 275 1250 Protein efficiency % 31 15 9 6
Table I 1-4 Cereals food protein Potatoes Food protein Pulses, nuts Food protein Fruit and vegetables Food protein Total vegetable protein Meat Food protein Egg Protein Fish Food protein Milk and milk products Food protein Total animal protein Totals 18
Food balance sheet for the Netherlands expressed in thousand tons, calculated over Available supply 5363 427 3537 56.6 102 11.4 2081 19.2 514.2 540 73.6 155 18.5 269 22.5 9550 421.1 535.7 1049.9 the period 1960 Waste, seeds, manufacture 259 21 1702 27.3 6 0.7 506 4.7 53.7 —.— 15.0 1.8 126 10.5 6637 292.7 305.2 358.9 -63 Animal feed 3873 308 686 10.9 35 3.9 — ^ 322.8 —.— ^ 15 1.1 770 33.9 35.0 357.8 <» Gross food 1231 98 1149 18.4 61 6.8 1575 14.5 137.7 540 73.6 140 16.7 128 10.7 2134 94.5 195.5 333.2 Food protein g/c/d 22.9 4.3 1.6 3.4 32.2 17.2 3.9 2.5 22.1 45.7 77.9
protein that cannot be, or is not, used for human consumption, such as grass protein, protein from meat scraps, inedible fish meals, etc. However, to a very large extent the protein used as fodder for animals could also serve as protein for human consumption. Table 1 1—4 surveys the total supply of foodstuff, the amounts used for animal feed, for wastes, seeds and manufacture and for human consumption, related to protein; it is taken from the food balance sheets (1960-63) for the Netherlands.
From Table I 1 —4 the following conclusions may be drawn.
The total available amount of food protein in the Netherlands was in the period mentioned 77.9 g/c/d, and from this amount 58.7% was of animal origin.
Of the supply of vegetable protein 62.8% was used for fodder, whereas for that purpose only 6.5% of the animal protein supply was utilized.
Cereals were the main source for the supply of fodder protein (96.1%), followed by protein from milk, skimmed milk and skimmed milk-powder (9.5%). On the other hand, the percentage of cereals used for fodder was 72.1% and for milk protein 8% only.
An interesting information that can be derived from Table I 1—4 is the total amount of protein used as animal feed in relation to the total amount of meat protein produced. This relation is (73.6 : 357.8) x 100% = 20.6%, which is much higher than the figures given by Wilcke (1966). Even when the food wastes that are used directly or indirectly as fodder are taken into consideration, the percentage is far above the generally accepted average percentage of 9% to 11%.
Finally, something has to be said about the protein need in the near future. It is expected (Abbot, 1973) that to meet the requirements the production of protein should increase by 6% yearly. However, in order to increase the animal protein production much more vegetable protein must be available as fodder. It would be a very important contribution to the protein supply if at least a part of this fodder protein could be provided by sources that are not contributing to human nutrition. This very important condition is fulfilled by single cell protein (SCP) products. For every 10% of the fodder protein in the Netherlands that would derive from this source, which means a production of 60,000 to 80,000 tons of SCP yearly, the gross food production would increase by 10%. Under these conditions, the amount of milk protein available would increase by 60,000 tons per year. Apart from any other application, it will be evident that SCP undoubtedly may be considered an essential contribution to the world protein production.
1.2. General aspects of SCP production
The term 'Single Cell Protein' or SCP today includes a product more or less rich in protein, obtained by cultivation of bacteria, yeasts and fungi. It has most probably
been chosen in order to enable the introduction of biosynthetic products of microbial origin as animal fodder and/or human food, without mentioning yeasts, bacteria or fungi by name and thus avoiding the prejudice people have in general against these organisms
The advantages of SCP production are extensively emphasized by those interested in promoting this biosynthetic protein production As compared with protein food from animal and vegetable sources, the very high growth rate of microbes and the possibility of producing large quantities of SCP of reproducible quality are the most striking advantages For example, an annual production of SCP of 20,000 tons with 9 6% N corresponds to the amount of protein produced by 100 000 cows giving a milk production of 3900 1/y containing 30,5 g/1 protein (Frens 1961) It is evident that milk production depends on climatological circumstances, whereas SCP production does not Moreover, there is a very high economic use of land space in the case of SCP production Worgan (1973) has calculated the land use of yeast grown on sugar and that of beef cattle, he arrives at 2800 kg per hectare per year for yeast protem and 42 kg per hectare per year for beef protein
As far as raw materials suitable for SCP production are concerned, a variety of waste materials can be used, not only of agricultural but also of industrial origin, e g. sulphite liquors, wood hydrolysates, etc
Very important sources for SCP production are or might be petrochemicals, e g methanol and ethanol, and mineral oil products, e g gas oil and n-alkanes
In the case of agricultural wastes, synthetic symbiosis between various microbes, e g yeasts, may be applied in production of SCP (The Symba—process, see Wiken,
1972)
Last, but not least, a minimal effluent, poor in organic substance, is often claimed for SCP production and this is of great importance for environmental hygiene (Wiken, 1972)
The rather impressive list of advantage of SCP production makes it understandable that research in the field of microbial food production has grown tremendously over the last few decades It is, therefore, rather astonishing that, in spite of all these efforts, at this very moment of writing, Summer 1974, the production of SCP on an economically profitable scale, i e 100,000 t/y, has not yet been effected In search of the reasons for this phenomenon, a great number of factors emerge that might be to a greater or lesser extent responsible
One of the most important reasons might be a difference in concept that exists between technologists and microbiologists, as regards the way of tackling problems connected with processes based on growth of microbial cells Although there has been some intensive collaboration between the two disciplines in the last twenty years, there is still a completely different approach as far as large scale production is concerned The technologist often still prefers to overlook the fact that micro-organisms are living cells and not simply enzyme bags that can adequately be defined by their contents of fat, protein, carbohydrate and ash This aspect of dealing with living cells gives the conditions and control of SCP production extra
dimensions. Contamination with undesirable micro-organisms, aeration problems and prevention of excessive foaming are some of the factors that are obstacles for scaling up to 100,000 t/y productions. But perhaps the most intriguing factor is the susceptibility of yeasts, bacteria and fungi to even small changes in production conditions. When emphasizing that a consistent quality is one of the advantages of SCP production, we should add that this is true as far as the chemical composition is concerned only, and as long as the operating conditions are strictly constant. Because even minor changes in the process conditions may have important consequences for the yield as well as for the nutritive value and the toxicological safety of the product. Apart from technological reasons, this very fact might indeed give an explanation for the present situation that as yet no 100,000 t/y plant is in operation either in Japan, or in USSR, UK, France, etc.
A large number of toxicological investigations have been carried out with yeast grown on gas oil and n—alkanes, and the results published thus far do not give any reason why this yeast should not be used as animal fodder. It does fulfil every toxicological qualification and, when supplied with methionine, it has a very good nutritive value (De Groot et al. 1970a, 1970b, 1971; Shacklady and Gatumel, 1972; Van der Wal, 1968; Shacklady, 1969). One should, however, not overlook the fact that the yeast concerned is an example of production on a much smaller scale than the economically desirable 100,000 t/y plant. The technical disadvantages i.e. the low concentration of the end product (2% max.), the expensive plant in terms of materials and equipment, the high power consumption for aeration and homogenizing, the necessity of great cooling capacity, etc., represent great difficulties. But they can be solved as purely technical problems, though, admittedly, these factors will have their economic consequences in terms of costs of the final product.
As far as the sanitary, nutritive and toxicological aspects of the quality of the yeast are concerned, much progress has meanwhile been made. The level of the aromatic compounds present in yeast grown on gas oil is far below the safety level, thanks to extensive extraction procedures. The amount of odd-numbered fatty acids, present in yeast grown on n-alkanes and considered to cause undesirable effects, has been reduced through a better procedure for the preparation of the n-alkanes. Various processes have been developed to reduce the nucleic acid content of microbial SCP, and its nutritional value has been raised by adding synthetic methionine. For the rupture of the cell walls, which are indigestible for animals because of the presence of 1 -4 linked polysaccharide derivatives, a number of methods have been worked out, but so far they all result in lower protein yield and higher production costs (Heydeman 1973). It must be admitted that, all in all, a relatively large number of problems have still to be solved before SCP produced in quantities of 100,000 t/y will be available for use as fodder.
1.3. Specific problems of fat fermentation
In addition to the more general problems of SCP production, briefly discussed in the previous paragraph, some special remarks may now be made in relation to fat fermentation. In the first place it be here emphasized that 'fat fermentation' is incorrect terminologically, in spite of the fact that the term is generally accepted. Actually the fats and fat products are not 'fermented' but used for anabolic or biosynthetic and catabolic, mainly respiratory, processes, involving O2 uptake. A better description, therefore, is 'the production of biomass, using fats and fat products as sole sources of carbon and energy'. Wlien, in what follows, the term 'fermentation' is nevertheless applied, it denotes this concept.
Secondly, SCP production calls for a homogeneous reaction mixture. The nitrogen source and other nutrients are water-soluble but the fats and fat products are not. This gives rise to an extra problem in the case of fat fermentation. In addition to the other substrate components, present in normal SCP production, the fat component has to be homogenized too. Considering this problem, the raw materials used can be divided in two groups: fats and fat products that have a melting-point at or below 30°C, and those that have a higher melting-point. The first group of products can be homogenized by mixing the fermentation liquid thoroughly, whereas for the second group an emulsifier has to be applied. The application of an emulsifier has, however, a number of disadvantages. The price of the emulsifier increases the ultimate costs. Furthermore, the fact that the fat emulsion will be broken when sterilized in the presence of mineral salts, makes it necessary to sterilize the emulsion and salt solutions separately. This results in extra equipment costs. Moreover, the presence of the emulsifier in the end product might influence the nutritive value and may even have toxicological effects, in spite of the fact that the emulsifier itself may well be guaranteed by the manufacturer to be non—toxic. There are two other ways to utilize fats and fat products melting above 30 C as raw materials for SCP production. The first would be to increase the fermentation temperature. Actually, the Uterature describes processes in which SCP is produced at higher temperature, even as high as 42°C, but so far not with strains with lipolytic activity.
Chapter IV will report some preliminary attempts with a selected strain and further work to find a solution in this direction is in progress.
The second way would be to make mixtures of a liquid fat product and a solid one that melt between 28°C and 32°C. This has been done in practice. However it implies, of course, a restriction for the application of the animal fats, since the procedure involves a certain amount of liquid products that might not always be available.
1.4. Availibility of fats and fat products
The trade in animal fats in the Netherlands concerns edible fat, under control of the Veterinary Inspection, and inedible fat. Under the supervision of the Ministry of Health and Environmental Hygiene, the inedible fat is imported, distributed and partly refined into products suitable for human consumption. In view of this situation, the import of inedible animal fats and fat products is much larger than the production of animal fat in the Netherlands.
This is illustrated in Table I 4—1
Table I 4-1 Production and import figures for animal fat in the Netherlands (1968-1972) in 10* kg. 1968 1969 1970 1971 1972 Import 197.6 208.1 236.2 237.3 240.1 Production 99.0 94.1 106.8 117.9 123.4 Total 296.6 302.2 343.0 355.2 363.5
The refined animal fats are partly exported as such or as a product of fat, and partly used in milk substitutes for calves. The quantity of imported fats has been more or less constant over the last three years but that of refined fat is fluctuating; it depends on export opportunity. Refining of inedible fats includes
de-acidifica-Table 1 4-2 Fatty acids, obtained in refining of various oils and fats in 1971 and 1972 in the Netherlands in 1000 kg.
1971 1972 Fatty acids from:
Coconut oil Palmoil Palmkernel oil Soya oil Marine oils
Various other vegetable oils Animal fats 2794 7825 5317 2834 5556 3220 7367 5175 9920 6040 3632 5333 2270 5092 Totals • 34913 37462
tion, which means that the transformation to edible fats results in a quantity of fatty acids as by-products. However, the same fatty acids are also obtained when vegetable oils and fats are refined. In fact, all vegetable oils are refined before they are used for human consumption or used as raw materials in food products. To give an impression of the quantities concerned, Table 1 4—2 shows the quantities of various fatty acids, obtained in the refining, hardening, etc. of animal and vegetable oils and fats in the Netherlands (1971 and 1972).
To avoid any misunderstanding, it be here pointed out that the totals for fatty acids produced in 1971 and 1972 were much larger than the quantities listed in Table 1 4—2. Actually, considerable quantities of beef fat, mostly ranged as tallows, are also used for the production of fatty acids. From the total thus available, a rather high percentage is usually exported, as will be seen in Table 14—3.
Table I 4 - 3 1969 1970 1971 1972 Production and 1972) in 10* kg. Production 106 93 106 105 export of fatty Export 87.7 84.3 79.4 81.2 acids in the Ne Difference 18.3 8.7 26.6 23.8
In fact, the by-products of the fat refineries (see Table I 4-2) are not pure fatty acids; they contain at least 25% fat or oil. Generally speaking, the composition of these products is: MIU max 3%; FFA 60-70% and fat 30-40%. •
In Table I 4—4 some characteristics are given for a number of fatty acids of vegetable and animal origin.
As will be explained in Chapter II, the calculation of the percentages of fat and fatty acids is based on the assumptions that the amount of mono- and diglycerides can be neglected, and that the average molecular weight of the free fatty acids is the same as that of the fatty acids bound in the triglycerides.
As has been discussed in item I 3, the most important property from a technical point of view is the temperature at which the fats or fat products are in the liquid phase. Table I 4 - 4 , therefore, shows the melting points, too. Table 1 4 - 4 , specifically reveals that the melting point of the fatty acids obtained in refining coconut oil is very low. This raw material is therefore suitable for mixing with fatty acids of higher melting points. For example, a mixture of 67.5% animal fatty acids
Table 14—4 Characteristics of some fatty acids, obtained from two refineries in the Netherlands
Fatty acids Coconut Palm Soya Arachis Animal from: oil oil oil oil fats Saponificat-ion value 246.2 201.5 155.8 192.3 201.9 mg KOH/g Acid value mgKOH/g 180.5 150.7 133.9 116.3 123.7 Average mol. weight 222.1 273.6 278.1 282.6 265.3 Fat content g/lOOg 27.6 25.9 31.5 40.1 38.8 FFA content g/lOOg 71.6 73.5 66.5 58.7 58.6 MIU content g/lOOg 0.8 0.6 2.0 1.2 2.6 melting point °C 23.5 50.0 30.5 31.5 35.0
and 32.5% of the fatty acids from coconut oil melts at 30 C, and so does a mixture of 20% fatty acids from palm oil and 80% of coconut fatty acids. These examples illustrate the possibility, mentioned in 1.3., of utilizing mixtures of raw materials in order to avoid the application of an emulsifier. They confirm, at the same time, that the use of animal fat and palm oil fatty acids may be restricted by the circumstance that sufficient amounts of coconut fatty acids will not be available for mixing in the required proportions.
CHAPTER n
VARIOUS ANALYTICAL METHODS
1. Fat analysis
1.1. Moisture, impurities and unsaponifiable matter (MIU)
The determination of these quaUty characteristics was performed according to the methods described in lUPAC Standard Methods (1966). The moisture percentage is in fact the matter volatile at 105 C, and is expressed as % m/m; the impurities constitute the percentage of the fat that is insoluble in petroleum ether (1:5) and the unsaponifiable matter is determined by means of the lUPAC diethyl ether method (1966).
1.2. Saponification value (SV)
The determination of this identity characteristic was carried out according to the method described in lUPAC Standard Methods (1966). The result is expressed as the number of mg KOH per g fat and will be referred to as SV.
1.3. Fatty acid content and fat content
These characteristics were derived from the acid value (AV), obtained by the method described in lUPAC Standard Methods (1966), the saponification value (SV) and MIU (see II 1.1. and 1.2.).
Supposing that a fat with an MIU value of m % contains a % fat and b % free fatty acids we can express the saponification value and the acid value in these percentages, assuming that the average molecular weight of the free and esterified fatty acids has the same value (M) and that the amount of mono- and diglycerides can be neglected.
The following formulas will be the result: 56.1 x a 56.1
^ ^ ~ M+12.67 "^ M (1)
AV = ^ (2) m = 1 0 0 - a - b (3)
Eliminating M and rearrangement gives:
( S V - A V ) x ( l O O - m ) ^ 12.7x A V x ( S V - A V ) SV 561 xSV
^ ^ A V x ( l O O - m ) 1 2 , 7 x A V x ( S V - A V )
SV 561 xSV
When the values of AV and b are known, the value of M can be calculated with formula (2) and, next, compared with the value derived from the fatty acid composition.
1.4. Fatty acid profile of fat products
The fatty acid profile of oils and fat products was estimated after conversion of the fatty acids into methyl esters according to the method described in lUPAC Standard Methods (1966).
These methyl esters were then determined by gas chromatography. The apparatus used has the following characteristics:
Column: glass 200 cm; i.d. 0.3 cm
Stationary phase: 5% PEGA on gas chromosorb Q, 100-200 mesh; Carrier gas: N2, 99,95% pure;
Temperatures: oven 170°C, injector 210°C and detector 210°; Detector: FID;
Measurement: electronic peak surface integrator.
From the fatty acids profile, the average molecular weight of the fatty acids, present in the fat product, can be derived and this molecular weight can be compared with the value obtained from the values of SV, AV and MIU.
1.5. Fat content of emulsions
For this determination, the method of Weibull (1892-1894) was used. The emulsion was boiled with 1.5 n hydrochloric acid for 1 hour and the mixture then filtered through a wet filter and washed until it was acid free (Congored paper as indicator). After drying overnight, extraction with petroleum ether according to Berntrop (1902) was performed for five hours. After evaporation of the solvent, the residue was dried until constant weight was attained.
1 6 Emulsifier in fat products
Tlie amount of emulsifier could be estimated on the basis of its solubility in water and in caibon tetrachloride 50 ml of the emulsion was boiled for 20 minutes with 3 35 grams of potassium hydroxide and 40 ml of ethanol, containing 0 4% v/v amylalcohol After cooling, and adding 20 ml of a 25% m/v hydrochloric acid solution, the mixture was extracted with diethyl ether After separation of the two phases the water was evaporated and the residue extracted with carbon tetrachloride The solution thus obtained was dried until constant weight was reached
1 7 Pesticides in fat products
The methods used for determination of the chlorinated pesticides in fats and fat products were based on the procedure described by Holden and Marsden (1969) The sample was extracted with hexane, containing an internal standard, and, after cleaning up on one or two alumina columns, the extract was analyzed by gas chromatography with electron capture detection
2. Yeast analysis
2.1. Yeast concentration of culture liquid 2 1.1. Introduction
The purpose of this determination, carried out at mtervals, is to follow and control the conversion of fat into yeast The simplest way is to count the viable yeast cells, but this method has two disadvantages The first is that the results of the counts are available only after three to five days The second difficulty is the formation of clusters of cells, but this can be overcome by strongly shaking the culture liquid and changing pipettes at least twice in making dilutions The determination of the cell concentration by measuring the light extinction of the diluted culture hquid at 660 nm is no doubt the most rapid method, but it has the disadvantage that other absorbing particles are interfering The problem that the fats are practically insoluble in water was overcome by using a mixture of propanol-2 (IPA), hexane and hydrochloric acid for making the dilutions
In view of the fact that, finally, it is the weight of the biomass that counts for the calculation of the yield, it was sensible to relate these methods to one another In doing so it was found that during the last hours of fermentation the number of cells did not increase whereas the biomass did increase in weight Therefore in the following the results obtained by the colorimetric method, related to counting,
will be given. The estimation of the biomass was performed by weighing the product obtained after centrifuging the culture liquid, and washing and freeze-drying of the wet yeast paste obtained.
2.1.2. Yeast count
Starting with 1 ml of the culture liquid, suitable decimal dilutions were made. In most cases the 10'*, 10'* and 10'^ dilutions were used. From each of these dilutions, 1 ml was plated in duplicate into an oxytetracycline glucose yeast extract agar cooled to approximately 47°C. The inoculated plates were then incubated for three to five days at 22°C to 24°C before counting. From the six observations made the average value of the yeast concentration, expressed as cells/ml, was derived.
0 7 0 6 OS 0 4 a3 0.2 ai 5 10 1^ 20 25 30 35 40 45 50 55 60 x 10* cells/ml
Figure II 2-1 Relation between extinction at 660 nm and cell concentration in cells/ml
Us/ml 10* 7 14 21 28 35 42 56 ^660 0.14 0.26 0.38 0.50 0.60 0.68 0.80 cells/ml x l 0 6 4 8 12 16 20 30 40 50 E660 0 06 0.14 0.22 0.30 0.37 0.52 0.66 0.77 cells/ml x l 0 6 10 15 20 25 30 35 40 45 Eeeo 0.18 0.28 0.37 0.45 0.53 0.60 0.68 0.72
2 1 3 Colorimetric method
The test solution was prepared by diluting 1 ml of the culture hquid with 9 ml of a mixture of 885 5 ml IPA, 111 ml hexane and 3 5 ml 4 n hydrochloric acid At very high concentrations, the culture liquid was diluted 20 times
Tlie blank was prepared by diluting the fermentation liquid before inoculation with the mixture of IPA, hexane and 4 n hydrochloric acid mentioned The measurements were made at 660 nm with an Engel colorimeter, combined with a hghtspot galvanometer AL 4 Figure 11 2 1 gives the relation between the light extinction and the cell concentration, on the basis of the results of a number of experiments in which the yeast concentration was estimated simultaneously by means of both methods
As will be seen in Figure II 2 - 1 , the deviations of the various results are not very great, especially not in the straight part of the curve For that reason most of the determinations were made with dilutions of yeast suspensions in that part of the curve (below Eeao = 0 5)
2 2 Moisture content and ash content
The determination of the water content was carried out by drying with phosphor pentoxide under vacuum at 70°C according to the method described by Van der Kamer (1949) The ash content was determined at 550°C according to the AOAC Method (1965) described in its item 13006
2 3 Crude protein content and lipid content
For the crude protein content (including nucleic acids, peptides free ammo acids etc ), the method of Kjeldahl for the determination of nitrogen was followed in a modification described by Bradshaat (1965) The crude protein was calculated from the nitrogen content by multiplying with 6 25
The lipid content was estimated by means of a modification of the Weibull method described in AOAC Methods (1965) After acid destruction of the sample with hydrochloric acid, it was washed, dried and extracted with light petroleum After evaporation of the light petroleum the residue was dried and weighed
2 4 Polysaccharide content and crude fibre content
The determmation of the polysaccharide, expressed as glucose, was based on the method of Sachse (1878) After acid destruction of the sample, and clearing with Carrez solutions I and 11 of the filtrate, the amount of reducing sugars was
estimated according to the Luff method (see Schoorl 1929). The filtrate was, in addition, examined on pentoses by means of tfiin layer chromatography (TLG). The crude fibre determination was performed by means of the method of Van der Kamer and Van Ginkel (1952). Starch, protein and Hpid were made soluble with the reagent of Scharrer and Kiirschner. After extraction with diethyl ether the residue was dried and weighed.
2.5. Amounts of emulsifier and pesticides
The determination of emulsifier was carried out as is described for fat emulsion, using a yeast suspension in water.
For the estimation of chlorinated pesticides, the dried yeast was ground to a fine powder and extracted with hexane (with internal standard) in a conical flask by shaking for one hour on a mechanical shaker. After cleaning up on a small alumina column, the extract was analyzed by gas chromatography with electron capture detection as described in II 1.7.
2.6. Elementary analysis and gross formula
The nitrogen content of the yeast was also estimated according to a modified Dumas method, using a microcombustion apparatus, type Hosli. The carbon and hydrogen were determined as is described by Buys and Schroder (1970) and the oxygen according to the method of Romer (1972). These determinations were carried out at the Institute for Organic Chemistry TNO. On the basis of the results, the gross formula of the yeast was calculated.
2.7. Amino acid spectrum and fatty acid profile
In his doctoral thesis. Slump (1969) describes the methods of analysis for the various amino acids and we used these methods, slightly modified, for determina-tion of the amino acid spectrum.
For the determination of the fatty acid profile, performed as described in II 1.4., the Hpids used were obtained by means of the method described in II 2.3.
2.8. Vitamin composition 2.8.1. Introduction
of a number of vitamins of the B group. In the literature cited, the respective microbiological methods are given in detail and in practice only slight modifications of these methods were applied.
2.8.2. Thiamine
The determination of vitamin Bl was made using the ATTC strain 9338 of Lactobacillus fermenti. The samples were heated with 85 ml 0.1 n sulphuric acid for 30 minutes at 120°C and after cooling treated with Taka diastase at 45°C for 105 minutes (see Sarett and Cheldelin, 1944).
2.8.3. Riboflavin
The estimation of vitamin B2 was performed by means of the ATTC strain 7469 of Lactobacillus easel after extraction of the samples with 0.1 n hydrochloric acid at 120°C for 15 minutes (see Snell and Strong, 1939).
2.8.4. Pyridoxine
The vitamin B6 was determined with the ATTC strain 9080 of Saccharomyces carlsbergensis after extraction of the samples with 0.4 n sulphuric acid at 120°C for
1 hour (see Atkin et al., 1943).
2.8.5. Niacin
The determination of nicotinamide was carried out with Lactobacillus plantarum strain ATTC 8014 after extraction of the samples with 1 n hydrochloric acid at 120°C for 20 minutes (see Barton-Wright, 1944, 1945).
2.8.6. Panthotenic acid
For the determination of this vitamin Lactobacillus plantarum strain 8014 was used after treatment of the samples with Taka diastase and papain in a buffer at pH 4.5 at 45° C for 4 hours (see Sheggs and Wright, 1944).
2.8.7. Folic acid
This vitamin was determined using the ATCC strain 7469 of Lactobacillus casei, after extraction of the samples with phosphate buffer pH 6.1 at 120°C for 10 minutes, followed by digestion with chicken pancreas at pH 7.2 and 37°C for 24 hours (see Flynn et al. 1951).
2.8.8. Inositol
The determination of inositol was performed with Saccharomyces carlsbergensis strain ATTC 9080, after extraction of the samples with 0.6 n sulphuric acid at 120°C for 2 hours. In the medium used by Atkin et al. (1943) the inositol was replaced by 50 meg pyridoxine in 100 ml of the basal medium.
2.9. Elements in ash 2.9.1. Introduction
The various elements were determined by means of atomic absorption spectro-photometry (AAS), using a Perkin Elmer 303 spectrophotometer. The test solutions were prepared with an internal standard after destruction of the samples. For some elements this destruction was effected by ashing at 550°C, using the method described in AOAC Methods (1965), and for others it was carried out by boiUng the sample with a mixture of concentrated sulphuric acid and nitric acid, followed by boiling with distilled water to remove the excess of nitric acid. The methods applied were modifications of the methods described in detail in the literature cited below.
2.9.2. Calcium and magnesium
After dry destrucfion the method of Belcher and Brooks (1963) was applied, using a solution of 5% lanthanum chloride in 25% hydrochloric acid for calcium but not for magnesium.
2.9.3. Copper and iron
These elements were determined after wet destruction, using the method described by Allen (1959).
2.9.4. Manganese and zinc
After wet destrucfion these elements were estimated according to the method elaborated by Buchanan and Muraoka (1964).
2.9.5. Nickel
After dry destruction of the sample this element was determined by the method described by Kinson and Belcher (1964).
2.10. Content of ribo-nucleic acid (RNA) and purine base content
The determination of yeast RNA was carried out by preparing a test solution, the extinction of which at 260 nm and 300 nm was compared with that of a standard solution of yeast RNA (Boehringer).
The test solution was obtained by the extraction procedure according to Schmidt and Tannhauser (1945). However, the fat extraction was omitted.
For the preparation of the purine bases, the sample was hydrolyzed with 1 n sulphuric acid in sealed tubes (Vischer and Chargaff, 1948).
The determination of adenine and purine was done spectrophotometrically after separation, using an ion exchange column according to the procedure described by Bonnelycke (1969).
3. Sanitary analysis of yeast 3.1. Introduction
The test described in the following are of two different types, namely a series of counting tests and a series of present/absent tests.
It is a rather arbitrary decision whether a test should be done by the former or latter method. For Enterobacteriaceae, for instance, a present/absent test is proposed in Guideline No 5 of the Protein Advisory Group (PAG) (1969 - 1972) but, of course, plate counting gives more information when bacteria of this type are present. In Guideline No 5, the following specifications or conditions are suggested for the present/absent tests:
Salmonella absent in 25 g Arizona absent in 25 g Shigella absent in 25 g Escherichia coli absent in 10 g Staphylococcus aureus absent in 1 g Enterobacteriaceae absent in 0.1 g
For the tests concerned, the amounts of samples have to be chosen in agreement with these specifications.
3.2. Total counts of aerobic bacteria and spores
Starting with 10 g/100 ml pepton—salt suspension (PES), suitable dilutions were made and 1 ml thereof was plated into a tryptone pepton agar, according to the method described by Wetzler et al. (1962), and incubated for 3 days at 3I± 1°C. In these tests the total number of aerobic bacteria in so far as these are present in the samples as single cells, and the number of spores of such bacteria in so far as the spores germinate under the experimental conditions applied, are obtained.
In the case of spore counting, the tubes, containing the suspensions, were heated in a waterbath until the thermometer in a parallel test tube, containing merely water, reached a temperature of 79°C. Then the tubes were transferred immediately to a waterbath of 80°C, in which they were kept for exactly 1 minute. The tubes were chilled rapidly to approximately 10°C in a beaker supplied with running tap water. With the suspensions in the tubes, the same procedure was performed as described for the aerobic bacterial count.
3.3. Enterobacteriaceae count
For this test we used the method described by Mossel (1970). A suspension containing 10 g yeast in 100 ml tryptone soya pepton glucose broth was shaken at 20°C for two hours to resuscitate sublethally impaired cells. Subsequently, a number of decimal dilutions were made and 1 ml of these was plated on violet red bile glucose agar, at a temperature of 47°C. After soHdification the plates were covered with a second layer of approximately 20 ml violet red bile glucose agar to suppress strictly aerobic gram-negative rods. After incubation at 36°C for 20 h, the number of purple colonies surrounded by purple haloes of precipitated bile salts was counted.
3.4. Escherichia coli present/absent test
This test was performed after resuscitation of sublethally impaired cells for 2 h at 20 C in a tryptone soya broth suspension of 10 g yeast/100 ml. The suspension thus obtained was enriched in 100 ml of double concentrated brilliant green bile lactose broth at 30°C for 24 h. Cultures, that did not show any gas production were discarded. The gas-positive enrichments were subcultured onto McGonkey agar no 3 and incubated overnight at 44±0.1°C (Thomas 1971). Typical lactose-positive colonies were examined by means of a modified Eijkman test (McKenzie et al. 1958).
3.5. Salmonella present/absent test
Following the method of Edel and Kampelmacher (1969), suitable aliquots of resuscitated cell suspensions were enriched in Muller—Kaufmann broth, incubated at 42±0.5°C. After 24 h and 48 h the suspensions were streaked onto brilliant green phenol red lactose sucrose agar. After incubation at 37°C for 24 to 30 h, the colonies surrounded by red haloes were examined for the following properties: fermentation of glucose, absence of j3—galactosidase and urease activities, presence of lysine decarboxylase and oxidase and, finally, specific agglutination reactions.
3.6. Counts of Clostridium group and Clostridium perfringens
For this test, plastic pouches were used as described by De Waart and Smit (1962). Suitable dilutions of the cell suspensions concerned were mixed with sulphite iron polymyxin agar and poured in the plastic pouches. These were sealed and incubated
for24to48hat3rc.
For selecting Clostridium perfringens in principle the same method was applied. This time, however, sulphite iron polymyxin neomycin agar was used and the incubafion occurred at 46±0.2°C for 24 to 48 h (Mossel and De Waart, 1968).
3.7. Lancefield group D Streptococci count
According to the method of Pike (1945), 0.1 ml of appropriate cell dilutions was spread onto crystal violet azide agar (Streptosel BBL, Md USA). After 2 days of incubation at 37°C, a number of typical colonies (at least three) were examined for the following characteristics: catalase negative cocci, usually in short chains, capable of rapid growth at 45°C and acid formation from glucose without gas.
3.8. Staphylococcus aureus present/absent test
1 ml of serial dilutions was added to 20 ml of tellurite glycine broth (Giolitti and Cantoni, 1966) in tubes; these were sealed with sterile paraffin and incubated at 37°C for 48 h. The presence of Staphylococcus aureus in blackened tubes was confirmed by plating 0.1 ml onto Baird-Parker's tellurite glycine egg—yolk agar, followed by testing both egg—yolk positive and negative isolates for coagulase activity (De Waart et al., 1968).
3.9. Moulds
Suitable decimal dilutions were plated on oxytetracycline glucose yeast extract agar. Incubation occurred for 3—5 days at 22°C ± 2°C before counting the mould colonies appearing on the plates.
4. Nutritional testing of yeast 4.1. Protein calories per cent (PCP)
The energy available to the human body is the gross energy of the foodstuff minus the pertinent losses in urine and faeces.
According to At water (Merill and Watt, 1955), these losses are allowed for by applying 17 kJ per g for protein, 38 kJ per g for fat and 18 kJ per g for carbohydrates, expressed as monosaccharides.
To calculate the PCP — which truly should be called 'protein Joule per cent' — the exact figures have to be determined for protein content, fat content and carbohydrate content expressed as monosaccharides.
When the carbohydrates are not determined by analysis, but calculated by difference, the factor should be 16 kJ instead of 18 kJ (FAO/WHA report No 52,
1973).
Southgate and Durnin (1970) have compared the calculated energy value with that obtained by bomb calorimetry analysis, and they found a quite good agreement between both values. The PCP can be calculated as the percentage protein energy of the total energy of the foodstuff.
4.2. Chemical score and EEA—Index 4.2.1. Introduction
The nutritional value of proteins is due to the amino acids they yield on digestion. The relation between the chemical composition of the amino acid mixture found after hydrolysis and the biological activity of the amino acids concerned may be disturbed by the fact that the amino acids in dietary proteins may only be partly available biologically. Particulariy after heat treatment this factor may be of great influence.
The determination of the amino acid spectrum is mentioned in Chapter II 2,7. The nutritional value based on this spectrum will be discussed below.
4.2.2. Chemical score
Mitchell and Block (1946) introduced the concept of a 'Chemical score' for proteins on the basis of the amino acid composition of the proteins concerned. They used whole egg protein as a standard, and expressed the concentration of each essential amino acid (g per 16 g N) in the product under investigation as a percentage of the concentration of the same amino acid in the whole egg protein. The lowest value obtained was taken as the 'Chemical score' because the limiting amino acid determines the nutritional value of the product.
On the basis of the determination of the Chemical score of a series of foods and the determination of the net protein utilization of the same foods, Mitchell and Block (1946) came to the following equation: y = 102 - 0,634X in which y means the biological value, and X the percentage amino acid deficit in comparison to the standard protein.
They also pointed out that there is no relation between amino acid deficits and coefficients of digestibility. Miller et al. (1965) found that a heat treatment of dried cod fillets reduced their Chemical score by 15%. However, the reduction of the nutritional value for rats was nearly 40%. It is therefore evident that the Chemical score has its limitations as a measure of the biological value of proteins. Since the pubUcation of Mitchell and Block (1946), other investigators have suggested to abandon whole egg protein as a standard and to replace it by a synthetic amino acid mixture, which is more efficiently used by young rats (Bender, 1958, 1965).
Table II 4—1 Essential amino acid patterns (g/16 g N) considered as a standard for calculating of the Chemical score
F A O - s t a n d a r d s Provi-sional 1957 4.3 4.9 4.3 Cow's milk 1973 4.7 9.5 7.8 Hen's egg 1973 5.4 8.6 7.0 Provi-sional 1973 4.0 7.0 5.5 5.8 4.3 2.9 1.4 4.3 10.2 3.3 4.4 1.4 6.4 9.2 5.6 4.7 1.7 6.6 6.0 3.5 4.0 1.0 5.0 Isoleucine Leucine Lysine Phenylalanine Total aromatic amino acids Total sulphur amino acids Threonine Tryptophan Valine Block and Mitchell 1946 8.0 9.2 7.2 6.3 10.8 6.5 4.9 1.5 7.3 Bender 1965 4.3 7.8 5.2 4.9 . -4.7 4.1 1.0 5.0 Oser 1951 7.7 9.2 7.0 6.3 . -6.4 4.3 1.5 7.2
The FAO (1957, 1970, 1973) finally has suggested to adopt a provisional amino acid pattern as a reference standard for calculation of the Chemical score. Table II 4—1 gives the various standards for the essential amino acids.
It is evident that the slope of the graph from which the equation BV = 102 - 0,634X is derived will change considerably when, instead of the values of Mitchell and Block (1946) the 1973 values of the FAO are used. In most cases the amino acid isoleucine as a limiting amino acid is replaced by the sulphur—containing amino acids when the FAO standards are applied. The ehmination of isoleucine as Umiting amino acid in human diets is in agreement with the biological evidence that the sulphur-containing acids are the most commonly limiting amino acids in these diets. The Chemical score of the yeast grown on fats and fat products will therefore be calculated with reference to the provisional amino acid scoring pattern proposed by the FAO (1973) as given in Table II 4 - 1 .
4.2.3. Essential amino acid index (EAA-Index)
Oser (1951) has introduced the Essential Amino Acid Index (EAA-Index); it is derived from the formula:
I n 100 a log EAA-Index = — 2 log (——) n
e
in which n is the number of the essential amino acids taken into consideration and a/a is the ratio of the individual amino acid concentrations, respectively, of the food protein (a) and the egg protein (ae). The maximum value of the ratio taken into account is 1.
The difference between the Chemical score and the EAA-Index is quite evident. For example, the amino acids concerned are not the same. Histidine and arginine are being considered in the case of the EAA-Index, whereas tyrosine is left out. However, the values of the EAA-Index do not vary markedly whether histidine and argnine are included or not.
An interesting equation is that giving the relation between the biological value (BV) and the EAA-Index of a protein: EAA-Index = 0.877 BV + 7.38 Oser (1951) claims that statistical analysis of the relation between the EAA-Indices and the biological values shows that the EAA-Index can be used to predict the BV-value of a protein within —5 to +11 per cent.
In Table II 4 - 1 the ae-values used by Oser (1951) are also given; it is evident that these figures differ considerably from the values suggested by the FAO (1973). Nevertheless, in addition to the Chemical score the EAA-Index can to a certain extent be considered as a measure of the nutritional value of a food protein.
4.3. Protein efficiency ratio (PER) 4.3.1. Introduction
Eggum (1965) proved experimentally that different amino acids from the same protein source can be digested in different ways. For that reason reliable figures for the biological availability of individual amino acids cannot be obtained from the digestibility estimated by analysis of total nitrogen. This makes a combination of the chemical analysis with biological tests imperative.
The most simple biological screening test is that of the protein efficiency ratio (PER), which uses the growth of laboratory animals as a parameter.
However, it is known that growth is not always a reliable measure of protein synthesis. Moreover, the results of the efficiency tests vary with the level of protein in the diet and, finally, the results differ with the food intake, which, in turn, is dependent on palatability and other factors. These facts present some drawbacks in the interpretation of the PER as a measure of the nutritional value of a foodstuff. There are two more difficulties. Firstly, the casein in the reference diet can be different in the various experiments performed all over the world. Secondly, the response of the rats suppUed by different breeders is not the same througliout, even if the animals are of the same species. For the reasons mentioned above, the experimentally found PER-value is multiplied by a certain factor relating it to a standard value for the casein diet (see II 4.3.2.).
4.3.2. The performance of the test
The determination of the PER-value is carried out as described by Derse (1958). The dry sample is incorporated as the only source of protein into a complete diet at a level providing 10% crude protein (N x 6.25). A diet which contains casein, also at 10% crude protein level, is used as a standard diet. Both diets are each fed to ten newly weaned male rats kept individually in wire screen cages. Food and tap water are provided ad libitum. Food consumption and body weight are recorded at one-week intervals during a period of 4 weeks.
From the amount of food consumed, and the gain in bodyweight of each rat, the PER is calculated. The value of the PER obtained with the casein diet is converted to 2.50, and the experimental value for the other diet is multiplied with the same conversion factor, thus relating the PER to the standardized value of 2.50 for casein. Because of the drawbacks mentioned in II 4.3.1., and the lack of sufficient amounts of yeast, the PER-value was not determined.
4.4. Net protein utilization (NPU), true digestibility (TD) and biological value (BV)
4.4.1. Introduction
In addition to the chemical composition and the results of biological screening tests more quantitative information is needed in order to characterize the nutritional value of a protein foodstuff. In fact, the percentage of absorbed nitrogen used for the synthesis of body protein is the true biological value (BV). This can be measured when metabolic and endogenous losses are taken into account.
Thomas (1909) and Mitchell (1923) have expressed this in the formula: _ I - ( F - F k ) - ( U - U k ) _ B - B k
I - ( F - F k ) I - ( F - F k ) where:
I = nitrogen intake F = faecal nitrogen
Fk = endogenous faecal nitrogen i.e. on nitrogen—free diet U = urinary nitrogen
Uk = endogenous urinary nitrogen i.e. on nitrogen—free diet B = body nitrogen after intake of test—protein diet Bk = body nitrogen after intake of nitrogen—free diet
A modified method, known as the carcass nitrogen method, was introduced by Bender and Miller (1953) in order to get a measure of the BV. According to this method, the BV is not measured directly but derived from the percentage of consumed nitrogen used for the synthesis of body protein, and the percentage of consumed nitrogen not excreted in the faeces. Applying this principle they have introduced two new formulas:
R — Ri
(a) NPU= —j—^xlOO I - (F-Fk) (b) TD = ^ xlOO where the designations are as given above.
The net protein utilizafion (NPU) value is obtained by comparing the nitrogen content in the carcasses of rats, fed with the test diet, with the nitrogen content in the carcasses of rats on a nitrogen-free diet.
In fact this value is a product of digestibility and biological value. After determination of the digestibility, the biological value can be calculated according
NPU to the formula: BV = - ^ x 100.
The digestibility is called true digestibiUty (TD) when the value of the faecal nitrogen is corrected for endogenous nitrogen losses; this can be done by measuring the amount of nitrogen excreted in the faeces of animals fed with a nitrogen-free diet.
It is generally assumed that rats have a standard endogenous nitrogen value and that their carcasses do not become oedematous within a test period often days.
4.4.2. Performance of the test
The determination of the NPU value is based on a publication of Miller and Bender (1955).
The dry test sample was incorporated as the only source of protein into a complete diet at a level providing 10% crude protein (N x 6.25). A diet containing no protein was used in order to take into account the endogenous nitrogen losses. The 27—29 days old rats were kept in groups of four in wire screen cages. During a pre-test period of seven days, the rats received a stock diet and tap water ad libitum. The body weights were recorded during these days and abnormally low weight—gaining animals were replaced.
Both diets were fed ad libitum to three groups of four pre—selected rats (usually two males and two females). The food consumption was recorded and the total amount of faeces was collected from each group of twelve. After an experimental period of 10 days, the following determinations were made:
(a) amount of nitrogen consumed; (b) amount of nitrogen present in carcasses; (c) amount of nitrogen excreted in the faeces.
We determined the nitrogen content of the carcasses indirectly from the total content of body—water (found as a loss of body weight after three days' drying at 105 C) because in previous experiments the relation between the contents of nitrogen and body water had proved to be constant.
The amount of nitrogen in the faeces was estimated after dehydration, weighing and grinding of the combined faeces pellets of the twelve rats on the same diet. The carcass nitrogen was derived from the water content of the three groups of four rats separately.
In order to obtain a value for endogenous nitrogen, which is necessary to calculate the true instead of the apparent digestibility of a food stuff, the faeces of two groups of rats, fed on different diets, have to be compared. It can be expected, however, that the food intake as well as the amounts of faeces produced are not the same for both groups. For that reason the nitrogen, present in the faeces of the group fed on a nitrogen-free diet, must be corrected before it can be substracted from the nitrogen present in the faeces of the test group. This correction is made by multiplication with a factor equal to the ratio of food intake of both groups.
The method is based on the consideration that in most experiments there is a constant relation between food intake and the amount of faeces, the latter being an
