research institute zealand · (37-38 c) used during part of the cheese makingprocess. theyproved to...

BACTERIOPHAGE IN CHEESE MANUFACTUREH. R. WHITEHEAD

The Dairy Research Institute (N. Z.), Palmerston North, New Zealand

In 1935 Whitehead and Cox (32) reported the observation that bacteriophage(bacterial virus) was responsible for the failure in acid producing activity of aculture of Streptococcus cremoris used as a cheese starter. The virus was shown tolyse the bacteria, thus terminating acid production in the culture as a whole.This discovery opened up a new and profitable line of research in dairy manu-facturing technique, more particularly in connection with the manufacture ofcheddar cheese where uncontrollable variations in rate of acid development fromday to day had long been a cause of difficulty and spoilage. Workers in Iowa (11)had previously observed that filtrates from certain butter cultures, i.e., milkcultures containing a mixed flora of lactic streptococci and betacocci (Leuconostocspp) sometimes contained an "inhibitory" factor which caused a check in acidproduction when added to other butter cultures. They had not, however, realizedthe true significance of their observation and only later agreed that the phenom-enon was due to the presence of a bacterial virus in the "inhibitory" filtrate (19).Since 1935 evidence has gradually accumulated that phage is responsible all overthe world for much of the irregular behavior observed in bacterial cultures usedin the manufacture of dairy produce (2, 3, 12, 15, 18, 28, 30, 47). The effects areperhaps most evident in the manufacture of cheddar cheese where a vigorousgrowth of lactic streptococci sufficient to produce the equivalent of about 1 percent of lactic acid is required within a period of five to six hours; but phageevidently is also capable of causing difficulties in the ripening of cream for buttermanufacture and in the production of various fermented milk preparations.

Characteristics of phage action in cheese manufacture. Until the present centurycheese makers relied upon contaminant bacteria, present by chance in the milk,to bring about the fermentation of lactose and gradual production of acid whichis an essential part of cheese manufacture. A gradual improvement in hygiene inthe production and handling of milk led to the necessity for addition of a cultureof lactic streptococci (starter) to the cheese milk since, in the absence of such anaddition, the milk would not now sour quickly enough. Pasteurization of thecheese milk, practiced most widely perhaps in New Zealand, made the use ofstarter still more essential. Cultures containing a mixed flora of lactic streptococciand betacocci were similarly introduced early in this century for the ripening ofcream for butter manufacture. The essential feature of this process was latershown to be the production of diacetyl, the butter-aroma substance, by thebetacocci under the acid conditions produced by the streptococci. The prepara-tion and supply of starter cultures to the dairy industry were for many yearsalmost entirely in the hands of commercial interests who were secretive about theprinciples followed in selecting the strains of bacteria used in the mixed cultures.It appears also to have been assumed that the mixtures used in butter starterswere equally suitable for cheese manufacture.

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110 H. R. WHITEHEAD [VOL. 17

The behavior of commerical mixed starter cultures in cheese milk and curd isnotoriously erratic. The cheese maker prepares in advance a milk culture of thestarter in a quantity amounting to one or two per cent of the volume of cheesemilk to be handled. At the beginning of the cheese making process this cultureis added to the cheese milk, and the subsequent progress of acid developmentis followed throughout the manufacturing process by titration of the milk (inlater stages the whey) with alkali. The various mechanical treatments appliedto the curd are synchronized with the attainment of various degrees of acidity.Unexpected delays in acid development are frequently experienced. A culturemay behave regularly day after day for long periods and then may suddenly losevigor between one day and the next. Sometimes a culture may be of normalvigor at the beginning of the process and then may suddenly show a decreaseor even a complete collapse in activity at any stage of cheese manufacture. Thisphenomenon constituted a major problem for scientific workers in the dairyindustry for many years. Investigation was complicated by the widespread usefor cheese manufacture of raw milk containing a variety of contaminant bacteria,and by the presence of a mixed flora in the starters themselves. The currentuniversal remedy for "starter trouble" was to procure a fresh starter.

In New Zealand the problem was simplified a little by the general use ofpasteurized milk in cheese factories. Furthermore, in 1934-35 a beginning wasmade in the use of cultures consisting of single strains of Streptococcus cremorisin place of the mixed culture starters of commerce. The strains used were selectedfor their high activity and ability to withstand the relatively high temperature(37-38 C) used during part of the cheese making process. They proved to haveseveral advantages over the mixed cultures for cheese manufacture, viz., thestarters had more stable characteristics since there was no possibility of fluctua-tion in relative numbers of different strains, the whole cheese making processcould be shortened by about one hour, and the cheese was of better quality andmore particularly of closer texture. The single strain cultures, however, had oneserious drawback: they were liable to fail completely without warning in a muchmore disastrous manner than mixed cultures ever did. Failures were evidencedeither by a complete lack of growth in the culture being prepared for addition tothe cheese milk or by a sudden and final cessation of acid production during thecheese making process.

It was from a sample of a single strain starter which suffered a sudden failurein the cheese vat that Whitehead and Cox originally isolated a phage or bacterialvirus capable of lysing the strain of S. cremons present in the original culture andof forming on a plate culture the classical "plaques" described for other phages.The lysing action of the virus on the organism adequately accounts for the suddencessation in acid production which occurs when such a virus exerts its actionduring the cheese making process. The subsequent finding that various strepto-coccal phage races tend to be strain specific explains why starter cultures con-taining several strains show sudden variations in activity from time to time butrarely fail completely. The difficulty sometimes experienced by dairy tech-nologists in demonstrating the presence of phage where all the circumstances


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1953] BACTERIOPHAGE IN CHEESE MANUFACTURE ill

indicate its presence is often due to attempts to work with mixed cultures inwhich the growth of nonsusceptible strains conceals the action of phage on sus-ceptible strains.

Origin of phage-The cycle of infection. The immediate origin of the bacterialviruses which always appear sooner or later in cheese curd and whey is unknown.Maz6 (16, 17) has suggested that they originate in the intestines of farm animalssuch as pigs, but since these animals often have access to cheese whey the virus,when present in the animal intestine, may have come from the whey and notvice versa. Nichols and Hoyle (21) came to the conclusion that pig intestine couldnot be considered a usual source of phage. Since the origin even of the host strep-tococci is unknown, it is unlikely that the origin of the viruses will be easy todefine.The circumstances attending the appearance of a phage race under commercial

dairying conditions are as follows. A strain of Streptococcus cremoris or of S.lactis isolated from sour milk or from a commercial mixed starter culture may beused daily in cheese manufacture for weeks or in rare cases even for monthswithout any indication that a phage capable of attacking the organism is presentin the environment. The culture may be handled in a nonaseptic manner withoutuntoward result. Samples of whey taken from the cheese vat and spread on platecultures of the organism will not give any plaques. After a longer or shorterperiod of daily usage of the culture in cheese vats, whey samples tested on a plateculture will begin to show a few plaques. Then, within a few days either the bulkculture prepared for use in the cheese vats will fail to coagulate owing to lysis ofthe organisms, or acid production in the cheese curd and whey will suddenlycease part way through the process. When failure has once occurred, infectionwith the virus is widespread throughout the cheese factory, and the particularculture involved cannot be successfully propagated in the vicinity unless verycomplete protection from all forms of infection is provided. Since attempts toinduce a similar appearance of phage for a given culture in the laboratory havenever been successful, at the present time the only known method for isolatingwith certainty a phage race for a given culture is to use the culture in a com-mercial cheese factory.The mechanism of the appearance of phage under commercial conditions is

unknown, but as a working hypothesis it is suggested that particles of dormantphage of a variety of races occur in nature as a widespread but usually verysparse ubiquitous dust-borne infection. The cheese vat during the manufacturingprocess offers a large exposed surface of an actively growing streptococcal culture-a much larger surface than can be exposed under laboratory conditions. Sooneror later, with day to day use of a given strain of streptococcus, a phage particlecapable of attacking the organism finds its way into the vat and through theprocess of lysis undergoes multiplication. Under commerical conditions the vatand cheese making equipment are never completely sterilized; consequentlyreinfection occurs daily with a gradual increase in phage titer in the whey up toa point determined by the efficiency of the equipment sterilizing processes em-ployed. Coincidently, the passage of the cheese whey through a high speed


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centrifugal separator for recovery of residual butter-fat and the splashing andspillage of whey during the course of its disposal, all help to create an air bornedroplet infection which permeates the air of the factory (4) (see figure 1). Suc-cessful maintenance of a culture of the starter susceptible to this phage racesoon becomes impossible in the immediate vicinity of the factory even with anormal aseptic technique unless special precautions to exclude the air-borneinfection are taken. Use of another streptococcal strain may be quite possiblewithout these precautions if it happens to be insensitive to the phage present inthe atmosphere. The same sequence of events will ultimately recur with thefresh culture.

This hypothesis fits the facts, but there is a possibility, so far not fully investi-gated, that the phage race which appears in the cheese vat sometimes originatesnot from a dust-borne dormant particle but by mutation from another phagepresent in the surroundings in high concentration as the result of lysis of a pre-viously used starter culture. As host-range mutations among bacterial viruses

Whey _ WheyTank ( separator

Splash from air-bornetaps droplets

Milk Cans tap bodroplets

--

Atmosphere\ | ~~C-hees~eVat|/\ Pg hPage developing each day

Fresh Milk Starterin Cans

FIG. 1. Cycle of bacteriophage infection in cheese manufacture

are known to occur (1), such an explanation on these lines would obviate theneed to postulate the existence of the very large number of phage races in nature.That is, should each new race that occurrs under cheese making conditions beconsidered a completely independent entity? This question of immediate origindoes not, however, bear on the practical problem so far as protective measuresnecessary to preserve cultures are concerned. It needs to be considered in anyscheme where strain specificity of phage races is made use of in systems involvingthe rotational use of cultures.

Characteristics of the streptococcal phases. Only a few observations on strepto-coccal phages (4, 6) had been made before phage races capable of attacking thelactic streptococci were first isolated. Since 1935 a considerable amount ofinformation on the nature and characteristics of the lactic streptococcal phageshas been obtained. Several observers have described the appearance of thephages under the electron microscope (24, 29, 43). The particles of all races exceptone so far examined are sperm shaped with spherical heads about 70 mju in-diameter and tails about 30 mu wide and 150 m1A long. The exception (44) is arace showing larger particles and in particular a much larger tail about 580 msAlong. For most of the phage races the plaques formed on mats of the host organ-


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19531 BACTERIOPHAGE IN CHEESE MANUFACTURE 113

isms on agar plates are between 0.25 mm and 1 mm in diameter, but a minorityforms smaller plaques down to pin-point size or larger plaques up to a maximumof about 3 mm. Purification of the individual phage races is most convenientlyaccomplished by picking single isolated plaques from the solid medium nto aculture of the host organism in a liquid medium. When, after incubation, lysishas taken place, filtration of the lysed culture through a Chamberland or Seitzfilter yields a bacteria-free filtrate containing a high concentration of a purepreparation of the phage race.The various races differ in their optimum growth temperature on the host

organism and the optimum temperature for development on the phage, andlysis of the bacterium does not necessarily coincide with the optimum tempera-ture for growth of the bacterium (7, 36). Thermal death points differ among thevarious phage races but only within a small range; all the phages are destroyedby exposure to temperatures ranging between 70 C and 75 C for 30 min at pH6 (20, 36). It follows that the streptococcal phages are not destroyed by the ac-cepted milk pasteurizing procedures (61.7-62.8 C for 30 min. or 72 C for 15 sec.).The phages are readily destroyed by chemical disinfectants (9a, 22, 23, 26, 27)the most suitable agents for use in a food industry being hypochlorites, per-manganates and quaternary ammonium compounds (table 1). Phage prepara-tions are quite stable when stored in the laboratory at temperatures around 5 C.They decrease slowly in titer during storage but have been proved to retain someactivity for more than ten years (unpublished observations). The various racesdiffer considerably in the rate at which the titer of a stored specimen falls away.Hunter (8) in a comprehensive examination of many races of phage and

strains of lactic streptococci found a general distinction between phages forS. cremoris and those for S. lactis. Phage races active against S. cremors were tosome extent strain specific or at least group specific, in their action, whereasthose active against S. kzctis were in general polyvalent. This characteristic wassufficiently clearly marked to lend quite significant support to other evidenceindicating that S. cremoris and S. lactis are distinct, although closely relatedspecies. From a practical point of view this finding means that strains of S.crentors are more desirable for use in cheese manufacture since there is a lesserlikelihood that adventitious phage in the milk supply will be capable of attackingthe cheese starter. Furthermore the partial strain specificity of the cremoris-typephage races makes for greater safety from phage failure in cheese manufactureif starters consisting of different strains of S. cremoris are used in a rotationalcycle over several days.When a streptococcal culture is lysed by a phage, continued incubation of the

culture generally results in the growth of a phage resistant form of the bacterium,similar in most respects to the original strain but less susceptible or totally in-susceptible to the lytic action of the phage. This phenomenon occurs with manygenera of bacteria under the action of bacterial viruses. There is considerableevidence that the phage resistant strains are mutants present normally in smallnumbers in the original culture and made evident by the lysis of all the sensitiveindividuals (13, 14). Attempts to make use of phage resistant cultures as cheese


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114 H. R. WHTEHEAD [VOL. 17

starters, in an effort to obviate failures in commercial practice, gave disap-pointing results (33) since the resistant cultures soon succumbed to the action ofnew (or mutant) phage races present in the surroundings of cheese factories.Furthermore, resistant cultures are rarely as active in acid production as theoriginal parent culture, and they sometimes gradually revert towards sensitivityto the original phage race on continued subculture in the laboratory.

TABLE 1Power of disinfectants in destroying phase

FINAL CONCENTRATION TIME REQUIRED FOR COMPLETEDISINFECTANT IN PEAGE-DISINFECTANT DESTRUCTION

MIXCTURE

per cent

Hypochlorite (available chlorine) 0.05 Less than 1 min

KMnOt 0.25 Less than 1 min0.05 Between 1 and 5 min0.025 Not in 2 days

H202 3.0 Between 15 and 60 min2.5 Between 1 and 24 hr0.5 Between 1 and 24 hr

0CHO 5.0 Between 5 and 30 min2.5 Between 30 and 60 min1.0 Between 1 and 24 hr0.5 Between 1 and 24 hr

HgCl2 2.5 Between 1 and 24 hr1.0 Between 2 and 3 days0.5 Not in 14 days

Alcohol 90.0 Between 3 and 4 days85.0 Between 2 and 3 days80.0 Between 2 and 3 days75.0 Between 3 and 4 days70.0 Not in 6 days

Phenol 2.5 Not in 14 days

Data from Journal of Dairy Research (9a).

Resistant or partially resistant cultures of S. cremoris sometimes are capable ofnormal growth in constant association with the phage race which lyses the parentbacterial strain. The phage once added to such a culture maintains itself fromtransfer to transfer in a low titer which can be detected in supernatant fluid fromthe otherwise normal coagulated milk culture of the resistant strain. Hunter (9)found that a "phage-carrying" culture was protected against attack by otherphage races which would normally lyse the culture in the absence of the asso-ciated phage. An attempt was made to use this protective effect in commercial


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practice (10) on the assumption that a "phage-carrying" strain should survivein an atmosphere where air-borne phage was present without the need for anyspecial protection against phage infection. The results showed that this assump-tion was correct up to a point. The "phage-carrying" cultures survived in dailyusage for months under crude cultural conditions in which the original strainshad been proved to suffer lysis in one or two days. But considerable difficultieswere found even in the laboratory in maintaining the organism-phage associationin a constant state of balance, and on the practical side, a new race of phageagainst which the culture was not proof ultimately appeared in the cheesefactory.

Further study of the reactions which take place between various races ofphage and strains of S. loctis and S. cremoris has shown that, as with other generaof bacteria, the relationships are very complicated. Nichols and Hoyle (21) at-tempted to type a large number of strains of lactic streptococci by their reactionsto a number of phage races. They obtained indications of family relationshipsbetween groups of strains and confirmed Hunter's observation (8) that phageraces active against S. cremoris tended to be more strain specific than thoseactive against S. lactis. They encountered, however, the difficulty experiencedby other workers (e.g., 44, 45), viz., that any group of strains isolated fromnatural sources contains phage resistant types and possibly lysogenic types whichmay not be recognized as such. On the basis of simple lytic tests these typesmay appear to be completely unrelated to the parent types from which theyoriginated. More recently it has been shown (41) that the lytic reaction is notthe only form of interaction between phage and organism so far as the lacticstreptococci are concerned. There are many instances in which phage in highconcentration inhibits the growth of an organism which it cannot lyse, and thereare indications in other instances that a phage race may contain small numbersof mutant particles which can extend its host range under certain conditions(unpublished work). All these observations probably will not be fully clarifieduntil more is known of the mechanism of the action of bacterial viruses.

Systems used for preventing phage action during cheese manufacture. Turningback to the practical issue the salient points, as they concern cheese manufac-ture, are:

(i) that phage races regularly become established in high concentration in thevicinity of cheese factories.

(ii) that some of the procedures in cheese manufacture result in the establish-ment of a potent air-borne phage infection.

(iii) that phage races active against S. cremoris tend towards strain specificity.The problem in commercial practice is to devise means for excluding phage fromthe starter culture and cheese milk or for inhibiting its action. The problem ismore acute where cultures of pure strains of S. cremoris or S. lactis are used.The following discussion refers primarily to such cultures.

Early attempts in New Zealand to overcome the trouble caused by phageinfection of starter cultures were colored by the belief that the virus originatedspontaneously within the culture under the influence of certain environmental


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conditions (34, 35). Only when it was finally discovered that a potent air-bornedroplet infection existed in the vicinity of a cheese factory was the problemclarified (37, 38). The degree of infection which can, on occasions, exist in theair of a cheese making room is perhaps best illustrated by the fact that removalof the plug from the mouth of a 1,000 ml Erlenmeyer flask containing a freshlyinoculated starter culture will sometimes result in an infection sufficient to causelysis of the culture within 24 hours. The extremely insidious nature of the infec-tion is still not appreciated by practical dairymen in some countries, and con-sequently one still sees advocated for phage control measures which are hopelesslyinadequate.The practical problem resolves itself into two parts:(a) How to provide adequate protection for starter cultures against air-borne

phage infection.(b) How to reduce to a minimum the phage infection which inevitably occurs

in the cheese vat.The second aspect is related to the first because the potency of the air-borne

infection depends essentially on the titer of phage developed in the cheese whey;consequently, a reduction of the infection in the vat makes the protectivemeasures used for the starter culture more effective.

Protection from air-borne infection. Several principles have been applied inefforts to protect cultures from air-borne infection. Elimination or reduction inintensity of the air-borne phage by disinfection might appear to be the logicalmethod of procedure, and attempts have been made to do this by means ofhypochlorite mists (39, 46). In a cheese factory, however, since an infection ofthe atmosphere occurs continuously throughout the day, a germicidal mist doesnot persist long enough to give sufficient safety margin. Protection of culturesrather than elimination of air-borne phage is thus the more practicable procedure.

In the first place, it is necessary to have some means for sterilizing by filtrationor other means the air which must necessarily re-enter culture vessels as theycool to incubation temperature after the milk sterilizing procedure. The classicalmethod of using a cotton-wool plug in the mouth of the vessel to prevent theentry of dust and liquid droplets carrying phage is quite effective. But experiencehas emphasized a point often not appreciated, that under conditions of intenseair-borne infection the size of the plug must bear a certain relationship to thevolume of air in the culture vessel if sterilization of the ingoing air is to be effec-tive. In early work this was not realized until failure to exclude phage was some-times traced to the use of too small a plug. The indications are that any degreeof air-borne infection likely to be encountered in practice can be combatedprovided that a big enough filter of cotton-wool is used. The maintenance ofsmall mother cultures in Erlenmeyer flasks plugged with cotton-wool is thus astraightforward procedure demanding only a normal aseptic technique andremoval of the culture to some distance'from the factory during the process ofsubculture.The closure of large vessels containing up to 150 gallons of milk in such a way

that they can be easily filled and emptied and yet can be made air-tight except


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1953] BACTERIOPHAGE IN CHEESE MANUFACTURE 117

for the opening through which sterilized air may pass presents problems whichhave been solved in various ways. The use of a water seal between lid and vesselis one of the most simple and effective methods (39) (see plate I). Since an aper-ture in the large starter vessel must be opened for the purpose of inoculation,special means have to be provided to exclude air-borne infection during thisprocess. Two methods have been used (a) a spirit flame and (b) a steam "blan-ket", both arranged to envelop the opening, normally closed by the cotton-woolfilter, through which the mother culture is poured.

PLATE I. Bulk starter vessel (100 hnperial gallons) with all openings fitted with water-seal

Sometimes the application of the just-described protective devices is sufficientto enable starter cultures to be prepared within the cheese factory (except forthe actual inoculation of mother cultures). But it has in general proved advisableto adopt further precautions in addition to those described. Two alternativeschemes have been adopted in commercial practice.

(i) A special building isolated from the main cheese factory is used for thewhole operation of starter preparation.

(ii) A piping system is used to draw air from a point some distance from thefactory. The air is sterilized in the system and then supplied to the starter vesselswhich are still located within the factory.


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118 II. R. WAIITEIIEAD [VOL. 17

The provision of a separate building was tried out very soon after the signiifi-cance of air-borne phage wvas first realized (38). Tests showed that the intenisitvof air-borne phage infection inl tle surroundinggs of the cultures cotuld be ieducedenormously by this removal from the factory, hut experience indicated (as wasto he expected) that the site of the bllilding in relation to prevailing willds, fac-tory drainage and other factors was also of importance (see figure 2). MAoreover,it was found that evein with a starter building in the most advantageous positionthe use of air-tight culture vessels sealed with cotton-wool plugs was still advis-able if failures were to be completely eliminated.

Prevailing

Cheese i/ wind

Factory makinga^^sw>rinage room L______________

Starter room

I Milk receivingI stage

Whey tanks

FIG. 2. Example of satisfactory location of starter-preparation building isolated fromfactory. From Journal of Dairy Research (39).

A desire to avoid the expense of erection of a separate building led to the de-velopment of various systems under which the bulk starter vessels were locatedwithin the factory but were connected to a piping system in such a way that theair drawn into the vessels as they cooled was treated inl one of the followviing ways:

(i) Drawn from a point some distance from the factory and filtered throughcotton wool. Sometimes the filter was fitted omi the end of a vertical pipeabove the roof of the factory.

(ii) 1)rawni through a filter and passed over ultraviolet lamps fitted ill thepiping system.

(iii) Drawgn through a filter and then passed through a cylinder containing asteam pipe which heated the air to over 300 F.

(iv) Treated as in (i), (ii) and (iii) and supplied uider slight pressure to thestarter vessels by means of a water-jet pump.

All these systems have been found satisfactory in practice, choice among thembeing based oni individual preference according to circumstances. It has iiot, been


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possible to prove under field conditions that all the individual phage trapping orphage destroying devices are completely effective. For instance although it isknown that ultraviolet light destroys phage (5, 31), it is very doubtful whetherlamps in a piping system give an irradiation of air-borne phage containing drop-lets sufficient to cause complete inactivation of phage. In practice, however, allthe systems embody more than one safeguard and generally a cotton-wool filter,one of the most effective known safeguards, is included.

Reduction of degree of phage infection. Wherever a certain strain of lactic strep-tococcus is used regularly as a cheese starter over a period of time, phage capableof attacking the culture is present daily in the cheese vat. So long as the infectionis held down to a certain level, however, the starter is able to survive during thefive to six hours necessary for cheese manufacture and thus to produce the acidityrequired. Multiplication of phage is in any event checked at high acidities eitherbecause of the effect of acidity on the phage itself or because further growth ofthe host organism is progressively inhibited. Where for any reason the cheesemaking process is unduly prolonged (as, for instance, where a small proportionof starter and a long "ripening" period is used), there is greater danger that tracesof phage present in the cheese vat will lyse the bacteria before the end of themanufacturing process (42). Since in practice there is not a great margin of safety,all possible means available for reducing the level of phage infection before andduring the process have to be exercised to the full. At the end of operations eachday all the equipment is infected with phage, an air-borne infection has beencreated through the action of the whey separator, and the main waste product,whey, has probably been spilled over near-by roads by farmers taking it to thefarms for feeding to pigs. It follows therefore that next morning, the starter,even if it has been successfully protected from infection during the course of itspreparation, inevitably becomes infected in the cheese vat either from the factoryequipment or from the milk itself which has been infected with whey on thefarm (40) (see figure 1). Experience has shown that treatment of the factoryequipment with a sterilizing agent such as hypochlorite immediately before thestart of the day's operations will on the great majority of occasions reduce infec-tion from this source to such a low level that the cheese making process can becarried through successfully unless the milk itself contains too much phage.The problem presented by the occasional presence of phage in milk delivered

to the cheese factory is more difficult to overcome. The phage is not destroyedby milk pasteurizing procedures. In New Zealand many farmers use milk cansfor the transport of whey, and the difficulty caused by the consequent presenceof traces of phage in the milk became at one time so acute that an investigationwas made into the practicability of destroying, either by heat or by chlorinetreatment, the phage in the whey as it left the cheese vats. Both methods wereshown to be effective, but they inevitably involved extra cost in materials andlabor (40). They were never actually applied in commercial practice because inthe meantime a cost-free method for circumventing the effect of phage in themilk was discovered, viz., the use of a series of cultures in a rotational cycle overseveral days (40).


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A search among many hundreds of strains of S. cremoris made possible theselection of about ten strains, active enough for use as cheese starters and appar-ently unrelated to one another so far as sensitivity to a large group of phageraces was concerned. The use of these strains enabled a factory manager tochange the starter culture each day according to a definite rotational system sothat each culture was used not oftener than once in four days. Thus, phage in-fection for a given strain present in the whey, on the factory equipment, and inthe air as a droplet infection was not capable of attacking the starters to be usedon the following three days. Phage which might be in the milk delivered to thefactory was also without effect on the culture in use on any given day. Thesystem thus had the double benefit of obviating trouble caused by phage con-tamination of the milk supply and also of giving a three day interval in whichthe phage for each strain of S. cremoris would be progressively eliminated fromthe factory environment before the culture was used again.

This rotational starter system has been in commercial use in New Zealand foreight years and has proved remarkably successful. Generally, the cultures areused in pairs in a four or five day cycle. Each culture is prepared separately upto the bulk starter stage, and the two starters are then both added to the cheesemilk. This provides yet another safeguard against failure since the two strainsconstituting each pair are unrelated, and if by accidental infection one culturefails, the chances are that the manufacturing process can still be carried throughwith the other culture.

Protection of starter cultures from contaminant bacteriophage as discussedabove is essential where single strain cultures are used, but it is also beneficialwith starters containing a mixed flora although the benefits are not so obvious.Mixed cultures even if protected from infection show changes in activity fromtime to time as a result of fluctuations in the relative numbers of the variousstrains present. Contamination of a mixed culture with phage causes suddenand unpredictable variations in activity as a result of the elimination by lysis ofone or more of the strains. Usually, however, because of the strain specificity ofphages, some strains are unaffected, and the culture continues to produce acidenabling cheese manufacture to be completed although perhaps at the cost ofsome delay. In most countries cheese makers still prefer to use mixed flora start-ers because they rarely fail completely whereas single strains of S. cremoris,unless adequately protected, fail in such a drastic and sudden manner that cheeseof only very poor quality can be made. In New Zealand the various benefits tobe gained from the use of single strain starters have resulted in a persistence intheir use and in the adoption of adequate protective measures against phageinfection in commercial practice.The problem presented by phage infection of the streptococci used in cheese

manufacture may be regarded as solved in the scientific sense in that a com-pletely aseptic technique would eliminate the trouble. In a commercial sensewhere various practical considerations prevent the attainment of complete asep-sis the problem has various aspects which differ over the world according to cir-cumstances and requirements. For instance much difficulty in New Zealand is


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caused by transport of whey in milk cans. From a scientific point of view thesolution is to cease this practice. From a commercial point of view this is notpracticable because transport of whey by other means for pig feeding wouldprobably be uneconomic. Circumstances with respect to disposal of whey varyin different countries and even within a country. Thus, the commercial problempresented by phage in whey has a different solution in different places. Anotherdifficulty in the way of complete solution of the phage problem in cheese manu-facture lies in the personal factor. In some countries a complete laboratorycontrol of starter preparation appears to be economic especially where largecommercial units are concerned. In other places because of long established cus-tom or the small size of commercial units the preparation of starter is in thehands of factory operatives. In this latter case an appreciation of aseptic tech-nique and of the insidious nature of phage infection has to be acquired by theoperatives-not an easy matter. Nevertheless in spite of these various difficultiesposed by commercial limitations the fundamental principles of control whichhave now been proved sound over the course of about twelve years can be appliedin various circumstances with success. This is evidenced by the universal use inNew Zealand of single strain starters, under the control of working managers andfactory operatives. Finally, there is still the possibility that investigations intothe nature of the streptococcal phages will disclose some method of inhibitingtheir action without interference with the growth of the host organisms. Thispossibility, although apparently remote, cannot be excluded.

REFERENCES1. ADAms, M. H. 1950 Methods of study of bacterial viruses. In Methods in medical

research. Vol. II, pp. 1-73. Edited by Comroe, J. H. The Yearbook Publishers,Chicago, Ill.

2. ANDERSON, E. B., AND MEANWELL, L. J. 1942 The problem of bacteriophage in cheesemaking. Part I. Observations and investigations on slow acid production. J.Dairy Research, 13, 58-72.

3. BABEL, F. J. 1946 Factors influencing acid production by cheese cultures. II.Influence of bacteriophage on acid production in the manufacture of Cheddar andcottage cheese. J. Dairy Sci., 29, 597-606.

4. EVANS, A. 1934 Streptococcus bacteriophage and its usefulness for the identificationof strains of haemolytic streptococci. J. Bact., 27, 49-50.

5. GREEN, G. I., AND BABEL, F. J. 1948 Effect of ultraviolet irradiation on bacteriophageactive against Streptococcus lactie. J. Dairy Sci., 31, 509-515.

6. HADLEY, P., AND DABNEY, E. 1926 The bacteriophagic relationships between B.coli, S.fecalis and S. lacticus. Proc. Soc. Exptl. Biol. Med., 24,13-18.

7. HUNTER, G. J. E. 1943 Bacteriophages for Str. cremoris-phage development at vari-ous temperatures. J. Dairy Research, 13, 136-145.

8. HUNTER, G. J. E. 1946 The differentiation of Streptococcus cremoris and Streptococcuslactie by means of bacteriophage action. J. Hyg., 44, 264-270.

9. HUNTER, G. J. E. 1947 Phage-resistant and phage-carrying strains of lactic strepto-cocci. J. Hyg., 46, 307-312.

9a. HUNTER, G. J. E., AND WHITEHEAD, H. R. 1940 The action of chemical disinfectantson bacteriophages for the lactic streptococci. J. Dairy Research, 11, 62-66.

10. HUNTER, G. J. E., AND WHITEHEAD, H. R. 1949 Phage carrying cultures as cheesestarters. J. Dairy Research, 16, 368-373.


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11. Iowa State College Agr. Exp. Sta., Ann. Rep. 1933, p. 63.12. JOHNS, C. K., AND KATZNELSON, H. 1941 Studies on bacteriophage in relation to

Cheddar cheesemaking. Can. J. Research, C, 19, 49-58.13. LURIA, S. E., AND DELBRUCK, M. 1943 Mutations of bacteria from virus sensitivity

to virus resistance. Genetics, 28, 491-511.14. LURIA, S. E. 1946 Spontaneous bacterial mutations to resistance to antibacterial

agents. Cold Spring Harbor Symposia Quant. Biol., 11, 130-138.15. MATTICK, A. T. R., NICHOLS, A., AND WOLF, J. Z. 1944 Bacteriophage and cheese-

making. Pub. Natl. Inst. Dairy, Reading, No. 794.16. MAZk, P. 1941 Origine des bact6riophages des ferments lactiques normaux du lait.

Compt. rend. soc. biol., 135, 807-808.17. MAZg, P. 1941 Origine des bacteriophages des ferments lactiques normaux du lait.

Compt. rend. soc. biol., 135, 1332-1335.18. MOSIMAN, W., AND RITTER, W. 1946 Bacteriophage as cause of loss of aroma in butter

culture. Schweiz. Milchztg., 72, 211-212.19. NELSON, F. E., HARRIMAN, L. A., AND HAMMER, B. W. 1939 Slow acid production by

butter cultures. Iowa Agr. Exp. Sta., Research Bull., no. 256, 219-287.20. NICHOLS, A. A., AND WOLF, J. Z. 1946 The heat resistance of the bacteriophages of

cheese starter with observations on the estimation of phage concentration. J.Dairy Research 14, 93-100.

21. NICHOLS, A. A., AND HOTLE, M. 1949 Bacteriophage in typing lactic streptococci.J. Dairy Research, 16, 167-208.

22. OVERCAST, W. W., NELSON, F. E., AND PARMELEE, C. E. 1951 Influence of pH onproliferation of lactic streptococcus bacteriophage. J. Bact., 61, 87-95.

23. PARKER, R. B., AND ELLIKER, P. R. 1951 Destruction of lactic acid streptococcusbacteriophage by hypochlorite and quaternary ammonium compounds. J. Milkand Food Technol., 14, 52-54.

24. PARMELEE, C. E., CARR, P. H., AND NELSON, F. E. 1949 Electron microscope studiesof bacteriophage active against Streptococcus lactis. J. Bact., 57, 391-397.

25. PETTE, J. W. 1946 Bacteriophage in starters. Versl. Rijkslandb. Proefsts, 's Grav.,51, (8) C, 133-156.

26. PROUTY, C. C. 1949 Inactivation of bacteriophage of the lactic acid streptococci ofstarters by quaternary ammonium compounds. J. Milk and Food Technol., 12,214-218.

27. PROUTY, C. C. 1950 Inactivation of bacteriophage of the lactic acid streptococci athigh and low pH levels. J. Milk and Food Technol., 13, 329-331.

28. RUNOV, E. V. 1949 The distribution of streptococcal bacteriophage in cheese.Mikrobiologiya (U.S.S.R.), 18, 174-176. Chem. Abs., 43, 7154 (1949).

29. SHEW, D. I., AND HODGE, A. J. 1950 Electron microscope studies on starter culturesand bacteriophage. Australian J. Dairy Technol., 5, 99-102.

30. SUTTON, W. S. 1939 Bacteriophage in a pure strain culture of Str. cremoris in NewSouth Wales. J. Australian Inst. Agr. Sci., 5, 168-169.

31. SUTTON, W. S. 1941 Irradiation of cheese moulds and bacteriophage by ultra-violetlight. J. Australian Inst. Agr. Sci., 7, 67-73.

32. WHITEHEAD, H. R., AND Cox, G. A. 1935 The occurrence of bacteriophage in culturesof lactic streptococci. New Zealand J. Sci. Technol., 16, 319-320.

33. WHITEHEAD, H. R., AND Cox, G. A. 1936 Bacteriophage phenomena in cultures oflactic streptococci. J. Dairy Research, 7, 55-62.

34. WHITEHEAD, H. R., AND HUNTER, G. J. E. 1937 Observations on the activity of bac-teriophage in the group of lactic streptococci. J. Path. Bact., 4, 337-347.

35. WHITEHEAD, H. R., AND HUNTER, G. J. E. 1939 Starter cultures for cheese manu-facture. Maintenance of acid-producing activity in cultures of lactic streptococci.J. Dairy Research, 10, 120-132.

36. WHITEHEAD, H. R., AND HUNTER, G. J. E. 1939 The bacteriophage-organism rela-tionships in the group of lactic streptococci. J. Dairy Research, 10, 403-409.


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37. WHITEHEAD, H. R., AND HUNTER, G. J. E. 1941 On the origin of bacteriophages forthe lactic streptococci. J. Path. Bact., 53, 440-441.

38. WHITEHEAD, H. R., AND HUNTER, G. J. E. 1941 Starter cultures for cheese manufac-ture. Further attempts to eliminate failures due to bacteriophage. J. Dairy Re-search, 12, 63-70.

39. WHITEHEAD, H. R., AND HUNTER, G. J. E. 1945 Bacteriophage infection in cheesemanufacture. J. Dairy Research, 14, 64-80.

40. WHITEHEAD, H. R., AND HUNTER, G. J. E. 1947 Bacteriophage in cheese manufac-ture. Contamination from farm equipment. J. Dairy Research, 15, 112-120.

41. WHITEHEAD, H. R., HUNTER, G. J. E., AND Cox, G. A. 1952 Inhibition of bacterialgrowth by bacteriophage as distinct from lytic action. J. Gen. Microbiol., 6, 21-29.

42. WHITEHEAD, H. R., AND HARKNESS, W. L. 1952 The influence of bacteriophage incheese manufacture. The effect of an extended ripening period. Australian J.Dairy Technol., 7, 3-5.

43. WILLIAMSON, K. I., AND BERTAUD, W. S. 1951 A new bacteriophage active againsta lactic streptococcus. J. Bact., 61, 643-645.

44. WILLIAMS SIUTH, H. 1948 Investigations on the typing of staphylococci by meansof bacteriophage. I. The origin and nature of lysogenic strains. J. Hyg., 46, 74-81.

45. WILLIAMS SMITH, H. 1948 Investigations on the typing of staphylococci by means ofbacteriophage. II. The significance of lysogenic strains in staphylococcal type desig-nation. J. Hyg., 46, 82-89.

46. WOLF, J. Z., NICHOLS, A. A., AND INESON, P. J. 1946 Mists containing hypochloriteas germicides in the destruction of air-borne bacteriophages attacking lactic strepto-cocci. J. Dairy Research, 14, 291-315.

47. YAKOVLEV, D. A. 1939 Bacteriophage of lactic acid streptococci. Mikrobiologiya(U.S.S.R.), 8,932-950. Chem. Abs., 35, 3674 (1941).


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