Anaerobic Spore-Formers

Anaerobic spore-forming bacteria are a potential threat to contaminate raw milk because of the spores' resistance to heat and other adverse environmental conditions. The source of most spore-forming bacteria is soil contamination of forages.

Whether a spore population will increase during ensiled storage is dependent on the specific bacteria and the micro-environments that exist in the silage mass.

It is known that spores consumed by cows will pass through the digestive tract unaffected and will be excreted in the manure. Manure-fertilized crops or dirty bedding are possible sources of contamination (Driehuis, 2013).

Clostridium species can be segmented based on their protein and carbohydrate fermentation properties. Clostridia sporogenes are the predominant proteolytic clostridia found in silages and can ferment both protein and carbohydrates. Clostridium butyricum can ferment a wide range of carbohydrates but are unable to utilize proteins. Clostridium tyrobutyricum can ferment some limited carbohydrates but are the classic "butyric acid" bacteria in their ability to ferment lactic acid to acetic acid and butyric acid at a low pH (Driehuis, 2013).

C. tyrobutyricum can be transferred from silage to milk and can grow equally well in the low-pH, low-water activity, lactic acid-rich, nitrate-poor environments of either silage or cheese. Their potential to produce off-flavors and gas formation that leads to texture defects is why silage feeding is prohibited in certain regions of Europe where milk is destined for making hard cheeses (Driehuis, 2013).

The pathogen Clostridium botulinum (botulism) is rarely found in silage since it is much more sensitive to low pH than C. tyrobutyricum. It usually is not a contaminant unless the silage is contaminated with carcasses of birds or small mammals or the crop was fertilized with poultry manure, which is notorious for containing C. botulinum spores (Driehuis, 2013).

European research shows that concentrations of butyric acid bacteria spores in fresh crops vary with the level of soil contamination from 10 to 100 spores per gram. Data from the Netherlands show that the average concentrations in corn silage are about 0.5 log units lower than in grass, likely due to less soil contamination during harvest and the low buffering capacity, rapid lactic acid formation and low pH (3.8-4.0) in corn silage that discourages the growth of C. tyrobutyricum (Driehuis, 2013).

European researchers now think that increased concentrations of butyric acid bacteria are related to aerobic instability problems rather than anaerobic instability caused by insufficient pH decline during the initial fermentation that allowed the growth of C. tyrobutyricum. This is because more recent investigations show that high spore counts are from sections of the silage where yeast and molds were already actively increasing the silage temperature and pH.

The growth of strictly anaerobic C. tyrobutyricum in aerobically deteriorated sections of the storage structure is likely due to the various aerobic and anaerobic micro-environments that can exist simultaneously in the silage mass.

In the cascade of events leading to aerobic instability, acid-tolerant, lactate-assimilating yeast grows slowly as oxygen penetrates the silage face (Feedstuffs, Dec. 10, 2007). As the yeast population increases, their consumption of oxygen also increases, resulting in anaerobic niches with increased pH where C. tyrobutyricum can thrive (Driehuis, 2013).

Aerobic Spore-Formers

Spores of aerobic, spore-forming bacteria can be isolated from a wide range of sources, including soil, silage, bedding and manure. Contamination in milk can be an issue if storage temperatures are not low enough to discourage the growth of psychrotrophic strains.

Bacillus cereus is the major spoilage organism of milk causing curdling and off-flavors. Psychrotropic strains are known to germinate and grow in milk and milk products down to 5°C (41°F). Highly heat-resistant strains such as Bacillus sporothermodurans and Geobacillus stearothermophilus can cause stability issues even in ultra-high-temperature pasteurized milk products (Driehuis, 2013).

Similar to anaerobic organisms, the primary source of these bacillus species is either soil contamination or high spore loads coming from manure used as fertilizer. They can also be isolated from the porous surface areas of aerobically unstable silage where yeasts have reduced inhibitory lactic acid levels.  

L. monocytogenes

L. monocytogenes are a facultatively anaerobic, Gram-positive bacteria responsible for causing listeriosis, particularly in young children, pregnant women, the elderly and people with health conditions that weaken their immunity.

An important feature of L. monocytogenes is that it can grow at temperatures as low as 0°C (32°F) and is rather stress tolerant, surviving for extended periods in environments where it is unable to actively grow.

Listeria can grow in cheese at both room and refrigerator temperatures and can also be spread to other cheeses that are cut and served on the same cutting board or stored in the same area as contaminated cheese. The good news is that L. monocytogenes is fairly sensitive to heat treatment and is effectively killed by pasteurization (Driehuis, 2013).

L. monocytogenes can be found in poorly fermented silages and also can be shed in the manure of animals fed contaminated silage. They are most commonly found in aerobically unstable, high-pH (greater than 4.2), low-density silages and inadequately sealed baled silages. Their presence in silage has been linked to contamination of raw milk, but if the milk has been pasteurized, food processing plant environments appear to be the major source of finished milk product contamination (Driehuis, 2013).

E. coli

The digestive tract of healthy ruminants is recognized as the main natural reservoir of E. coli, and the presumed route of possible transmission to raw milk is fecal contamination.

Fortunately, E. coli lose viability and do not grow at a pH below 4.5-5.0 and are effectively killed by pasteurization.

The presence of oxygen prolongs their survival, and some studies have shown that E. coli O157:H7 can theoretically survive and grow in poorly fermented silage and in aerobically deteriorated silage. However, it is believed that these pathogenic bacteria do not typically survive normal ensiling conditions.

Aflatoxin

This column has discussed molds and mycotoxins in the past (Feedstuffs, Oct. 8, 2007), but a few comments on aflatoxin seem appropriate given the focus of this month's column.

Aflatoxins are produced primarily by Aspergillus flavus and Aspergillus parasiticus. They appear as a grayish-green, powdery mold that may begin at the tip of the corn ear or follow insect injury tracks. Infected corn kernels are typically brownish and shrunken. Aspergillus species grow best at 14-30% moisture and 25°C (77°F). They don't grow well at less than 12°C (53°F) or greater than 41°C (106°F).

Aflatoxin M1 presence in milk is considered a potential risk to human health because of its possible carcinogenicity. The toxin has been classified as a group 1 human carcinogen. Aflatoxin M1 is the 4-hydroxylated metabolite of aflatoxin B1 that can be found in animal (and human) milk and dairy products. Aflatoxin M1 contamination of milk results mainly from the conversion of aflatoxin B1 that is metabolized by enzymes found primarily in the liver (Chase et al., 2013).

After aflatoxin M1 is formed, it is excreted in the urine and milk of the cow. It appears that 1-3% of the feed aflatoxin consumed is excreted in the milk. Research indicates that increased levels of aflatoxin in milk can be detected within 12-24 hours of consuming feeds with high aflatoxin levels. When these feeds are removed from the ration, milk aflatoxin levels decrease within one to four days (Chase et al., 2013).

Aflatoxins are the only mycotoxins currently regulated by the Food & Drug Administration, with actionable levels of no greater than 20 parts per billion in dairy feed or no greater than 0.5 ppb in milk (e.g., 20 ppb = 20 kernels in 297 metric tons of corn grain).

Corn with Aflatoxins can be used for ethanol production. Aflatoxins do not accumulate in the ethanol but will be concentrated in the distillers grain co-products. In wet-mill processing, Aflatoxins concentrate in the gluten co-products. A rough estimate is that aflatoxin levels in feed co-products will be three times the level in whole corn. Therefore, processors may not accept corn with aflatoxin if their co-product markets are sensitive to aflatoxin levels, such as dairy feed or pet food.

Silage additives (acids, inoculants or enzymes) cannot degrade preformed aflatoxin and should not be viewed as a possible solution for contaminated feeds. Dilution of the contaminated feed into the diet is still the best solution to the problem. It has been reported that aflatoxin B1 found in corn silage does degrade during storage with upwards of a three-fold decline over nine months of fermented storage (Driehuis, 2013).

The Bottom Line

It is our responsibility to ensure the biosecurity of milk and farm-produced dairy products, whether that involves the health of animals or adopting measures to provide pathogen-free and toxin-free feeds.

THE most important prevention strategies for managing the biosecurity of forage and silage crops are to reduce soil and fecal contamination, retard yeast growth (inoculants containing Lactobacillus buchneri strains), reduce oxygen porosity (compaction, oxygen-barrier film) and discard any visibly moldy or unstable silage.

References

Chase, L.E., D.L. Brown, G.C. Bergstrom and S.C. Murphy. 2013. Aflatoxin M1 in milk. Cornell University Cooperative Extension Dairy Nutrition Fact Sheet. Revised January 2013.

Driehuis, F. 2013. Silage and the safety and quality of dairy foods: A review. Agricultural & Food Science 22:16-34.

This article was originally published in the August 2013 Feedstuffs issue, and is reproduced with their permission.