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Lactic acid fermentation in silage preserved with formic acid
ANIMAL FEED SCIENCE AND TECHNOLOGY
ELSEVIER
Animal Feed Science and Technology 47 (1994) 107-124
Lactic acid fermentation in silage preserved with formic acid T. Moisio*, M. H e i k o n e n Valio Ltd., Research and Development, Meijeritie 4, 00370 Helsinki, Finland
(Received 10 June 1993; accepted 26 October 1993 )
Abstract A study has been made of silages preserved with formic acid (4.6 g kg- 1), in which the content of residual sugar in the aqueous phase was at least 10 g 1-1, thus permitting lactic acid fermentation. The mean ( + SD) pH of the direct-cut silages was 3.87 +_0.21. The factors studied accounted for most of the variation in silage acidity. In lactic acid fermentation in silage, 0.20 g acetic acid, 0.031 g ammonia-nitrogen and 0.090 g soluble nitrogen are produced with each gram of lactic acid, and 1.8 g reducing sugars are consumed. The other silage microorganisms, referred to here as spoilage microorganisms, also produce acetic acid, ammonia-nitrogen and soluble nitrogen. The production of acetic acid by the spoilage microorganisms is largely independent of the degradation of protein. For every gram of acetic acid produced by spoilage microorganisms, 3.6 g of sugar are consumed; the production of ammonia-nitrogen and soluble nitrogen does not result in a loss of sugar. For the ionisation of the amino acids released in protein degradation, 2.3 g of lactic acid are required for each gram of amino acid nitrogen. A surprising result was that the production of acetic acid increases that of lactic acid by 0.55 g for each gram of acetic acid, whereas the production of ammonia does not increase the content of lactic acid in well preserved silage. This study is based on the analysis of 61 036 silage samples which the farmers have sent to the silage laboratories.
1. Introduction An article entitled 'A T i t r a t i o n M e t h o d for Silage Assessment' (Moisio and H e i k o n e n , 1989 ) was published in this journal in 1989. By the s u m m e r o f 1991, *Corresponding author. 0377-8401/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved SSDI 0377-8401 (93)00573-E
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about 100 000 silage samples had been analysed by this method in Finland, and a digest of the results was made in the form of a description of fermentation in silage. This was published as a book (Moisio and Heikonen, 1992), in Finnish, entitled AIV-Rehun Perusteet (The Principles of AIV Silage). This article summarises several of the main sections of the book, restricting the material to well preserved silages only. Silage has been the subject of numerous books (Watson, 1939; Barnett, 1954; Eichholtz, 1960; Gross and Riebe, 1974; Woolford, 1984; Bolsen and Heidker, 1985; McDonald et al., 1991 ) and a very large number of scientific reports. The present article, however, deals only with the fermentation of good quality silages, preserved with formic acid (4.6 g kg-L). It is almost impossible to compare the results of our study with those of other authors. The results could be compared only if a large number of parallel silage samples were included in the experimental studies of other workers. Then it would be possible to calculate the interdependencies between different variables. However, reports based on sufficiently large material do not exist. For this reason, all other references to the literature, and a comparison of the present results with those of other studies, have been omitted from this article. Lactic acid fermentation in silage is a feedback-controlled regulation of acidity. The bacteria produce lactic acid, and the hydrogen ions formed when it ionises decrease the pH value of the buffer comprised of the organic acids of the grass and the compounds produced in the fermentation. When a low pH value has been reached, the bacteria are unable to produce more lactic acid, whereupon the acidification of the silage ceases. However, a slight lactic acid fermentation still continues, since basic compounds are formed continuously in the silage and they scavenge hydrogen ions. By producing hydrogen ions required for the neutralisation of the basic compounds in silage, lactic acid fermentation keeps the pH constant. The description of silage fermentation given in the present study is based completely on measurements of chemical and physical quantities. The microbial flora of the silage samples were not studied. The article examines only those silage samples in which there was a continuous potential for lactic acid fermentation, that is, with a minimum of 10 g 1-1 reducing sugars in the aqueous phase. Using this criterion, all silage samples with an unfavourable microbial population in the raw material or with a low sugar content were excluded. In this way, the remaining samples provide a slightly distorted, optimised picture of fermentation in silage. This anomaly cannot be avoided. Those samples in which the sugar remains in adequate supply and those in which it is consumed, diverge to completely different fermentation pathways. The agent in the regulation of acidity in correct fermentation is lactic acid. Acetic acid and the other volatile fatty acids are such weak acids that at the pH of good silage (3.8-4.0) only about 10% are ionised.
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2. Materials and methods
2.1. Silage In Finland there are eight silage laboratories serving milk producers. Farmers are advised to take a representative sample of the silage and then to send this to one of the eight laboratories. All these laboratories use exactly the same analytical methods, and the accuracy of the analyses is controlled continuously. For this study, all the data from the eight laboratories were collected between September 1988 and May 1991. During this time, 98 475 silage samples were analysed, of which 84 485 were preserved with formic acid (AIV II solution). Of these, 61 036 silage samples were suitable for the study of lactic acid fermentation, i.e. their aqueous phase had a sugar content of at least 10 g 1-1. Our description of lactic acid fermentation is based on these 61 036 silage samples. The raw material is usually timothy, meadow fescue and cocksfoot. The first harvest is taken in mid-June, the second during the second half of July and the third during the first half of September. All the silage samples in this study were preserved with AIV II solution, which at the recommended application rate provides 4.6 g formic acid kg-~ grass. Agricultural statistics show that in general, farmers use the correct application rates. The mean composition of the silage samples used in this study is given in Table 1.
2.2. Analytical methods The following parameters were determined by the titration method (Moisio and Heikonen, 1989 ): pH, lactic acid, acetic acid, amino acids, proportion of free (un-ionised) acids, ammonia, reducing sugars. The titration provides the sum of the lactic acid (pK= 3.8 ) and the formic acid (pK= 3.7) in moles and the result is reported as lactic acid. In the same way, the sum of the acetic acid (pK= 4.8 ) and the other volatile fatty acids (pK= 4.8-4.9) is reported as acetic acid. The Table 1 The mean composition of the silage samples analysed Dry matter (DM) (g kg- 1) pH Contents in DM (g kg- ' ) Crude protein Crude fiber Lactic acid Acetic acid Free (un-ionised) acids Amino acid nitrogen Ammonia-nitrogen Reducing sugars
223 3.87 161 276 57 9.2 58 7.2 1.2 59
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ammonia figure includes the amines. The result is reported as ammonia ( 1 tool diamine is equivalent to 2 tool ammonia). The amino acids are determined through their carboxyl groups. The amino acid nitrogen and ammonia-nitrogen together account for the soluble nitrogen. The dry matter contents were measured by oven drying, and crude protein and crude fibre by the near-infrared method (Helliim~iki and Moisio, 1983 ). The potassium, calcium and phosphorus contents of some of the silages were measured by the X-ray fluorescence method (Helliim~iki et al., 1988 ).
2.3. Calculations The distributions of the contents of fermentation products in silages are skewed and the interdependencies of the contents are complex. Getting a mental picture of a three-dimensional distribution is already extremely difficult. However, many quantities have to be calculated as functions of at least five variables. Unfortunately, the statistical data often do not satisfy the conditions specified by mathematical statistics for the use of regression and correlation analysis, for example. Multivariable linear regression analysis has to be used with special care even when mathematical preconditions for its use are satisfied. The values of variables far from the mean value have a strong effect on the regression coefficients. There is no reason to calculate partial regression coefficients from the material, in which silage quality varies extremely, because exceptionally fermented silages affect the result too much. This would yield a mathematically clear result (small standard deviation of coefficients); however, the result would represent a fermentation that might not exist at all. There is probably no continuous change from one type of fermentation to another. Because of this, the average values may be meaningless. An exceptionally large data base permits the use of simple, if somewhat less powerful from the point of view of statistical mathematics, methods of calculation. The number of variables is reduced by selecting a suitable subgroup from the statistical data. An example illustrates this best. To elucidate the effect of amino acid content on lactic acid content no selection is made with regard to these two variables. Instead, only those samples in which the other factors affecting lactic acid content (dry matter, protein content, pH, acetic acid and ammonia) have constant values, are selected. In practice, this constancy cannot be absolute, but a slight variability can be permitted. It is sufficient that the contents of irrelevant variables change so little that they have an insignificant effect on the factor being studied. In this work, the mean values of irrelevant variables were used as constant values to describe typical fermentation. The interrelationship of relevant variables (content of lactic acid and amino acids) was first examined graphically. It was observed that the relationship was linear in moderate range of variables ( _+1 SD), and therefore the linear model was chosen. The slope of the linear part of the curve describes the dependency of lactic acid content on amino acid content when other variables are constant. If lactic acid content were calculated directly as a function of six variables, it
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would be difficult to observe possible non-linearities in the absence of a visual picture. Thus, it would be easy to accept an erroneous result. In all the figures of this report, values represented by points in graphs are not derived from single silage samples but are the means of tens or hundreds of sampies. Owing to the large number of samples analysed, the standard deviations of the means are so small that they have not been presented in the figures or the text.
3. Results and discussion
3.1. Regulation of silage pH 3.1.1. Initial condition Immediately after cutting and application of AIV II, the pH of the grass is typically 4.2. Fermentation of the acidified grass does not commence immediately. The enzymes of the grass degrade protein to amino acids, which scavenge hydrogen ions. Apparently, the acid degrades the oligosaccharides and polysaccharides, because the sugar content of the grass rises before the start of the fermentation. This process probably binds hydrogen ions also. The pH value of the grass rises before the fermentation starts. A typical pH value at the start of lactic acid fermentation is 4.3. During the stall-feeding period, the average pH value of silage is 3.87 and the average content of lactic acid, produced through fermentation, is 28 g kg -1 dry matter (DM). The total lactic acid equivalent, including formic acid, is 57 g kg- 1.
3.1.2. pH distribution Direct-cut silages have a mean ( _+SD) pH of 3.87 _+0.21. The distribution is skewed: the curve at the lower pH values is steeper than at higher pH values.
3.1.3. pH and DM content Figure 1 shows the effect on pH of the DM content. Only those samples which yielded press-juice for pH measurement are included. The pH values of aqueous extracts of silage samples of DM content 300-350 g kg -1 may, depending on silage composition, differ considerably from the pH values of their press-juices. The other known factors affecting the pH value were given constant values. The activity of water at a DM content of 360 g kg- 1 is still sufficient for lactic acid fermentation. Most of the osmotic pressure and electrolytic strength are due to fermentation products, whose concentration falls as the DM content increases and the acidity decreases. Water activity, osmotic pressure and electrolytic strength do not account for the decrease in acidity which occurs as the DM content increases in the range 260-360 g kg-1. In dry silage, the aqueous phase does not flow, so that the increasing numbers of lactic acid bacteria do not spread throughout the silage. The diffusion of fermentation products is very slow. In dry silage there is no continuous three-dimensional aqueous phase in which involatile compounds could diffuse. The material surrounding the bacterial colonies becomes
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4.2
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Fig. 1. The dependency of silage acidity on dry matter content.
so acid that fermentation ceases. In the material between the colonies, however, the pH continues to rise as plant enzymes degrade protein. The average (homogenised silage) pH depends on the number of colonies and the speed of diffusion in the silage. The higher the DM content the slower the diffusion of fermentation products and substrates and the higher the average pH of the crushed silage.
3.1.4. pH and protein content As the crude protein content of the grass DM increases from 110 to 220 g kg- 1, the pH value rises from 3.81 to 3.93, when other factors affecting acidity are kept constant. However, a statistical examination of this result shows that it might be due to the content of minerals, which correlates with protein content, and in turn with the contents of organic acids and phosphoric acid derivatives, which correlate with the minerals. The results of the 3000 mineral analyses performed to date are insufficient to clarify the dependency of pH on protein content. 3.1.5. pH and temperature The average silage temperature during fermentation depends on the type of silo used (as shown in measurements made by the authors). When the other factors affecting acidity are held constant it is observed that the pH of the silage decreases by 0.02 pH unit for each degree Celsius increase. Thus, as the silage temperature decreases in winter the pH should rise. The slight rise, however, is masked by changes described in the following section.
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3.1.6. pH and time In this analysis it is impossible to avoid the effects of variable temperature. If it remained constant the changes of pH with time would be larger than those actually observed. The mean pH of both first- and second-harvest silage in September is 3.90 and in May 3.80. Thus, continual acidification is not due to slow fermentation. What probably happens is that the lactic acid bacteria of the silage become more tolerant of acidity. Pre-wilted silage (300-360 g DM kg -~ ) behaves differently. The pH of firstharvest silage in September averages 4.09 and that of second-harvest silage 4.18. The difference disappears by the end of the winter and in May the pH of both groups of silage is 4.00. In dry silage the main factors appear to be slow fermentation (slow diffusion of products of fermentation) and the development of the bacterial population. In some types of silo, the temperature of the silage remains well above freezing point even in late winter. In such silos the pH of pre-wilted first-harvest silage averages 3.81 in May, 11 months after preservation. 3. I. 7. pH and the products of fermentation It is difficult to determine statistically whether fermentation products affect the acid tolerance of lactic acid bacteria. In technical terms, the question is: do the fermentation products change the set point of the pH control system? The following conclusions are supported by the statistics. Lactic acid is merely an agent from which hydrogen ions are produced for the control of acidity. The contents of neither lactate ion nor un-ionised lactic acid have a noticeable effect on the final pH. Amino acids and ammonia have no effect on the final pH of silage. A high content of acetic acid, however, restricts lactic acid fermentation. Silage pH rises from 3.87 to 4.10 as the content of acetic acid rises from 5 to 10 g 1-1. However, acetic acid has no absolute effect on the lactic acid bacteria. Even when the content of acetic acid is 10 g 1- ~the pH range starts from a value of 3.6. 3.1.8. Examination o f the pH distribution As noted earlier, the standard deviation of the pH of well preserved direct-cut silage is 0.21, i.e. the variance is 0.044. When all the variables, discussed earlier, which affect the regulation of silage pH are held constant we obtain a variance of 0.015. Thus most of the variation of silage pH is due to variation in chemical composition, environmental conditions and time. 3.2. By-products of lactic acid fermentation The higher the pH of the silage the lower its content of lactic acid and the greater its content of acetic acid, while that of ammonia remains largely unaffected. It should not be concluded from this, however, that the lactic acid bacteria produce, in relation to lactic acid, more acetic acid and ammonia the weaker acidifiers they are. There is substantial evidence that the other microorganisms (denoted spoilage microorganisms) have greater activity in silage with increasing pH.
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In order to simplify the statistical analysis in this section, the dry matter and protein contents of the silage are held constant. We then examine three classes of silage with different acidities corresponding to pH values of 3.75, 3.95 and 4.15. The silage samples of each class are then divided up into five groups according to their lactic acid content. Titration analysis provides the sum of the lactic acid and formic acid contents. The formic acid contents have been subtracted from the titration result, so that the x-axis is a scale of fermentation-produced lactic acid only. The contents are in the units provided by the titrator, i.e. grams per litre of juice expressed from the silage. For each pH value and for each lactic acid group, the contents of acetic acid, ammonia nitrogen and soluble nitrogen have been calculated. Changes in these contents at constant pH may be assumed to depend only on changes in lactic acid content, since the activity of spoilage microorganisms, which depends on the pH, is the same on average in each lactic acid group. It can be seen (Figs. 2-4) that the contents of acetic acid, ammonia-nitrogen and soluble nitrogen are linearly related to lactic acid content at each pH value. The slopes of the lines indicate how much acetic acid, ammonia and soluble nitrogen is formed in parallel with the lactic acid. These relationships are shown in Figs. 2-4. Our calculations showed that in lactic acid fermentation in silage, 0.20 g acetic acid, 0.031 g ammonia-nitrogen and 0.090 g soluble nitrogen are produced for each gram of lactic acid, irrespective of how acid the lactic acid bacteria have made the silage. It is interesting to observe that in the decomposition of protein, amino acids and ammonia are
6 pH 3.75 pH 3.95 pH 4.15
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Fig. 2. The aceticacidcontentof silageas a functionof lacticacidcontentat differentacidities.
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0.8 pH 3.75 pH 3.95 pH 4.15 0.6 v C O
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formed in the ratio 2:1 (soluble protein is composed of amino acids and ammonia). The amounts of by-products calculated above are almost independent of the DM and protein contents of the silage, but they appear to be affected slightly by temperature and rate of acidification. The systematic variations in the amounts of by-products of lactic acid fermentation are so small, however, that they have no effect on the development of our analysis of fermentation. In Figs. 2-4, the higher the pH of the silage the higher are the lines relating byproduct to lactic acid. The position of the lines in relation to the vertical axis indicates the dependency of the activity of spoilage microorganisms on the acidity of the silage. The quantities of by-product calculated above are averages of a large number of samples. The statistical data do not permit calculation of how much the amount of by-product varies on a single sample basis. When direct-cut silage is preserved and the amount of lactic acid produced in fermentation is 28 g kg-1, acetic acid at 5.6 g kg-1, ammonia-nitrogen at 0.87 g kg- ~ and soluble nitrogen at 2.5 g kg- ~ are produced at the same time. The contents here are expressed on a DM basis. 3.3. Surplus contents The contents of acetic acid, ammonia-nitrogen and soluble nitrogen reported in the previous paragraph as originating in lactic acid fermentation are subtracted from the total contents. The remainders are reported as surplus acetic acid, surplus ammonia-nitrogen and surplus soluble nitrogen. They are not involved in the lactic acid fermentation in silage. The surplus acetic acid and most, if not all, of the ammonia-nitrogen originate from the activity of spoilage microorganisms. In this way their contents may be considered as indices of the hygienic quality of the silage. The surplus soluble nitrogen is mainly a product of enzyme activity in the grass. Figures 5-7 show how the total contents and contents of surplus acetic acid, ammonia-nitrogen and soluble nitrogen depend on silage acidity. The continuous line shows the total contents and the dashed line the surplus contents. In silage with a low pH, almost all the acetic acid and ammonia originate from lactic acid fermentation. Such silage results from vigorous lactic acid fermentation, and the high acidity prevents any activity by spoilage microorganisms. High pH silage has mainly surplus acetic acid and surplus ammonia. In such silage, the acidity has not prevented effectively the activity of spoilage microorganisms. Further, high pH silage has low contents of lactic acid and by-products of lactic acid fermentation. With regard to soluble nitrogen the position is different. Plant enzymes convert the protein to a soluble form even in very acid silage, so that the content of surplus soluble nitrogen at any acidity level is never very small. The values presented in the figures are averages of large numbers of samples. There is considerable variation. Even in the pH range 4.2-4.5 there are samoles in which neither surplus acetic acid nor surplus ammonia can be found. In con-
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trast, the most acid samples sometimes contain large quantities of surplus acetic acid and ammonia. All three surplus contents decline with increasing silage DM contents. This is
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surprising as air readily penetrates dry silage, and one would expect these conditions to encourage the growth of spoilage microorganisms. The content of surplus acetic acid also decreases with an increasing crude protein content--another surprising result. The satisfactory preservation of grass is generally thought to be more difficult with rising protein content. The contents of surplus ammonia-nitrogen and soluble nitrogen both rise with increasing crude protein content. However, they increase by a factor of only 1.5 as the protein content doubles. The content of surplus acetic acid in the DM of direct-cut silage averages 3.3 g kg-~, that of surplus ammonia-nitrogen 0.39 g kg-~ and that of surplus soluble nitrogen 6.1 g kg- ~. The surplus soluble nitrogen is comprised of 5.1 g kg- ~produced by grass enzymes, and 1.0 g kg- 1 produced by spoilage microorganisms.
3.3.1. Interdependence of surpluses Resolving the interdependence of the various surpluses is problematic, since each one is closely related to the same factors--acidity, DM and protein content. In order to elucidate interrelationships, these common factors are made invariant. When this is done, it is found that the content of surplus acetic acid correlates very weakly with the contents of surplus ammonia-nitrogen and surplus soluble nitrogen. The variance of the content of surplus acetic acid decreases by only 20% when the contents of surplus ammonia-nitrogen and soluble nitrogen are made invariant. The same is found in an approach from the reverse direction. The variances of the ammonia-nitrogen and soluble nitrogen hardly decline, even when the content of surplus acetic acid is held invariant. Thus the formation of
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surplus acetic acid depends, statistically and apparently microbiologically, only slightly on the formation of surplus ammonia-nitrogen and soluble nitrogen. The relationship between surplus ammonia-nitrogen and surplus soluble nitrogen is more complex (Fig. 8). When silages have no measurable surplus ammonia-nitrogen and no surplus acetic acid, the mean content of surplus soluble nitrogen in the DM is 5.1 g kg- 1. This soluble nitrogen is a product of enzyme function in the grass tissue, because there is no evidence of any spoilage microbial activity in these silages. When the content of surplus soluble nitrogen in the DM exceeds 5.1 g kg-1 it correlates closely with the content of surplus ammonia-nitrogen. When either one of these contents is held invariant the variance of the other decreases by 60% of its original value. The correlation is now due to the fact that spoilage microbes decompose protein to soluble nitrogen and further to ammonia-nitrogen. Figure 8 shows that amino acids and ammonia are formed in the ratio 2: 1. It is interesting to note that this ratio is approximately the same as in lactic acid fermentation (see 'By-products of lactic acid fermentation').
3.4. The content of lactic acid in silage 3.4.1. Principles In this section, the sum of the lactic acid and formic acid (calculated as lactic acid) is considered. As acids of the same strength, their physico-chemical equilibria are very similar and they are equally responsible for the neutralisation of the 'T
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Fig. 8. T h e relationship between surplus soluble nitrogen a n d surplus ammonia-nitrogen.
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basic compounds produced in the acid silage. In this connection the reader should recall Henderson-Hasselbalch's equation pH = p K - l o g ( [acid ] / [ s a l t ] ) that is [acid]/[salt ] = 10 ~pK-pH) The square brackets indicate contents in moles per litre. If 1 tool of basic compound is formed in silage o f p H 3.8 ( 1 t for instance), the pH of the silage rises. Two moles of lactic acid are required to return the pH to its original value of 3.8. This is because at this acidity, only half of the lactic acid is ionised. The mean ( _ SD) lactic acid content in the DM of direct-cut silage is 57 _+20 g kg -1, that is with a variance of 400 (g kg -1 )2. 3.4.2. Lactic acid and dry matter The lactic acid content of the DM of direct-cut silage declines slightly with increasing DM content (other silage components being held invariant). This is because of the flow of effluent, raising the DM content and removing ionising compounds from the silage. 3.4.3. Lactic acid and pH Figure 9 shows how the lactic acid content depends on acidity. In calculating the curve the contents of the by-products of lactic acid fermentation were allowed to follow the content of lactic acid, as described in the section 'By-products of lactic acid fermentation'. The contents of DM, crude protein, surplus acetic acid, surplus ammonia-nitrogen and surplus soluble nitrogen were held constant (at their respective average values). From the shape of the curve it can be calculated that silage cannot be regarded merely as a lactic acid buffer. When examining the buffering properties of silage the amino acids at least should be taken into account, and in high pH silage acetic acid also. When this is done, the properties of silage as a pH buffer may be calculated. When the pH of direct-cut silage is held invariant while the other factors are allowed to vary, the lactic acid variance falls to a value of 256 (g kg- 1)2, the content unit being on a DM basis. The original variance was 400 (g k g - l ) 2, so that pH accounts for a considerable proportion of the variation in lactic acid content, though not the major proportion. 3.4.4. Lactic acid and acetic acid IfDM, crude protein, lactic acid, amino acids and ammonia contents of silages made under similar conditions are given constant values and silages are classified according to acetic acid content, it will be found that the pH increases with rising acetic acid content. If the pH is held constant and the lactic acid content is al-
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80
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lowed to vary, the lactic acid content in the DM increases by 0.55 g kg- ~ as the acetic acid content increases by 1 g kg- 1. This is a surprising phenomenon. Even though only about one-tenth of the acetic acid is ionised at the pH of silage, acetic acid should increase the silage acidity slightly. The formation of acetic acid, however, decreases the acidity. One explanation could be that acetic acid is formed from the stronger organic acids of grass. In direct-cut silages, the average acetic acid content in the DM is 9.2 g kg-
3.4.5. Lactic acid, amino acids and peptides Under the acid conditions of good quality silage, pH 3.8-4.0, all of the amino groups of the amino acids and peptides are ionised, that is bind hydrogen ions. In the monocarboxyl amino acids there is about 97% ionisation or release of hydrogen ions. In the second carboxyl group of glutamic and aspartic acid, as with the carboxyl group of 7-aminobutyric acid, the ionisation is less than 50%. There is a paucity of information on the pK values of the carboxyl groups of the peptides, but according to the available data the pK values are so high that perhaps only about one-third of the carboxyl groups are ionised. The hydrogen ions released by the carboxyl groups are not enough to ionise the amino groups. The required hydrogen ions are obtained from the lactic acid fermentation. The same statistical procedure as was used for acetic acid leads to the result that 1 g kgamino acid plus peptide nitrogen requires 2.3 g kg-~ lactic acid to maintain a constant pH value (3.87). Direct-cut silages have an average amino acid nitrogen content in their DM of 7.3 g kg -1 .
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3.4.6. Lactic acid and ammonia When ammonia-nitrogen is submitted to the same statistical examination as was used for acetic acid and amino acids the result is surprising. In good quality silage, lactic acid is not required for the neutralisation of ammonia. It appears possible that the residue after the deamination of an amino acid is an organic acid, the pK value of which is approximately the same as that of the carboxyl group of an amino acid. When the ammonia-nitrogen (ammonia plus amines) content in the silage DM rises above the threshold value of 1.3 g kg- ~, it begins to require the theoretically specified neutralisation with lactic acid. This may be related to the replacement of amino acid deamination by decarboxylation. The ammonia-nitrogen content in the DM of direct-cut silages is on average 1.1 gkg - l . 3.4.7. Lactic acid and crude protein The lactic acid content in silage increases with increasing crude protein content, even when the amino acid and ammonia contents are held invariant. A more rigorous study shows that along with the protein content the mineral and organic acid contents generally increase, so that the buffering capacity of the grass rises. If the content of minerals is held constant it is found that the lactic acid content does not rise as the protein content increases. For the present, the data available are insufficient for the exact calculation of the effect of minerals on lactic acid content. 3.4.8. Lactic acid fermentation is self-maintaining As lactic acid fermentation proceeds the by-products acetic acid and amino acids are formed, and these require further lactic acid for neutralisation. When the overall effect of this chain reaction is calculated, it is found that the original lactic acid requirement of 1.0 g kg- ~builds up to a value of 1.4 g kg- ~ when the complete sequence of reactions is taken into account. The conditions are probably much worse in silage with no acid added as preservative. The lactic acid content of the DM of direct-cut AIV II silages averages 57 g kg- ~ (pH 3.87 ). In silage prepared from similar grass by means of enzymes and inoculants the lactic acid content, when the supply of sugar is adequate, averages about 100 g kg- ~ at the same level of acidity. Such an increase in requirement for lactic acid can be explained only by concluding that fermentation has degraded considerable quantities of grass material into basic compounds which require neutralisation. It is significant that in the silages examined the concentration of lactic acid in the aqueous phase has a mean of 0.18 mol 1- ~, whereas that of hydrogen ions is only 0.00014 mol 1-~. The basic compounds of the silage create the requirement for acid. In spite of this the silage compounds most often studied are acids, and an analysis of basic compounds is rarely made.
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3.5. Sugar consumption In this section, those silages with dry matter, crude protein and crude fibre contents close to the specified mean composition are examined. Accordingly, their raw materials were very similar. When the silages are grouped according to other factors it is probable that the average sugar contents in the raw material were the same in each group. First, the contents of all three surpluses in the silages are held invariant. In this way it can be assumed that the activities of spoilage microorganisms, which utilise the sugar of the silage, are also invariant. When the silages are classified according to lactic acid content it can be expected that the between-group differences in sugar content are due to differences in lactic acid content alone. A close linear relationship between lactic acid and sugar content is found: the sugar content declines by 1.8 g kg- 1when the lactic acid content increases by 1 g kg- 1. This consumption of sugar includes the sugar consumed in the synthesis of the byproducts of lactic acid fermentation. Second, the lactic acid content of the silages is made invariant, that is, the amount of sugar consumed in the lactic acid fermentation is held constant. The silages are then grouped three-dimensionally in relation to surplus acetic acid, surplus ammonia-nitrogen and surplus soluble nitrogen. Each of the three variables is divided up into four classes according to content. The number of classes is thus 4 × 4 X 4 = 64. The sugar content is calculated for each class, and it is observed that the sugar content decreases by 3.6 g kg-1 as the surplus acetic acid content increases by 1 g kg -1. The same quantitative relationship holds for all surplus ammonia-nitrogen classes and all surplus soluble nitrogen classes. In this three-dimensional classification, the entire consumption of sugar is involved in the production of acetic acid. According to this classification the production of surplus ammonia-nitrogen and surplus soluble nitrogen does not result in the consumption of any sugar. In this connection it should be borne in mind that the production of surplus acetic acid is only weakly related to the production of surplus ammonia-nitrogen and soluble nitrogen. Thus the microorganisms which degrade protein and amino acids do not obtain energy from sugar through the production of acetic acid. Their energy source is probably protein. In the direct-cut silages examined here, the mean content in the DM of lactic acid produced in fermentation is 28 g kg- 1 and that of surplus acetic acid 3.3 g kg-l. The total consumption of sugar resulting from the production of both silage constituents averages 62 g kg- 1.
Acknowledgements The authors thank Professor Kari Salminen, Vice President, Research and Development, Valio Ltd., for making this project possible. They also thank David Homer for the translation into English and for many valuable discussions.
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References Barnett, A.J.G., 1954. Silage Fermentation. Butterworths, London, 208 pp. Bolsen, K. and Heidker, J.I., 1985. Silage Additives USA. Chalcombe Publications, Marlow, 201 pp. Eichholtz, F., 1960. Silage und ~ihnliche G~irerzeugnisse, Friedr. Vieweg & Sohn, Braunschweig, 144 PP. Gross, F. and Riebe, K., 1974. G~irfutter. Eugen Ulmer, Stuttgart, 283 pp. Hell~im~iki,M. and Moisio, T., 1983. Prediction of protein and fiber contents in silage by near infrared reflectance analysis. Milchwissenschaft, 38:14-15. Hell~im~iki, M., Moisio, T. and Heikonen, M., 1988. S~iil6rehujen kivenn~iisille nopea m~i~irityslaite. Karjatalous, 55: 33-34. McDonald, P., Henderson, N. and Heron, S., 1991. The Biochemistry of Silage. Chalcombe Publications, Marlow, 340 pp. Moisio, T. and Heikonen, M., 1989. A titration method for silage assessment. Anita. Feed Sci. Technol., 22: 341-353. Moisio, T. and Heikonen, M., 1992. A1V-Rehun Perusteet. Kirjayhtym~i Oy, Helsinki, 170 pp. Watson, S.J., 1939. The Science and Practice of Conservation: Grass and Forage Crops. Fertiliser, Feeding Stuffs and Farm Supplies Journal, London, 820 pp. Woolford, M.K., 1984. The Silage Fermentation. Marcel Dekker, New York, 350 pp.
ELSEVIER
Animal Feed Science and Technology 47 (1994) 107-124
Lactic acid fermentation in silage preserved with formic acid T. Moisio*, M. H e i k o n e n Valio Ltd., Research and Development, Meijeritie 4, 00370 Helsinki, Finland
(Received 10 June 1993; accepted 26 October 1993 )
Abstract A study has been made of silages preserved with formic acid (4.6 g kg- 1), in which the content of residual sugar in the aqueous phase was at least 10 g 1-1, thus permitting lactic acid fermentation. The mean ( + SD) pH of the direct-cut silages was 3.87 +_0.21. The factors studied accounted for most of the variation in silage acidity. In lactic acid fermentation in silage, 0.20 g acetic acid, 0.031 g ammonia-nitrogen and 0.090 g soluble nitrogen are produced with each gram of lactic acid, and 1.8 g reducing sugars are consumed. The other silage microorganisms, referred to here as spoilage microorganisms, also produce acetic acid, ammonia-nitrogen and soluble nitrogen. The production of acetic acid by the spoilage microorganisms is largely independent of the degradation of protein. For every gram of acetic acid produced by spoilage microorganisms, 3.6 g of sugar are consumed; the production of ammonia-nitrogen and soluble nitrogen does not result in a loss of sugar. For the ionisation of the amino acids released in protein degradation, 2.3 g of lactic acid are required for each gram of amino acid nitrogen. A surprising result was that the production of acetic acid increases that of lactic acid by 0.55 g for each gram of acetic acid, whereas the production of ammonia does not increase the content of lactic acid in well preserved silage. This study is based on the analysis of 61 036 silage samples which the farmers have sent to the silage laboratories.
1. Introduction An article entitled 'A T i t r a t i o n M e t h o d for Silage Assessment' (Moisio and H e i k o n e n , 1989 ) was published in this journal in 1989. By the s u m m e r o f 1991, *Corresponding author. 0377-8401/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved SSDI 0377-8401 (93)00573-E
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about 100 000 silage samples had been analysed by this method in Finland, and a digest of the results was made in the form of a description of fermentation in silage. This was published as a book (Moisio and Heikonen, 1992), in Finnish, entitled AIV-Rehun Perusteet (The Principles of AIV Silage). This article summarises several of the main sections of the book, restricting the material to well preserved silages only. Silage has been the subject of numerous books (Watson, 1939; Barnett, 1954; Eichholtz, 1960; Gross and Riebe, 1974; Woolford, 1984; Bolsen and Heidker, 1985; McDonald et al., 1991 ) and a very large number of scientific reports. The present article, however, deals only with the fermentation of good quality silages, preserved with formic acid (4.6 g kg-L). It is almost impossible to compare the results of our study with those of other authors. The results could be compared only if a large number of parallel silage samples were included in the experimental studies of other workers. Then it would be possible to calculate the interdependencies between different variables. However, reports based on sufficiently large material do not exist. For this reason, all other references to the literature, and a comparison of the present results with those of other studies, have been omitted from this article. Lactic acid fermentation in silage is a feedback-controlled regulation of acidity. The bacteria produce lactic acid, and the hydrogen ions formed when it ionises decrease the pH value of the buffer comprised of the organic acids of the grass and the compounds produced in the fermentation. When a low pH value has been reached, the bacteria are unable to produce more lactic acid, whereupon the acidification of the silage ceases. However, a slight lactic acid fermentation still continues, since basic compounds are formed continuously in the silage and they scavenge hydrogen ions. By producing hydrogen ions required for the neutralisation of the basic compounds in silage, lactic acid fermentation keeps the pH constant. The description of silage fermentation given in the present study is based completely on measurements of chemical and physical quantities. The microbial flora of the silage samples were not studied. The article examines only those silage samples in which there was a continuous potential for lactic acid fermentation, that is, with a minimum of 10 g 1-1 reducing sugars in the aqueous phase. Using this criterion, all silage samples with an unfavourable microbial population in the raw material or with a low sugar content were excluded. In this way, the remaining samples provide a slightly distorted, optimised picture of fermentation in silage. This anomaly cannot be avoided. Those samples in which the sugar remains in adequate supply and those in which it is consumed, diverge to completely different fermentation pathways. The agent in the regulation of acidity in correct fermentation is lactic acid. Acetic acid and the other volatile fatty acids are such weak acids that at the pH of good silage (3.8-4.0) only about 10% are ionised.
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2. Materials and methods
2.1. Silage In Finland there are eight silage laboratories serving milk producers. Farmers are advised to take a representative sample of the silage and then to send this to one of the eight laboratories. All these laboratories use exactly the same analytical methods, and the accuracy of the analyses is controlled continuously. For this study, all the data from the eight laboratories were collected between September 1988 and May 1991. During this time, 98 475 silage samples were analysed, of which 84 485 were preserved with formic acid (AIV II solution). Of these, 61 036 silage samples were suitable for the study of lactic acid fermentation, i.e. their aqueous phase had a sugar content of at least 10 g 1-1. Our description of lactic acid fermentation is based on these 61 036 silage samples. The raw material is usually timothy, meadow fescue and cocksfoot. The first harvest is taken in mid-June, the second during the second half of July and the third during the first half of September. All the silage samples in this study were preserved with AIV II solution, which at the recommended application rate provides 4.6 g formic acid kg-~ grass. Agricultural statistics show that in general, farmers use the correct application rates. The mean composition of the silage samples used in this study is given in Table 1.
2.2. Analytical methods The following parameters were determined by the titration method (Moisio and Heikonen, 1989 ): pH, lactic acid, acetic acid, amino acids, proportion of free (un-ionised) acids, ammonia, reducing sugars. The titration provides the sum of the lactic acid (pK= 3.8 ) and the formic acid (pK= 3.7) in moles and the result is reported as lactic acid. In the same way, the sum of the acetic acid (pK= 4.8 ) and the other volatile fatty acids (pK= 4.8-4.9) is reported as acetic acid. The Table 1 The mean composition of the silage samples analysed Dry matter (DM) (g kg- 1) pH Contents in DM (g kg- ' ) Crude protein Crude fiber Lactic acid Acetic acid Free (un-ionised) acids Amino acid nitrogen Ammonia-nitrogen Reducing sugars
223 3.87 161 276 57 9.2 58 7.2 1.2 59
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ammonia figure includes the amines. The result is reported as ammonia ( 1 tool diamine is equivalent to 2 tool ammonia). The amino acids are determined through their carboxyl groups. The amino acid nitrogen and ammonia-nitrogen together account for the soluble nitrogen. The dry matter contents were measured by oven drying, and crude protein and crude fibre by the near-infrared method (Helliim~iki and Moisio, 1983 ). The potassium, calcium and phosphorus contents of some of the silages were measured by the X-ray fluorescence method (Helliim~iki et al., 1988 ).
2.3. Calculations The distributions of the contents of fermentation products in silages are skewed and the interdependencies of the contents are complex. Getting a mental picture of a three-dimensional distribution is already extremely difficult. However, many quantities have to be calculated as functions of at least five variables. Unfortunately, the statistical data often do not satisfy the conditions specified by mathematical statistics for the use of regression and correlation analysis, for example. Multivariable linear regression analysis has to be used with special care even when mathematical preconditions for its use are satisfied. The values of variables far from the mean value have a strong effect on the regression coefficients. There is no reason to calculate partial regression coefficients from the material, in which silage quality varies extremely, because exceptionally fermented silages affect the result too much. This would yield a mathematically clear result (small standard deviation of coefficients); however, the result would represent a fermentation that might not exist at all. There is probably no continuous change from one type of fermentation to another. Because of this, the average values may be meaningless. An exceptionally large data base permits the use of simple, if somewhat less powerful from the point of view of statistical mathematics, methods of calculation. The number of variables is reduced by selecting a suitable subgroup from the statistical data. An example illustrates this best. To elucidate the effect of amino acid content on lactic acid content no selection is made with regard to these two variables. Instead, only those samples in which the other factors affecting lactic acid content (dry matter, protein content, pH, acetic acid and ammonia) have constant values, are selected. In practice, this constancy cannot be absolute, but a slight variability can be permitted. It is sufficient that the contents of irrelevant variables change so little that they have an insignificant effect on the factor being studied. In this work, the mean values of irrelevant variables were used as constant values to describe typical fermentation. The interrelationship of relevant variables (content of lactic acid and amino acids) was first examined graphically. It was observed that the relationship was linear in moderate range of variables ( _+1 SD), and therefore the linear model was chosen. The slope of the linear part of the curve describes the dependency of lactic acid content on amino acid content when other variables are constant. If lactic acid content were calculated directly as a function of six variables, it
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would be difficult to observe possible non-linearities in the absence of a visual picture. Thus, it would be easy to accept an erroneous result. In all the figures of this report, values represented by points in graphs are not derived from single silage samples but are the means of tens or hundreds of sampies. Owing to the large number of samples analysed, the standard deviations of the means are so small that they have not been presented in the figures or the text.
3. Results and discussion
3.1. Regulation of silage pH 3.1.1. Initial condition Immediately after cutting and application of AIV II, the pH of the grass is typically 4.2. Fermentation of the acidified grass does not commence immediately. The enzymes of the grass degrade protein to amino acids, which scavenge hydrogen ions. Apparently, the acid degrades the oligosaccharides and polysaccharides, because the sugar content of the grass rises before the start of the fermentation. This process probably binds hydrogen ions also. The pH value of the grass rises before the fermentation starts. A typical pH value at the start of lactic acid fermentation is 4.3. During the stall-feeding period, the average pH value of silage is 3.87 and the average content of lactic acid, produced through fermentation, is 28 g kg -1 dry matter (DM). The total lactic acid equivalent, including formic acid, is 57 g kg- 1.
3.1.2. pH distribution Direct-cut silages have a mean ( _+SD) pH of 3.87 _+0.21. The distribution is skewed: the curve at the lower pH values is steeper than at higher pH values.
3.1.3. pH and DM content Figure 1 shows the effect on pH of the DM content. Only those samples which yielded press-juice for pH measurement are included. The pH values of aqueous extracts of silage samples of DM content 300-350 g kg -1 may, depending on silage composition, differ considerably from the pH values of their press-juices. The other known factors affecting the pH value were given constant values. The activity of water at a DM content of 360 g kg- 1 is still sufficient for lactic acid fermentation. Most of the osmotic pressure and electrolytic strength are due to fermentation products, whose concentration falls as the DM content increases and the acidity decreases. Water activity, osmotic pressure and electrolytic strength do not account for the decrease in acidity which occurs as the DM content increases in the range 260-360 g kg-1. In dry silage, the aqueous phase does not flow, so that the increasing numbers of lactic acid bacteria do not spread throughout the silage. The diffusion of fermentation products is very slow. In dry silage there is no continuous three-dimensional aqueous phase in which involatile compounds could diffuse. The material surrounding the bacterial colonies becomes
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4.2
4.1
-Q.. r
4.0
3.9
3.8
E
50
I
J
I
200 250 300 Dry matter content (g kg'l)
L
350
Fig. 1. The dependency of silage acidity on dry matter content.
so acid that fermentation ceases. In the material between the colonies, however, the pH continues to rise as plant enzymes degrade protein. The average (homogenised silage) pH depends on the number of colonies and the speed of diffusion in the silage. The higher the DM content the slower the diffusion of fermentation products and substrates and the higher the average pH of the crushed silage.
3.1.4. pH and protein content As the crude protein content of the grass DM increases from 110 to 220 g kg- 1, the pH value rises from 3.81 to 3.93, when other factors affecting acidity are kept constant. However, a statistical examination of this result shows that it might be due to the content of minerals, which correlates with protein content, and in turn with the contents of organic acids and phosphoric acid derivatives, which correlate with the minerals. The results of the 3000 mineral analyses performed to date are insufficient to clarify the dependency of pH on protein content. 3.1.5. pH and temperature The average silage temperature during fermentation depends on the type of silo used (as shown in measurements made by the authors). When the other factors affecting acidity are held constant it is observed that the pH of the silage decreases by 0.02 pH unit for each degree Celsius increase. Thus, as the silage temperature decreases in winter the pH should rise. The slight rise, however, is masked by changes described in the following section.
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3.1.6. pH and time In this analysis it is impossible to avoid the effects of variable temperature. If it remained constant the changes of pH with time would be larger than those actually observed. The mean pH of both first- and second-harvest silage in September is 3.90 and in May 3.80. Thus, continual acidification is not due to slow fermentation. What probably happens is that the lactic acid bacteria of the silage become more tolerant of acidity. Pre-wilted silage (300-360 g DM kg -~ ) behaves differently. The pH of firstharvest silage in September averages 4.09 and that of second-harvest silage 4.18. The difference disappears by the end of the winter and in May the pH of both groups of silage is 4.00. In dry silage the main factors appear to be slow fermentation (slow diffusion of products of fermentation) and the development of the bacterial population. In some types of silo, the temperature of the silage remains well above freezing point even in late winter. In such silos the pH of pre-wilted first-harvest silage averages 3.81 in May, 11 months after preservation. 3. I. 7. pH and the products of fermentation It is difficult to determine statistically whether fermentation products affect the acid tolerance of lactic acid bacteria. In technical terms, the question is: do the fermentation products change the set point of the pH control system? The following conclusions are supported by the statistics. Lactic acid is merely an agent from which hydrogen ions are produced for the control of acidity. The contents of neither lactate ion nor un-ionised lactic acid have a noticeable effect on the final pH. Amino acids and ammonia have no effect on the final pH of silage. A high content of acetic acid, however, restricts lactic acid fermentation. Silage pH rises from 3.87 to 4.10 as the content of acetic acid rises from 5 to 10 g 1-1. However, acetic acid has no absolute effect on the lactic acid bacteria. Even when the content of acetic acid is 10 g 1- ~the pH range starts from a value of 3.6. 3.1.8. Examination o f the pH distribution As noted earlier, the standard deviation of the pH of well preserved direct-cut silage is 0.21, i.e. the variance is 0.044. When all the variables, discussed earlier, which affect the regulation of silage pH are held constant we obtain a variance of 0.015. Thus most of the variation of silage pH is due to variation in chemical composition, environmental conditions and time. 3.2. By-products of lactic acid fermentation The higher the pH of the silage the lower its content of lactic acid and the greater its content of acetic acid, while that of ammonia remains largely unaffected. It should not be concluded from this, however, that the lactic acid bacteria produce, in relation to lactic acid, more acetic acid and ammonia the weaker acidifiers they are. There is substantial evidence that the other microorganisms (denoted spoilage microorganisms) have greater activity in silage with increasing pH.
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In order to simplify the statistical analysis in this section, the dry matter and protein contents of the silage are held constant. We then examine three classes of silage with different acidities corresponding to pH values of 3.75, 3.95 and 4.15. The silage samples of each class are then divided up into five groups according to their lactic acid content. Titration analysis provides the sum of the lactic acid and formic acid contents. The formic acid contents have been subtracted from the titration result, so that the x-axis is a scale of fermentation-produced lactic acid only. The contents are in the units provided by the titrator, i.e. grams per litre of juice expressed from the silage. For each pH value and for each lactic acid group, the contents of acetic acid, ammonia nitrogen and soluble nitrogen have been calculated. Changes in these contents at constant pH may be assumed to depend only on changes in lactic acid content, since the activity of spoilage microorganisms, which depends on the pH, is the same on average in each lactic acid group. It can be seen (Figs. 2-4) that the contents of acetic acid, ammonia-nitrogen and soluble nitrogen are linearly related to lactic acid content at each pH value. The slopes of the lines indicate how much acetic acid, ammonia and soluble nitrogen is formed in parallel with the lactic acid. These relationships are shown in Figs. 2-4. Our calculations showed that in lactic acid fermentation in silage, 0.20 g acetic acid, 0.031 g ammonia-nitrogen and 0.090 g soluble nitrogen are produced for each gram of lactic acid, irrespective of how acid the lactic acid bacteria have made the silage. It is interesting to observe that in the decomposition of protein, amino acids and ammonia are
6 pH 3.75 pH 3.95 pH 4.15
-0
3
1
0
5
10 15 Lactic acid (g I "1)
20
Fig. 2. The aceticacidcontentof silageas a functionof lacticacidcontentat differentacidities.
7". Moisio, M. Heikonen / Animal Feed Science and Technology 47 (1994) 107-124
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0.8 pH 3.75 pH 3.95 pH 4.15 0.6 v C O
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,
I
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i
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j
3 v C
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Fig, 4. The soluble nitrogen content of silage as a function of lactic acid content at different acidities.
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T. Moisio, M. Heikonen /Animal Feed Science and Technology 4 7 (1994}10 7-12 4
formed in the ratio 2:1 (soluble protein is composed of amino acids and ammonia). The amounts of by-products calculated above are almost independent of the DM and protein contents of the silage, but they appear to be affected slightly by temperature and rate of acidification. The systematic variations in the amounts of by-products of lactic acid fermentation are so small, however, that they have no effect on the development of our analysis of fermentation. In Figs. 2-4, the higher the pH of the silage the higher are the lines relating byproduct to lactic acid. The position of the lines in relation to the vertical axis indicates the dependency of the activity of spoilage microorganisms on the acidity of the silage. The quantities of by-product calculated above are averages of a large number of samples. The statistical data do not permit calculation of how much the amount of by-product varies on a single sample basis. When direct-cut silage is preserved and the amount of lactic acid produced in fermentation is 28 g kg-1, acetic acid at 5.6 g kg-1, ammonia-nitrogen at 0.87 g kg- ~ and soluble nitrogen at 2.5 g kg- ~ are produced at the same time. The contents here are expressed on a DM basis. 3.3. Surplus contents The contents of acetic acid, ammonia-nitrogen and soluble nitrogen reported in the previous paragraph as originating in lactic acid fermentation are subtracted from the total contents. The remainders are reported as surplus acetic acid, surplus ammonia-nitrogen and surplus soluble nitrogen. They are not involved in the lactic acid fermentation in silage. The surplus acetic acid and most, if not all, of the ammonia-nitrogen originate from the activity of spoilage microorganisms. In this way their contents may be considered as indices of the hygienic quality of the silage. The surplus soluble nitrogen is mainly a product of enzyme activity in the grass. Figures 5-7 show how the total contents and contents of surplus acetic acid, ammonia-nitrogen and soluble nitrogen depend on silage acidity. The continuous line shows the total contents and the dashed line the surplus contents. In silage with a low pH, almost all the acetic acid and ammonia originate from lactic acid fermentation. Such silage results from vigorous lactic acid fermentation, and the high acidity prevents any activity by spoilage microorganisms. High pH silage has mainly surplus acetic acid and surplus ammonia. In such silage, the acidity has not prevented effectively the activity of spoilage microorganisms. Further, high pH silage has low contents of lactic acid and by-products of lactic acid fermentation. With regard to soluble nitrogen the position is different. Plant enzymes convert the protein to a soluble form even in very acid silage, so that the content of surplus soluble nitrogen at any acidity level is never very small. The values presented in the figures are averages of large numbers of samples. There is considerable variation. Even in the pH range 4.2-4.5 there are samoles in which neither surplus acetic acid nor surplus ammonia can be found. In con-
T. Moisio, M. Heikonen / Animal Feed Science and Technology 47 (1994) 107-124
117
15
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trast, the most acid samples sometimes contain large quantities of surplus acetic acid and ammonia. All three surplus contents decline with increasing silage DM contents. This is
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T. Moisio, M. Heikonen / Animal Feed Science and Technology 47 (1994) 107-124
10
-7" v
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surprising as air readily penetrates dry silage, and one would expect these conditions to encourage the growth of spoilage microorganisms. The content of surplus acetic acid also decreases with an increasing crude protein content--another surprising result. The satisfactory preservation of grass is generally thought to be more difficult with rising protein content. The contents of surplus ammonia-nitrogen and soluble nitrogen both rise with increasing crude protein content. However, they increase by a factor of only 1.5 as the protein content doubles. The content of surplus acetic acid in the DM of direct-cut silage averages 3.3 g kg-~, that of surplus ammonia-nitrogen 0.39 g kg-~ and that of surplus soluble nitrogen 6.1 g kg- ~. The surplus soluble nitrogen is comprised of 5.1 g kg- ~produced by grass enzymes, and 1.0 g kg- 1 produced by spoilage microorganisms.
3.3.1. Interdependence of surpluses Resolving the interdependence of the various surpluses is problematic, since each one is closely related to the same factors--acidity, DM and protein content. In order to elucidate interrelationships, these common factors are made invariant. When this is done, it is found that the content of surplus acetic acid correlates very weakly with the contents of surplus ammonia-nitrogen and surplus soluble nitrogen. The variance of the content of surplus acetic acid decreases by only 20% when the contents of surplus ammonia-nitrogen and soluble nitrogen are made invariant. The same is found in an approach from the reverse direction. The variances of the ammonia-nitrogen and soluble nitrogen hardly decline, even when the content of surplus acetic acid is held invariant. Thus the formation of
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surplus acetic acid depends, statistically and apparently microbiologically, only slightly on the formation of surplus ammonia-nitrogen and soluble nitrogen. The relationship between surplus ammonia-nitrogen and surplus soluble nitrogen is more complex (Fig. 8). When silages have no measurable surplus ammonia-nitrogen and no surplus acetic acid, the mean content of surplus soluble nitrogen in the DM is 5.1 g kg- 1. This soluble nitrogen is a product of enzyme function in the grass tissue, because there is no evidence of any spoilage microbial activity in these silages. When the content of surplus soluble nitrogen in the DM exceeds 5.1 g kg-1 it correlates closely with the content of surplus ammonia-nitrogen. When either one of these contents is held invariant the variance of the other decreases by 60% of its original value. The correlation is now due to the fact that spoilage microbes decompose protein to soluble nitrogen and further to ammonia-nitrogen. Figure 8 shows that amino acids and ammonia are formed in the ratio 2: 1. It is interesting to note that this ratio is approximately the same as in lactic acid fermentation (see 'By-products of lactic acid fermentation').
3.4. The content of lactic acid in silage 3.4.1. Principles In this section, the sum of the lactic acid and formic acid (calculated as lactic acid) is considered. As acids of the same strength, their physico-chemical equilibria are very similar and they are equally responsible for the neutralisation of the 'T
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Fig. 8. T h e relationship between surplus soluble nitrogen a n d surplus ammonia-nitrogen.
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basic compounds produced in the acid silage. In this connection the reader should recall Henderson-Hasselbalch's equation pH = p K - l o g ( [acid ] / [ s a l t ] ) that is [acid]/[salt ] = 10 ~pK-pH) The square brackets indicate contents in moles per litre. If 1 tool of basic compound is formed in silage o f p H 3.8 ( 1 t for instance), the pH of the silage rises. Two moles of lactic acid are required to return the pH to its original value of 3.8. This is because at this acidity, only half of the lactic acid is ionised. The mean ( _ SD) lactic acid content in the DM of direct-cut silage is 57 _+20 g kg -1, that is with a variance of 400 (g kg -1 )2. 3.4.2. Lactic acid and dry matter The lactic acid content of the DM of direct-cut silage declines slightly with increasing DM content (other silage components being held invariant). This is because of the flow of effluent, raising the DM content and removing ionising compounds from the silage. 3.4.3. Lactic acid and pH Figure 9 shows how the lactic acid content depends on acidity. In calculating the curve the contents of the by-products of lactic acid fermentation were allowed to follow the content of lactic acid, as described in the section 'By-products of lactic acid fermentation'. The contents of DM, crude protein, surplus acetic acid, surplus ammonia-nitrogen and surplus soluble nitrogen were held constant (at their respective average values). From the shape of the curve it can be calculated that silage cannot be regarded merely as a lactic acid buffer. When examining the buffering properties of silage the amino acids at least should be taken into account, and in high pH silage acetic acid also. When this is done, the properties of silage as a pH buffer may be calculated. When the pH of direct-cut silage is held invariant while the other factors are allowed to vary, the lactic acid variance falls to a value of 256 (g kg- 1)2, the content unit being on a DM basis. The original variance was 400 (g k g - l ) 2, so that pH accounts for a considerable proportion of the variation in lactic acid content, though not the major proportion. 3.4.4. Lactic acid and acetic acid IfDM, crude protein, lactic acid, amino acids and ammonia contents of silages made under similar conditions are given constant values and silages are classified according to acetic acid content, it will be found that the pH increases with rising acetic acid content. If the pH is held constant and the lactic acid content is al-
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lowed to vary, the lactic acid content in the DM increases by 0.55 g kg- ~ as the acetic acid content increases by 1 g kg- 1. This is a surprising phenomenon. Even though only about one-tenth of the acetic acid is ionised at the pH of silage, acetic acid should increase the silage acidity slightly. The formation of acetic acid, however, decreases the acidity. One explanation could be that acetic acid is formed from the stronger organic acids of grass. In direct-cut silages, the average acetic acid content in the DM is 9.2 g kg-
3.4.5. Lactic acid, amino acids and peptides Under the acid conditions of good quality silage, pH 3.8-4.0, all of the amino groups of the amino acids and peptides are ionised, that is bind hydrogen ions. In the monocarboxyl amino acids there is about 97% ionisation or release of hydrogen ions. In the second carboxyl group of glutamic and aspartic acid, as with the carboxyl group of 7-aminobutyric acid, the ionisation is less than 50%. There is a paucity of information on the pK values of the carboxyl groups of the peptides, but according to the available data the pK values are so high that perhaps only about one-third of the carboxyl groups are ionised. The hydrogen ions released by the carboxyl groups are not enough to ionise the amino groups. The required hydrogen ions are obtained from the lactic acid fermentation. The same statistical procedure as was used for acetic acid leads to the result that 1 g kgamino acid plus peptide nitrogen requires 2.3 g kg-~ lactic acid to maintain a constant pH value (3.87). Direct-cut silages have an average amino acid nitrogen content in their DM of 7.3 g kg -1 .
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3.4.6. Lactic acid and ammonia When ammonia-nitrogen is submitted to the same statistical examination as was used for acetic acid and amino acids the result is surprising. In good quality silage, lactic acid is not required for the neutralisation of ammonia. It appears possible that the residue after the deamination of an amino acid is an organic acid, the pK value of which is approximately the same as that of the carboxyl group of an amino acid. When the ammonia-nitrogen (ammonia plus amines) content in the silage DM rises above the threshold value of 1.3 g kg- ~, it begins to require the theoretically specified neutralisation with lactic acid. This may be related to the replacement of amino acid deamination by decarboxylation. The ammonia-nitrogen content in the DM of direct-cut silages is on average 1.1 gkg - l . 3.4.7. Lactic acid and crude protein The lactic acid content in silage increases with increasing crude protein content, even when the amino acid and ammonia contents are held invariant. A more rigorous study shows that along with the protein content the mineral and organic acid contents generally increase, so that the buffering capacity of the grass rises. If the content of minerals is held constant it is found that the lactic acid content does not rise as the protein content increases. For the present, the data available are insufficient for the exact calculation of the effect of minerals on lactic acid content. 3.4.8. Lactic acid fermentation is self-maintaining As lactic acid fermentation proceeds the by-products acetic acid and amino acids are formed, and these require further lactic acid for neutralisation. When the overall effect of this chain reaction is calculated, it is found that the original lactic acid requirement of 1.0 g kg- ~builds up to a value of 1.4 g kg- ~ when the complete sequence of reactions is taken into account. The conditions are probably much worse in silage with no acid added as preservative. The lactic acid content of the DM of direct-cut AIV II silages averages 57 g kg- ~ (pH 3.87 ). In silage prepared from similar grass by means of enzymes and inoculants the lactic acid content, when the supply of sugar is adequate, averages about 100 g kg- ~ at the same level of acidity. Such an increase in requirement for lactic acid can be explained only by concluding that fermentation has degraded considerable quantities of grass material into basic compounds which require neutralisation. It is significant that in the silages examined the concentration of lactic acid in the aqueous phase has a mean of 0.18 mol 1- ~, whereas that of hydrogen ions is only 0.00014 mol 1-~. The basic compounds of the silage create the requirement for acid. In spite of this the silage compounds most often studied are acids, and an analysis of basic compounds is rarely made.
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3.5. Sugar consumption In this section, those silages with dry matter, crude protein and crude fibre contents close to the specified mean composition are examined. Accordingly, their raw materials were very similar. When the silages are grouped according to other factors it is probable that the average sugar contents in the raw material were the same in each group. First, the contents of all three surpluses in the silages are held invariant. In this way it can be assumed that the activities of spoilage microorganisms, which utilise the sugar of the silage, are also invariant. When the silages are classified according to lactic acid content it can be expected that the between-group differences in sugar content are due to differences in lactic acid content alone. A close linear relationship between lactic acid and sugar content is found: the sugar content declines by 1.8 g kg- 1when the lactic acid content increases by 1 g kg- 1. This consumption of sugar includes the sugar consumed in the synthesis of the byproducts of lactic acid fermentation. Second, the lactic acid content of the silages is made invariant, that is, the amount of sugar consumed in the lactic acid fermentation is held constant. The silages are then grouped three-dimensionally in relation to surplus acetic acid, surplus ammonia-nitrogen and surplus soluble nitrogen. Each of the three variables is divided up into four classes according to content. The number of classes is thus 4 × 4 X 4 = 64. The sugar content is calculated for each class, and it is observed that the sugar content decreases by 3.6 g kg-1 as the surplus acetic acid content increases by 1 g kg -1. The same quantitative relationship holds for all surplus ammonia-nitrogen classes and all surplus soluble nitrogen classes. In this three-dimensional classification, the entire consumption of sugar is involved in the production of acetic acid. According to this classification the production of surplus ammonia-nitrogen and surplus soluble nitrogen does not result in the consumption of any sugar. In this connection it should be borne in mind that the production of surplus acetic acid is only weakly related to the production of surplus ammonia-nitrogen and soluble nitrogen. Thus the microorganisms which degrade protein and amino acids do not obtain energy from sugar through the production of acetic acid. Their energy source is probably protein. In the direct-cut silages examined here, the mean content in the DM of lactic acid produced in fermentation is 28 g kg- 1 and that of surplus acetic acid 3.3 g kg-l. The total consumption of sugar resulting from the production of both silage constituents averages 62 g kg- 1.
Acknowledgements The authors thank Professor Kari Salminen, Vice President, Research and Development, Valio Ltd., for making this project possible. They also thank David Homer for the translation into English and for many valuable discussions.
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References Barnett, A.J.G., 1954. Silage Fermentation. Butterworths, London, 208 pp. Bolsen, K. and Heidker, J.I., 1985. Silage Additives USA. Chalcombe Publications, Marlow, 201 pp. Eichholtz, F., 1960. Silage und ~ihnliche G~irerzeugnisse, Friedr. Vieweg & Sohn, Braunschweig, 144 PP. Gross, F. and Riebe, K., 1974. G~irfutter. Eugen Ulmer, Stuttgart, 283 pp. Hell~im~iki,M. and Moisio, T., 1983. Prediction of protein and fiber contents in silage by near infrared reflectance analysis. Milchwissenschaft, 38:14-15. Hell~im~iki, M., Moisio, T. and Heikonen, M., 1988. S~iil6rehujen kivenn~iisille nopea m~i~irityslaite. Karjatalous, 55: 33-34. McDonald, P., Henderson, N. and Heron, S., 1991. The Biochemistry of Silage. Chalcombe Publications, Marlow, 340 pp. Moisio, T. and Heikonen, M., 1989. A titration method for silage assessment. Anita. Feed Sci. Technol., 22: 341-353. Moisio, T. and Heikonen, M., 1992. A1V-Rehun Perusteet. Kirjayhtym~i Oy, Helsinki, 170 pp. Watson, S.J., 1939. The Science and Practice of Conservation: Grass and Forage Crops. Fertiliser, Feeding Stuffs and Farm Supplies Journal, London, 820 pp. Woolford, M.K., 1984. The Silage Fermentation. Marcel Dekker, New York, 350 pp.