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Testing procedures for determination of nutritional value
E.J. van Weerden
Institute for Animal Nutrition Research (ILOB), Wageningen, Netherlands
Micro-organisms such as yeast, bacteria, fungi, or algae are the single-cell proteins used for most bioconversion of wastes or other substrates to make food or feed. A crucial question is: What is the nutritional value for man or animals of the final product of the bioconversion process? A second important aspect is the toxicological status of the product. This is the subject of papers by Scrimshaw and Shacklady appearing elsewhere in these proceedings, and my presentation is based on the assumption that the materials are acceptable toxicologically. I will consider some points that must be taken into account when evaluating a bioconversion product for animal feeding.
The main reasons for using micro-organisms in the conversion of agricultural residues are: First, to degrade that part of the residue that is not available for absorption by animals or man when the material is fed as such. In most cases this means that the enzymes secreted in the animal or human gastro-intestinal tract cannot, or are insufficiently able to, break down the material into components that can be absorbed. This pertains to cellulosic, hemicellulosic, and ligno-cellulosic components. The second purpose is to upgrade the nutritional quality of the residue by increasing its protein content, or, for monogastric animals and man, raising its content of essential amino acids.
Of the four categories of micro-organisms involved in bioconversion processes (yeasts, bacteria, fungi, and algae), a considerable amount of information is available about the nutritional value of yeasts. Species of yeast have been used for many years as a valuable component of animal feeds, supplying proteins and certain vitamins. In addition, some of the large-scale industrial SCP processes developed over the past ten years use yeasts that utilize hydrocarbons (i.e., paraffins) as an energy source and carbon and hydrogen for growth and synthesis of cell constituents. The results of extensive evaluation programmes show that these yeasts form a highly valuable source of protein for monogastric animals.
The second category of SCP, the bacteria, have, for many centuries, contributed to food supplies for man in an indirect manner: the protein supply of the ruminant is largely dependent on the bacteria and protozoa abundantly present in the fore-stomach of the animal, which forms, in principle, a large in vivo fermentation vessel.
Bacteria will be used in several large units being constructed for industrial protein production where methane or methanol will provide the energy. The data available show that bacterial material produced in this way also forms a highly valuable protein source.
The last two categories of SCP, the fungi and algae, have until now not been used to any extent in animal feeding, and this is why very little is known about the nutritional value of these products. The scarce data in the literature show variable results and indicate that, for monogastric animals, digestibility may be a problem. ILOB experiments with a fungal product showed reasonable results for digestibility and growth performance in pigs, but the results in poultry were unsatisfactory. Because fungi and algae will most likely be the microorganisms of choice for the small-scale bioconversion units considered in this work shop, a thorough look at the nutritional value of the material produced is essential. I would especially stress the necessity of testing nutritional value at an early stage of process development in order to be able to provide some sort of guidance for that development, for example, the choice of the micro-organism or the relevance of including a special treatment, if possible.
I shall discuss the procedures that can be applied when testing the nutritional value of new components meant for inclusion in animal feeds. Table 1 illustrates three different possible approaches. The left column shows tests used: analysis, acceptability, digestibility, and comparative feeding trials. These four different types of tests form a chronological sequence of steps in the evaluation procedure. In the columns on the right the efforts involved in each of the steps are given. The figures mentioned in these columns are calculated from the real costs of the experiments under Western European conditions. For the animal experiments they reflect the type of equipment needed, number of animals involved, degree of sophistication, etc. All figures mentioned are calculated as percentages of the total of "Efforts" column I - that is, the total of the efforts involved in a complete, elaborate nutritional evaluation of an SCP developed for production in large industrial operations in highly industrialized countries. It must be realized that the number 100 means, according to standards of the wealthy Western countries, a sum on the order of 400,000 - 500,000 Dutch guilders or US$200,000 - 250,000, and only involves the nutritional evaluation per se, not special toxicological determinations, or time-consuming, expensive multi-generation studies.
"Efforts I" is the most sophisticated evaluation procedure. An evaluation always starts with an elaborate analysis step, including determination of the major components: protein, fat, ash, carbohydrate, but preferably also amino acids, macro elements, and the more important minor elements. Because of the very modest costs involved, it is advisable to perform as many of the relevant analyses as possible, as a complete analytical profile can provide much early information about the potential nutritional value of the product to be tested.
The second step, acceptability trials, includes small, short-run tests with chicks and pigs. Sheep are not included in column I because, in highly developed countries, SCP is usually too expensive a source of protein for ruminants. These acceptability studies determine whether the inclusion of a moderate and a high dose of the test-product in the diet affects feed intake, faeces consistency, etc., as indicators of digestibility and general state of health. Weight gain is also measured as a first, very rough estimate of nutritional value.
The third step in the evaluation programme, determination of digestibility, is a very important one because the result is, to a great extent, a determination of the nutritional value of the product under test. In column 1, complete digestibility trials with chicks and pigs are anticipated. In these trials energy value (metabolizable energy for chicks, digestible energy for pigs) is also determined.
After the results of these first three phases of the programme have become available, a reasonably reliable prediction of nutritional value can be given, provided the test product does not contain specific negative factors not discovered in the acceptability and digestibility trials. The prediction is verified in the last, fourth phase: the comparative feeding experiments. In addition to chicks and pigs, these trials also include an experiment with laying hens. In these experiments, the test product replaces part or all of the usual high-protein components in the diet in order to see how it affects weight gain, egg production, feed conversion efficiency, and product quality.
I should like to emphasize that phase 2 (acceptability) and especially phase 4 (comparative feeding) allow inclusion of toxicological determinations, because the target animals are consuming moderate to high levels of the test product over a prolonged period. In some programmes evaluating commercial products (ILOB work on B.P. yeast, Imperial Chemical Industries, Ltd. [ICI] in the evaluation of the ICI bacterial product), multi generation feeding studies with laying hens and breeding pigs were also included in order to assess the reproductive capacity of these farm animals, and to detect any long-term effects.
TABLE 1. Three Approaches for Determination of Nutritional Value of New Components for Animal Feed
|Type of test||I||II*||III*|
Percentage of the total cost of scheme I attributable to individual components. 100% represents 400,000 500,000 guilders or us$200,000 - 250,000 at 1978 prices.
Moderately simplified and greatly reduced evaluation schemes are given in columns 11 and l l l respectively, although these are based on the same principles and follow the same sequence of trials as in the sophisticated, elaborate evaluation scheme of column 1. In all stages of the animal experiments, pigs and sheep are given as an alternative, depending on local conditions. It is assumed that the efforts involved in experiments with pigs and sheep are approximately equal. In both reduced schemes, phases 1 (analysis) and 2 (acceptability trials) are maintained to the same extent because they are relatively simple and cheap and yield much useful information. In phase 3, determination of digestibility, the study is considerably simplified because the test is carried out with only one level of the test product in the diet in one test period instead of the two test periods used in column 1. The result of this simplification is that the figures for digestibility become less reliable, but when the study is carefully done, a good measurement of nutritional value can still be obtained. It is possible that an even simpler test for digestibility could be used by an in vitro method, but it remains to be determined whether existing in vitro methods give reliable results with the kinds of SCP products under discussion in this paper.
TABLE 2. Effects on Growth and Mortality in Chicks Fed Fishmeal and Yeast Diets with and without Vitamin E and Arginine
0 - 5 weeks (%)
|30% Fishmeal + vitamin E||95||5|
|30% Fishmeal + vitamin E + arginine||122||6|
|30% Yeast + vitamin E||58||47|
|30% Yeast + vitamin E + arginine||68||63|
The experiments mentioned in phase 4, comparative feeding trials, are also simpler in terms of number of animals per trial and duration of the trials. In the moderately reduced scheme 11 a six-month experiment with laying hens fed one level of test product is still included; in scheme III it is omitted, and it is assumed that the results in chicks will give a sufficient predictability of the effects on laying hens.
The total effort involved in scheme 11 is only 30 per cent of that in scheme I and it is reduced to 11 and 19 per cent, respectively, in schemes III a and b. The difference between schemes III a and b is as follows. In scheme III a it is assumed that chicks will provide satisfactory information on digestibility. A further testing of the product in pigs or sheep can be omitted because it is very likely that a product showing satisfactory results in poultry will have an equally good, or even better, nutritional value for pigs or sheep. In scheme III a the chick test of digestibility is followed only by a five week comparative (chick experiment) feeding study with chicks.
When the chick digestibility test does not show promising results, we use scheme III b and continue with a check of digestibility in pigs or sheep and, if relevant, follow this with a comparative feeding trial in pigs or sheep.
The schemes shown in the table, of course, do not mean that these are strictly defined programmes that must be used, but they can be considered as guidelines that can be followed, depending on local conditions and circumstances. As has been noted before, the scheme indicated in column I is a full-scale, elaborate nutritional evaluation programme of an SCP from a large industrial operation. The moderately simplified scheme 11 can be used to test a product from a small industrial plant, for example, a regional co-operative.
TABLE 3. Effects on Growth and Mortality in Chicks Fed Fishmeal and Yeast Diets with and without Selenium
0 - 3 weeks (%)
|30% Fishmeal + 0.2 ppm selenium||101||3|
|30 % Yeast||70||53|
|30% Yeast + 0.2 ppm selenium||80||2|
The markedly trimmed schemes III a and b may be used in the evaluation of material from a village or multi-village unit.
In conclusion, I should like to present one example to illustrate that, when evaluating a new product, care must be taken not to confuse nutritional defects with toxicity. During the nutritional evaluation of yeast grown on e-paraffins or gas oil, an experiment was carried out in which chicks were fed semi-synthetic diets with fishmeal or Yeast, respectively, as the sole source of protein. After four weeks, about 50 per cent of the yeast-fed chicks had died, whereas only one of the fishmeal-fed chicks had dies. Because at that stage of the yeast evaluation we already knew from the results of rat and other chick experiments that true toxicity was a very unlikely explanation for the phenomenon observed, we undertook a closer look at the nutritional characteristics of the yeast, as summarized in Tables 2-4.
In the experiment just described, many birds showed symptoms pointing to vitamin E and/or arginine deficiency, so the effect of the addition of vitamin E and arginine was studied. In the fishmeal-fed controls, mortality was always low but adding arginine improved weight gain distinctly. In the unsupplemented yeast group, mortality was 70 per cent and weight gain 50 per cent of that in fishmeal-fed controls. Addition of vitamin E improved weight gain only slightly, but mortality was considerably lower. Addition of both vitamin E and arginine effected a further improvement in growth, but mortality rose once again.
The problem was for the most part solved in the following experiment by the addition of 0.2 ppm selenium. Vitamin E and arginine were added to all rations. Whereas selenium had hardly any effect on chicks consuming the fishmeal diet, it dramatically reduced mortality of chicks on the yeast diet. In addition, weight gain was improved by 10 per cent, although still lagged 20 per cent behind the fishmeal-fed controls (Table 3). This fairly considerable growth depression proved to be caused by the extremely fine, sandy structure of the yeast, which apparently limited feed intake when the diet was fed as meal.
TABLE 4. Effects on Growth and Mortality in Chicks Fed Fishmeal and Yeast Diets in the Form of Meal or Pellets
0 - 3 weeks (%)
The problem could be completely overcome by pelleting the final feed, as shown in Table 4, which gives the results of an experiment in which meal and pelleted diets were compared. Selenium, arginine, and vitamin E were added to both meal and pelleted diets.
This example was chosen because it gives such a clear illustration of the dramatic effects of a combination of nutritional imbalances, in this case, selenium, vitamin E, and arginine, and effects of the structure of the feed, that might easily have been considered to be symptoms of real toxicity. In this example, the effects were somewhat exaggerated because of the use of semi-synthetic diets in which the test product was the only protein source. In the usual, less
comprehensive evaluation schemes, where the experimental conditions are less extreme - i.e., more realistic test diets and mixtures of proteins - the effects would be less marked, but nonetheless important.
The moral of the story is that, in the process of evaluating a new product, it is essential for the nutritionist and the toxicologist to co-operate as closely as possible. A reliable toxicological evaluation of a product can only be done when the main nutritional characteristics of that product are known; on the other hand, the nutritionist must know all the available toxicological data before he can complete the nutritional evaluation.
Finally, I should like to emphasize once more that very little is known about the nutritional and toxicological properties of fungi and algae, the micro-organisms most relevant for bioconversion processing of agricultural residues. In my opinion, we should urge research in the field as strongly as possible. As important research targets regarding nutritional value, especially for monogastric animals, I would include: Determination of the nutritional value of different relevant species of fungi and algae grown on different substrates and under different conditions; investigation of the influence of the cell wall on this nutritional value and, in connection with this, the effect of special processing methods such as drying, milling, and pelleting.
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