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* Department of Health and Safety, and
Department of Processing, NIZO Food Research, PO Box 20, 6710 BA Ede, the Netherlands
Dairy Science, Wageningen University and Research Centre, PO Box 8129, 6700 EV Wageningen, the Netherlands
1 Corresponding author: marc.vissers{at}nizo.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: butyric acid bacteria • late-blowing • silage • aerobic deterioration
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Spores of BAB in milk originate from the farm environment. Reducing the concentration of BAB spores in farm tank milk (FTM) is an important option to prevent late-blowing of cheeses (Stadhouders and Spoelstra, 1990). Additional measures are the removal of spores from milk by bactofugation, the addition of inhibitors, such as nitrate and lysozyme, and the use of nisin-producing cheese starter cultures (Stadhouders, 1990; Waes et al., 1990; Delves-Broughton et al., 1996). To encourage farmers to produce milk with low concentrations of BAB spores, Dutch dairies have FTM analyzed monthly for BAB spores. The milk payment by the dairies to farmers is partly dependent on the result of these tests. The objective of dairy processors is to have fewer than 1,000 BAB spores/L of FTM.
Silage is the most important source of BAB spores. The spores survive the passage through the alimentary tract of the cow and are excreted with the feces. Transmission to milk occurs via fecal contamination of the cow’s teats (Bergere et al., 1968). Control of the concentration of BAB spores in silage is essential to control the concentration in FTM (Vissers et al., 2006). The preservation of forage by ensiling is achieved via the attainment of a low pH by lactic acid fermentation and the maintenance of anaerobic conditions within the silo. A rapid and sufficient decline in pH after ensiling decreases the chance of clostridial growth in silage. The rate and extent of pH decline are influenced by many factors, such as DM content, concentration of fermentable sugars and buffer capacity of the crop, and activity of the lactic acid bacteria flora (Weissbach, 1996).
The creation and maintenance of anaerobic conditions in silage is important to prevent the growth of aerobic microorganisms. However, in practice, exposure of silage to air is unavoidable. During storage, small amounts of air will penetrate the silage, for instance, because silage covers (usually plastic sheets) are not completely airtight. Calculations showed that during storage, oxygen can penetrate up to a depth of 0.2 m from the top of the silage (McGechan and Williams, 1994). After opening for feeding, air will usually penetrate via the silage face. Parsons (1991) calculated that after opening, oxygen can penetrate silage via the feed-out face up to distances of 4 m from the feed-out face. During storage and after opening of the silage, surface layers are the most sensitive to the penetration of air. The main factors influencing the extent of air penetration are the porosity and density of the silage and the rate of silage feeding (Honig, 1991). As a result of air infiltration, acid-tolerant (facultative) aerobic microorganisms start to proliferate. This aerobic deterioration process is usually initiated by yeasts, which use residual sugars and lactic acid as substrates. As this process proceeds, the silage pH rises and other less acid-tolerant microorganisms, such as molds and Bacillus species, start to proliferate (Pahlow et al., 2003).
Traditionally, high concentrations of BAB spores in FTM were associated with anaerobically unstable silages made of crops with high buffer capacity, such as grass and alfalfa (Stadhouders and Spoelstra, 1990; Weissbach, 1996). Anaerobically unstable silages are generally characterized by a high pH and high levels of butyric acid and ammonia. Corn silages are considered a less significant source of BAB spores. Because of a combination of DM, fermentable sugars, and buffer capacity, silages made of corn generally have a fast pH decline and a low final pH (<4.0). These conditions do not allow significant growth of clostridia (Stadhouders and Spoelstra, 1990). Furthermore, Stadhouders and Spoelstra (1990) found low concentrations of BAB spores in unopened corn silage silos. However, Driehuis and Te Giffel (2005) recently identified corn silage as the main source of BAB spores on 5 dairy farms that produced FTM with an elevated level of BAB spores. A relationship was suggested between the occurrence of high concentrations of BAB spores in the corn silages and aerobic deterioration because concentrations of BAB spores above 5 log10 most probable number (MPN)/g were detected in surface layers. The objectives of this study were to determine the importance of grass silages and corn silages as a source of BAB spores in the ration of dairy cows on farms in the Netherlands (survey 1) and to further investigate the relationships between high concentrations of BAB spores in corn silage and aerobic deterioration (survey 2).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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) and from areas with visible molds. From both the surface layer and the core, 10 subsamples were taken at different positions and mixed to provide composite samples. Areas with visible molds (molded spots) and the 15 cm surrounding these areas were excluded from the core and surface layer samples. Sub-samples from a maximum of 10 molded spots per silo were taken and mixed to provide a composite sample for molded spots. All samples were stored at 4°C until determination of the pH and microbial analysis within 24 h. Silage pH was determined using a Metrohm 532 pH meter (Metrohm, Herisau, Switzerland) in extracts prepared by adding 180 g of demineralized water to 20 g of silage sample and homogenizing for 2 min in a laboratory blender (Stomacher 400 Circulator; Seward, London, UK)
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). In each section, the temperature and penetration resistance (explained below) were measured in duplicate. The temperature was measured at a depth of 0.1 m using a Pt-100 probe (Ebro, Ingolstadt, Germany). In addition, the ambient temperature was measured in °C. The area characteristic temperature difference (dT) was defined as the difference between the temperature measured in the silage section and ambient temperature. Sampling for microbiological analyses and pH measurement was conducted as described for survey 1.
Penetration resistance was used as an indicator of the density of the silage silo; a higher penetration resistance indicated a higher degree of compaction and a higher density. Penetration resistance was measured using a manual penetrometer (Eijkelkamp Agrisearch Equipment, Giesbeek, the Netherlands), consisting of a manometer, a probing rod 1 m in length, and a cone of 0.0005 m2 (Figure 2). The device was pushed horizontally into the feed-out face with a constant speed of approximately 2 cm/s until the probing rod had penetrated the silage for 0.9 m. The maximum resistance during the measurement was read out from the manometer and corrected for the surface of the cone to obtain a value in N/cm2.
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The concentration of BAB spores was determined by the MPN method according to the Dutch Standard (NEN-ISO-6877; NEN, 1994). A volume of 0.1 mL of diluted extract was added to tubes containing 10 mL of sterilized milk supplemented with glucose and lactic acid. The tubes were heated for 5 min at 80°C to inactivate vegetative cells and to trigger the germination of spores. The tubes were sealed with paraffin and incubated for 4 d at 37°C. A tube scored positive if gas formation was visible after incubation.
Statistical Analyses
All microbial counts were log10 transformed to obtain log-normal distributed data. To calculate averages, the values below the detection level (detection levels: 30 BAB spores/g and 100 Y&M/g) were assigned a value corresponding to half of the detection level (i.e., 15 BAB spores/g and 50 Y&M/g). Student’s t-test was used to detect significant differences among samples (Snedecor and Cochran, 1989).
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RESULTS |
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lists the concentrations of BAB spores, the concentrations of Y&M, and the pH measured in the samples obtained in the first survey. The average concentration of BAB spores in the samples from the core of both the grass and corn silages was 3.0 log10/g. The concentrations in the core of grass and corn silages were, respectively, in 52 and 62% of the silages below 3 log10/g, the initial contamination level of silage (Pahlow et al., 2003). Based on a mathematical model of the contamination of milk with BAB spores, Vissers et al. (2006) determined that concentrations of BAB above 5 log10/g in silages fed gives a probability of more than 5% that the concentration in FTM will exceed 1,000 BAB/L. Based on this criterion, all silage cores, except for one corn silage, were considered to be of sufficient quality. The average concentrations of BAB spores in the surface layers of grass and corn silage were not significantly different (P > 0.10). However, concentrations above 5 log10/g were detected more frequently in the surface layers of corn silage (19%) than in the core of corn silages (5%), the surface layer of grass silages (5%), and the core of grass silages (0%; Figure 3).
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1 m from the surface. In general, the areas with visible molds were roughly 5 to 10 times larger in corn silages than in grass silages. Most farmers did not remove these areas from the silage ration offered to the cows.
The BAB spore concentration in the mixed silages in the barn was less than 3 log10 MPN/g at 29% of the farms and above the critical value of 5 log10 MPN/g at 10% of the farms. Remarkably, the average concentration in the mixed silage in the barn was significantly higher than the average concentrations measured in the grass core (difference = 1.2 log10 MPN/g, P < 0.001), grass surface layer (difference = 1.1 log10 MPN/g, P < 0.001), and corn core (difference = 1.2 log10 MPN/g, P < 0.001; Table 1). Also, the average concentration in the corn surface layer seemed to be lower (difference = 0.6 log10 MPN/g, P = 0.09). This is a remarkable result because the mixed silage in the barn was, for more than 90%, composed of cores and surface layers of the grass and corn silages fed.
Samples with the highest concentration of BAB spores also contained the highest number of Y&M. In both silage types, the highest concentrations of Y&M were measured in molded spots and the lowest were measured in the core. Concentrations of Y&M in samples from grass silages were lower than in samples from corn silages. Concentrations above 5 log10 cfu/g predispose a silage to aerobic stability problems (Honig, 1991). None of the core samples and 57% of the surface layer samples of grass silage contained more than 5 log10 cfu/g of Y&M. In corn silage, concentrations above 5 log10 cfu/g of Y&M were detected more often: in 76% of the core samples and in 90% of the surface layer samples.
In both silages, the highest pH values were detected in the molded spots and the lowest values were detected in the core. The pH values were higher in the grass silage than in the corn silage. Among other factors, the pH of silage after the fermentation period depends on the DM content. In general, silage pH increases with increasing DM (Weissbach, 1996). The average DM content of the grass silage sampled in survey 1 was 460 g/ kg (minimum of 250 g/kg, maximum of 600 g/kg). The range of pH values measured in the core of the grass silage (4.0 to 5.6) was typical for grass silages with this range of DM contents (Spoelstra, 1990). However, no significant correlations (P > 0.2) were observed between DM and pH or microbial concentrations measured in core samples from the grass silages. The average DM of the corn silages was 330 g/kg and varied less than the DM content of the grass silages (minimum of 300 g/kg, maximum of 340 g/kg).
Relationships Between Aerobic Deterioration and BAB Spores in Corn Silage (Survey 2)
Table 2 shows the concentration of BAB spores, concentration of Y&M, penetration resistance, dT between silage and ambient temperature, and pH measured in the 16 sections of the 8 corn silages sampled in the second survey. Significant differences were observed among the different layers (surface, second, and third; Figure 1) but not between the different segments (side, shoulder, and middle); therefore, the results are summarized per layer.
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Penetration resistance was measured as an indicator of density. The penetration resistance increased by approximately 25 N/cm2 per layer, going from the surface layer (average 25 N/cm2) to the core (average 96 N/cm2). The differences observed between the different layers were significant (P < 0.001). Within layers, no significant differences were observed, although the resistance in the shoulders (sections 4 and 10) of the outer layer tended to be lower than the resistance in the sides (sections 1 and 13, P = 0.07) and middle (section 10, P = 0.08). The results indicate that the lowest penetration resistances were measured in the layers in which the highest concentrations of BAB spores occurred.
Similar to the concentration of BAB spores, the concentration of Y&M, the dT, and the pH values were highest in the surface layer and lowest in the core. For dT and pH, the range of values measured was largest in the surface layer and lowest in the core. However, for Y&M the largest range of concentrations was measured in the core and the smallest range was in the surface layers. All sections in the surfaces layers of the 8 corn silages contained more than 5 log10 Y&M/g.
The results demonstrate that high concentrations of BAB spores and indicators of aerobic deterioration (high Y&M counts, increased dT and pH) occurred in the same layer. To confirm the relationship between high BAB spore concentrations and aerobic deterioration, the results of survey 2 were analyzed using 3 criteria for aerobic deterioration:
First, the number of sections that met each criterion or none of the criteria was counted. Second, for the sections meeting a criterion, the distribution of the concentration of BAB spores was determined. The results are presented in Figure 4.
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The relative frequency of concentrations of more than 5 log10 BAB spores/g increased with increasing signs of aerobic deterioration. The distribution of the concentration of BAB spores in the sections with a concentration of Y&M above 5 log10 cfu/g was similar to the distribution observed in all sections sampled; approximately 13% of the samples contained more than 5 log10 BAB/g and 19% more than 3 log10 BAB/g. The relative frequency of a concentration of BAB spores above 5 log10 MPN/g increased to 21% when dT was more than 5°C and to 50% when the pH of the section was above 4.4. Concentrations below 3 log10 BAB spores/g were never observed in sections with an increased pH, whereas sections that met none of the criteria always contained less than 5 log10 BAB spores/g.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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), the contribution of corn silage appears larger than that of grass silage on the farms sampled.
Aerobic Deterioration of Corn Silage and Growth of BAB
A generally accepted view is that high BAB spore concentrations in silage are associated with anaerobic instability of silage because of an insufficient pH decline during the primary fermentation phase (Pahlow et al., 2003). The results of the present study, however, show that increased BAB spore concentrations in both grass and corn silages were related to aerobic instability problems rather than to anaerobic instability problems. High BAB spore concentrations were detected particularly in corn silage samples showing signs of aerobic deterioration, such as high Y&M concentrations, high dT, and elevated pH (Tables 1 and 2). These samples were almost exclusively located in surface layers (top 50 cm) and in sections with a low density (i.e., in parts that were easily infiltrated by oxygen). Obvious signs of aerobic deterioration (e.g., visibility of molds, increased pH) were always accompanied by increased (above 3 log10 MPN/g) or high concentrations of BAB spores (above 5 log10 MPN/g; Figures 3 and 4).
The growth of strictly anaerobic BAB in aerobically deteriorated parts of silage may seem contradictory. However, microbial ecosystems with aerobic and anaerobic zones are found in many environments (e.g., in sediments, biofilms, and intestines; Brune et al., 1995; Stoodley et al., 2002; Fourcans et al., 2004). The underlying mechanism of growth of BAB in air-exposed parts of silage is presumably related to the succession of steps occurring in the process of aerobic deterioration (Pahlow et al., 2003). Oxygen penetrating the silage initiates the growth of aerobic, acid-tolerant yeasts and acetic acid bacteria. These bacteria oxidize residual sugars and organic acids, leading to an increase in pH. Because the initial concentration of aerobic microorganisms is low, oxygen initially penetrates relatively deep into the silage. But as the concentration of the aerobic microorganisms increases, the consumption of oxygen increases. As a result, oxygen penetrates less deeply into the silage, and deeper parts of the silage return to anaerobic conditions (Muck and Pitt, 1994). Consequently, just below the surface, anaerobic niches with an increased pH may develop. We hypothesize that the high concentrations of BAB spores detected in the top 50 cm of the corn silage and in molded spots were due to the frequent occurrence of anaerobic niches as a result of the mechanism described above. The growth of BAB associated with aerobic deterioration has been found previously in grass silage (Jonsson, 1991).
Oxygen can penetrate the surface layers of corn silage during the storage period and after the silo is opened (Parsons, 1991; McGechan and Williams, 1994). Additional research should be undertaken to establish whether high concentrations of BAB spores already occur during the storage period or whether the growth of BAB is initiated only after the silage is opened. The process by which oxygen penetrates into the silage and ultimately leads to the growth of BAB requires sufficient amounts of oxygen penetration into the silo and requires time. The growth rate of BAB is relatively low, 0.12/h at the optimal temperature of 37°C and pH of 5.6 (Thylin et al., 1995). The growth of BAB in silage after oxygen penetration is limited by this slow growth rate, especially because growth conditions are not optimal. During the storage period, the amount of oxygen that penetrates the silage per unit of time will be relatively low, but the time the silage is available to undergo the entire process described above is several months. After the silage is opened, presumably larger amounts of oxygen can penetrate it, speeding up the process of aerobic deterioration, but the time available for the formation of anaerobic niches with an increased pH and growth of BAB is less because the silage face is removed regularly (in the Netherlands a feed-out speed of 1.5 m per week is advised).
In the current study, silage samples have not been analyzed for organic acids. Therefore, it is unknown whether the corn silage samples with high BAB spore concentrations contained significant amounts of butyric acid, as is usually the case, for instance, in grass silage with a high BAB spore concentration caused by anaerobic instability. This subject needs further investigation in future studies.
Impact on BAB Spores in the Ration
The results of this study demonstrate that the concentration of BAB spores within the same corn silage varies strongly (from <1.5 log10/g to >7.0 log10/g). Extremely high concentrations (>5 log10 MPN/g) were found only in parts that constituted a relatively small fraction of the total mass (1 to 10%). However, these small fractions are the main source of BAB spores in the ration. If, for example, 10% of the silage fed contains 5 log10 MPN/g, the silages fed to cows will contain at least 4 log10 MPN/g. This explains why, in the first survey, the average concentration found in the mixed silage in the barn was significantly higher than the average concentrations measured in the core and surface layers of grass and corn silage, which represent the bulk of the material fed (Table 1).
One could argue that the elevated concentration of BAB spores in mixed silage in the barn may be due to growth of BAB in the period during which the feed is in the barn (generally less than 1 d). However, calculations using the theoretical growth rate of C. tyrobutyricum (Vissers et al., 2006), the most important BAB, as well as additional experiments investigating the change in the concentration of BAB spores in the feeds in the barn (results not shown) indicate that growth of BAB does not occur at this stage.
Control Measures
To control the concentration of BAB spores in the mixed silages in the barn, it is most important to prevent local growth of BAB in grass and corn silage. Growth can probably be prevented by limiting the penetration of oxygen or inhibiting the detrimental effect of oxygen penetration, for instance by addition of an inhibitor of aerobic deterioration by Y&M, such as propionic, sorbic, and benzoic acids and Lactobacillus buchneri (Driehuis et al., 1999; Kung et al., 2003). Achieving a high density of silage is the most important way to limit the penetration of oxygen. Muck and Holmes (2000) performed an extensive study to identify factors that affect the density of corn and alfalfa silages. They found that using heavier packaging tractors, lower initial layer thicknesses, and longer packing times affected silage density. A lower initial layer thickness means that each delivered load of freshly harvested corn is spread over a large area, allowing for a higher compaction of the freshly added corn before a new load is delivered. In recent years the capacity of corn harvesting machines has increased, increasing the mass of harvest silage delivered to the silo per unit of time. However, ensiling practices have not been adjusted accordingly, resulting in decreased packing times per delivery of harvested corn. This could explain why high concentrations of BAB spores have only recently been detected in corn silages. After the silage is opened, a high feed removal rate may be important because it limits the time available for aerobic deterioration, formation of anaerobic niches with an increased pH, and growth of BAB. However, in practice, complete prevention of oxygen penetration and growth of Y&M will be impossible. The highest concentration of BAB spores was found in visibly molded and deteriorated parts of the silage. During feed-out, removal of these areas is probably an effective measure to reduce the overall concentration in the mixed silages offered to the cows.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication June 7, 2006. Accepted for publication September 21, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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