In this article we will discuss about the staphylococci in milk:- 1. Occurrence of Staphylococci in Milk 2. Growth of Staphylococci in Milk 3. Thermal Destruction of Staphylococci in Milk 4. Interactions between Lactic Acid Bacteria and Staphylococci.
Occurrence of Staphylococci in Milk:
Raw milk may become, contaminated with staphylococci from several sources but the principal one is probably the mastitic bovine udder. Mastitis is often caused by Staphylococcus aureus and continues to be a major problem in dairy cattle. For example- in Wisconsin (U.S.A.) in 1974 at least 16% of all dairy cows suffered from mastitis; to be sure, not all of these cows were infected with S. aureus.
Mastitis also seems to appear more regularly in large than in small (38%) dairy herds. Since the trend is toward maintenance of ever larger herds on dairy farms, it is almost certain that mastitis will continue as a problem in animal health and also will result in the presence of S. aureus in some lots of raw milk.
The occurrence of staphylococci in raw milk has been verified by results of numerous surveys. Among the first surveys to do so was the one by Williams (1941). He studied 10 herds and found that more than 50% of the cows in these herds were shedding staphylococci in their milk.
Staphylococcal counts in excess of 1000/ml were not uncommon and isolates obtained were generally coagulase-positive staphylococci. Williams (1941) did his work before antibiotics were used regularly to treat mastitis; hence the problem of antibiotic resistance in staphylococci of bovine origin and the increase in frequency of staphylococcal mastitis had not yet occurred.
Twenty years after the work of Williams, samples of raw milk were tested by Clark and Nelson (1961). These investigators found that their samples contained 25 to 3300 coagulase-positive staphylococci/ml. Undoubtedly, present-day samples would yield similar results.
Occurrence of staphylococci in raw milk would be of less concern if enterotoxigenic strains were never present. Unfortunately, this is not true; data obtained by several researchers have demonstrated that some staphylococci from the bovine udder are enterotoxigenic. Bell and Veliz (1952) found that enterotoxin was produced by 25 of 35 cultures of staphylococci isolated from the udder.
Enterotoxigenicity of staphylococci obtained from raw milk was determined by Casman (1965). He found that 4.2% of 190 staphylococcal cultures from raw milk were able to produce enterotoxin A and B. About 75% of the toxigenic strains produced enterotoxin A, whereas the remainder formed enterotoxin B. More recently Olson et al. (1970) tested 157 cultures of staphylococci from mastitic udders. Eleven of the cultures produced enterotoxin C, 11 others yielded enterotoxin D, 1 formed both C and- D, and none produced enterotoxins A and B.
Data cited here are adequate to demonstrate that- (1) staphylococci are likely to be present in raw milk, and (2) some of the staphylococci in raw milk are likely to be enterotoxigenic.
Growth of Staphylococci in Milk:
The foregoing considerations would be of less consequence if staphylococci could not grow in raw or processed milk since growth is needed for synthesis of enterotoxins. In general, heated milks are excellent substrates, but raw milk is less favorable for staphylococcal growth.
Clark and Nelson (1961) held raw milk for 7 days at 4° and 10°C. Staphylococci failed to grow at 4°C but grew, although generally slowly, at 10°C. The initial number of staphylococci was not indicative of whether or not growth would occur during the holding period.
Although the aerobic plate count of all test milks was low initially, the count was uniformly high after 4 days at 10°C. In one instance, marked growth of staphylococci was evident, which suggests that the kind of bacteria present and growing in milk may affect proliferation of staphylococci.
Both growth and enterotoxin production by staphylococci in milk were investigated by Donnelly et al. (1968). They inoculated low- and high-count raw milk with S. aureus and then held the milks at different temperatures. It is again evident that behavior of staphylococci can differ in milks with similar initial numbers of bacteria. Although the indigenous flora grew in both samples of milk, staphylococcal growth was more pronounced in one than in the other milk. This more extensive growth by the staphylococcus resulted in enterotoxin production in one milk but not in the other. Similar results were obtained when incubation was at 25° and 35°C.
The same investigators also determined the amount of time needed for enterotoxin production when staphylococci were added to low- and high- count milks. Enterotoxin production in low-count milk was more rapid at all temperatures when the largest number (106/ml) of staphylococci was added When high-count milk was used, a large inoculum of staphylococci was needed for enterotoxin production, and then enterotoxin appeared only at 35°C
The data that have just been described indicate that:
(1) Staphylococci can grow and produce enterotoxins in raw milk,
(2) Raw milk with few bacteria is more suitable for growth and synthesis of enterotoxin by staphylococci than is milk with many bacteria, and
(3) There are differences among milks with few bacteria in their suitability for growth of staphylococci.
These facts must be recognized as one strives to produce raw milk with ever fewer bacteria and as such milk is held for ever longer periods before it is processed. Enterotoxins, if formed at this time, would probably appear in a finished product.
Raw milk often is pasteurized or receives some other heat treatment before it is used to make cheese. Is such milk suitable for growth of and enterotoxin production by staphylococci? Donnelly et al. (1968) tested pasteurized milk and found that it supported growth and enterotoxin production by staphylococci.
Pasteurized milk was even more suitable for enterotoxin production than was raw milk with few bacteria, particularly when the inoculum of staphylococci was small. When a larger inoculum of staphylococci was used, both pasteurized milk and raw milk with few bacteria were equally suitable for enterotoxin production. Tatini et al. (1971) did a similar experiment but gave milk several different heat treatments before staphylococci were added.
Their data also indicate that heat-treated milk was more suitable for enterotoxin production than was the same milk in the raw state. The amount of heat, which ranged from that needed for pasteurization to that required for sterilization, had little apparent effect on the capacity of milk to support enterotoxin production.
From the foregoing discussion, it is evident that both raw and heated milk can support growth of and enterotoxin production by staphylococci. In spite of this only very few reported outbreaks of staphylococcal food poisoning have been attributed to fluid milk and related products. There are good reasons why this is true.
They include- (1) the great degree of control exerted in production, processing, and distribution of these products (2) the extent of abuse needed to create a problem is greater than occurs because of the controls just mentioned, and (3) adventitious organisms can grow if milk is abused and thus they retard growth of staphylococci and also spoil the milk so that it would not be consumed.
Thermal Destruction of Staphylococci in Milk:
A heat treatment of some sort is commonly given to milk before cheese is made. Consequently, it is appropriate to-briefly consider use of heat to inactivate staphylococci in milk. According to data by Thomas et al. (1966), the D value (time in minutes at a given temperature to reduce the population by 90%) for S. aureus in skim milk at 60°C is 3.44 and at 65.6°C it is 0.28.
The z value (degrees Fahrenheit needed for the thermal death curve to traverse one log cycle), according to Thomas et al. (1966) was 9.17. Heinemann (1957) tested the same strain of S. aureus that was used by Thomas et al. (1966). When the bacterium was heated in raw milk, it was completely inactivated after 80 min at 57.2°C, 24 min at 60°C, 6.8 min at 62.8°C, 1.9 min at 65.6°C, and 0.14 min at 71.7°C. Heinemann (1957) determined the z value to be 9.2, a figure that agrees well with that of Thomas et al. (1966).
Zottola et al. (1965, 1969) isolated staphylococci from milk and cheese and then tested them for heat resistance in raw milk. When the test population exceeded 106/ml, 116 of 236 strains survived heating at 63.9°C for 21 sec; whereas when populations were below 106/ml, 108 of the strains survived exposure to 65.6°C for 21 sec.
These heat treatments were used to simulate conditions which sometimes exist in cheese-making when milk receives a sub-pasteurization treatment. This is done because many cheese-makers believe ripened cheese such as Cheddar is more flavorful when it is made from milk that has received a sub-pasteurization rather than a full pasteurization heat treatment.
Interactions between Lactic Acid Bacteria and Staphylococci:
Use of an active lactic starter culture contributes to production of safe cheese.
Why this is so will be evident from the discussion that follows – Reiter et al. (1964) worked with a lactic starter culture that was to be used for cheese-making. This culture inhibited growth of S. aureus in raw, pasteurized, and steamed milk. When lactic acid developed by the starter culture was neutralized as it was formed, inhibition was found to be a function of more than just pH.
The quantity of lactic acid bacteria added to milk can influence growth, enterotoxin production, and even survivial of S. aureus. For example, Richardson and Divatia (1973) noted that adding 0.001 and 0.01% of a milk culture of lactic streptococci to milk containing approx. 106 S. aureus/ml served to appreciably retard growth of the staphylococcus. Lack of growth by the staphylococcus during the first 8 hr of incubation and subsequent inactivation were observed when the inoculum was 0.1, 0.5, or 1.0%. Enterotoxin appeared when the inoculum was 0.001 and 0.01% but not when any of the 3 larger inocula was used. When the number of staphylococci in milk was reduced, a smaller inoculum of lactic acid bacteria was needed to inhibit or inactivate S. aureus.
According to a report by Jezeski et al. (1967), an actively growing culture of Streptococcus lactis inhibited and sometimes inactivated S. aureus when both organisms were together in sterile or steamed skim milk. Enterotoxin was produced by S. aureus when it grew alone or in the presence of the streptococcus and a homologous bacteriophage.
Absence of the bacteriophage enabled the lactic acid bacterium to prevent enterotoxin production by the staphylococcus. Haines and Harmon (1973) inoculated APT (All Purpose Tween) broth with S. aureus and S. lactis to determine when growth and enterotoxin production by S. fiureus would be inhibited.
They learned that- (1) no enterotoxin was produced and growth of S. aureus was retarded similarly when the initial pH values were 6.0, 6.5, and 7.0; (2) no enterotoxin was produced at either 25° or 30°C, but growth of S. aureus was inhibited somewhat more at 25° than 30°C; (3) enterotoxin was produced at 30°C when the number of S. lactis to S. aureus was 10:90, but not when it was 50:50 or 90:10; (4) different strains of S. lactis were equally inhibitory and prevented toxin production; and (5) different strains of S. aureus were inhibited in a similar way by S. lactis.
Further evidence for the inhibitory properties of lactic streptococci comes from a report by Gilliland and Speck (1972). They tested 6 different lactic streptococcus cultures for their ability to inhibit S. aureus. Inhibition of S. aureus was almost complete regardless of the starter culture used or the time, within limits, required for acid formation. These authors suggested that repression of staphylococci by lactic streptococci may involve production of antibiotics, hydrogen peroxide, and volatile fatty acids in addition to lactic acid. Of these, undoubtedly acid production is the single most important factor which causes the staphylococci to be inhibited.
Minor and Marth (1970) did a series of studies to more clearly define the role of acid in controlling growth of staphylococci in milk. They gradually added acid to milk which was inoculated with S. aureus. Acid was added over a 4, 8, or 12 hr period. When acid was gradually added over a 12 hr period, 90% reduction in growth of S. aureus was achieved if the final pH values were 5.2 for acetic, 4.9 for lactic, 4.7 for phosphoric and citric, and 4.6 for hydrochloric acid.
To achieve a 99% reduction during a 12 hr period, the final pH value had to be 5.0 for acetic, 4.6 for lactic, 4.5 for citric, 4.1 for phosphoric, and 4.0 for hydrochloric acid. A final pH value of 3.3 was required for 99.9% reduction in growth with hydrochloric acid, whereas the same result was obtained at a final pH value of 4.9 with acetic acid. Correspondingly lower final pH values were required to inhibit growth within 8 and 4 hr periods.