Phage Detection

Production of cheese today is a big business involving the processing of large quantities of milk every day. In order to attain the desired production level it is necessary that the milk clot in a specific period of time. Before clotting can occur there must be lactic acid production. Lactic acid is produced by the action of starter cultures which are lactic acid bacteria which both produce the lactic acid and provide enzyme systems for flavor development during curing. Failure to produce enough lactic acid within the time schedule results in economic loss either through dumping the vat full of milk or by degradation of the cheese quality.

Starter cultures are blends of genetically distinct strains or subclasses or organisms primarily within two classes of lactic acid producing bacteria, including Streptococcus cremoris and Streptococcus lactis. All culture strains are subject to attack by bacteriophages which are bacterial viruses which can attack bacterial cells, multiply within the bacterial cell and ultimately destroy it. These bacterial viruses are also genetically distinct and this enables the viruses to differentiate between the various strains of starter organisms . . . thus certain viruses will attack only particular strains of S. cremoris and S. lactis.

Phage is always present. Obviously a starter culture must be resistant to the phage in order to function properly. A given blend which is today resistant cannot be used continuously, however, since the phage continuously undergoes genetic changes and culture strains that were not susceptible can become susceptible as new viruses develop against them. Successful starter culture selection becomes a matter of staying ahead of the change in the phage in the plant. Current methods of guarding against failure involve use of a rotation of blends of strains. The cheesemaker uses a number of different blends on different days. The blends are purchased from suppliers. The relationship between the strains in a blend has usually been determined by experimental methods and by trial and error so the phage produced (built up) against one strain or blend used one day will not attack the blend (or strains therein) used on the following day or the day thereafter. This is called rotation. The basis is trial and error and there is no assurance of success . . . and in practice the rotation system is only about 96% successful. In a large cheese plant 4% is a very significant loss.

A further factor to be considered is the effect of slight loss of starter culture efficiency due to phage attack on one or two of the strains sufficient to lower lactic acid production but still passable (as opposed to dumping the milk). This lowers the cheese quality and the change in acid (pH) could necessitate changes in the processing of the whey (a by-product) causing economic loss.

There are known tests for determining the phage susceptibility. The M17 agar plate method (the Australian Journal of Dairy Technology, June 1977, pp 63-64) is used in certain laboratories but is generally regarded as requiring too much skill and experience for the cheese factory. A simpler test involves the use of Bromcresol Purple (BCP) Milk in which the direct acidification of milk is wholly or partially inhibited by phage so that after incubating the resistant (usable) culture has turned yellow while the inhibited (not usable) culture is blue or green. Neither the BCP method nor the M17 agar plate method has been adopted for use in the factory.

The object of this invention is to allow the cheesemaker to choose his starter culture on the basis of the phage environment in his plant at any particular time.

In carrying out this method the cheesemaker preferably has available to him a multiplicity of genetically distinct strains in each of the three groups of lactic acid producing bacteria which will make up the starter culture. A simple test kit containing each of the strains in his culture bank in a sterile medium and with BCP permits a simple test using whey from current production (the whey will contain the curent phage in the plant) to determine which strains in each group are resistant, thus indicating which strains can be used without fear of phage attack. If desired, blends may be used (tested) instead of working with single strains. The final starter culture can be a single strain (as sometimes done in Europe) or a blend (as used in the United States).

We have demonstrated by use of the M17 method that the success of the starter culture can be raised to 100% and we have demonstrated that the BCP method coincides with the M17 results 99.94%. This not only achieves significant direct reduction of losses over the 96% success of the rotation method but achieves significant economics in whey processing. No undergrade cheese was produced when following this method.

The test apparatus is simple and inexpensive. Each culture is in its own "test tube" along with BCP and sterile milk. All recepticles are sealed to maintain sterile conditions. A typical test kit will have 8 "test tubes" in each of three groups of culture strains . . . typically one group will be strains of S. lactis and there are 2 groups of S. cremoris (8 fast and 8 slow-acting strains as typical in culture blends). Each tube contains sterile milk, BCP and a strain. In use each tube is inoculated with filtered whey from current production and the tubes are then incubated and observed for color change at 6-7 and 16 hours. The strains which have turned yellow can be used safely. The cheesemaker uses any one safe strain from each of the 3 groups to make up a blend which is now known safe. As noted above the same procedure can be applied using different blends in the test tubes. In that event the test will indicate which blends are resistant to the current phage.

Instead of working with frozen (liquid) solution containing the culture, BCP, and sterile medium, the culture and sterile non-fat dried milk (NFDM) can be freeze dried and reconstituted by a solution containing sterile water, BCP and the whey sample. In either event, the method of determining the resistant culture is essentially the same.

FIG. 1 is an exploded perspective view of a test kit.

FIG. 2 is a detail view showing injection of the whey into a test tube and also showing how a solution whey, sterile water and BCP can be added to the freeze dried culture and NFDM.

FIG. 3 shows how the test tray is floated in an incubating bath or supported in a dry incubator.

The test kit supplied to the cheesemaker is a plastic tray 10 having test tubes 12 pressed into and depending therefrom in 3 rows, A, B, C of 8 tubes each. Row A may be 8 distinct strains of S. lactis, Row B may be 8 fast strains of S. cremoris and Row C would be 8 slow strains of S. cremoris. "Fast" and "slow" terminology is used in the sense reported in the Dairy Industries 1972, Vol. 37, Page 73, where a fast strain grows well at 38.degree. C. and slow strains grow slowly above 36.degree..

The strains are in a BCP milk solution which is prepared by first sterilizing a 10% (V/W) reconstituted non-fat dry milk (NFDM) solution at 121.degree. C., 15 psi for 15 minutes, cooling the solution to room temperature and adding sterile (121.degree. C., 15 psi, 15 min.) 5% Bromcresol Purple solution at the rate of 0.75 cc/1,000 cc of milk solution. Each test tube is filled with 2 ml. of the BCP milk solution and then inoculated with 0.03 cc (1 drop) of a freshly grown (16 hours) milk tube of the appropriate culture strain. A suitable plastic film 14 is then sealed to the top surface of the tray. Suitable coding or indicia for the respective cultures in rows A, B and C corresponding to the cultures inventoried by the cheesemaker can be applied to the tray or film as shown in FIG. 1 in which the various tubes are numbered according to the culture suppliers code. For handling and shipping purposes the rigid plastic cover 16 is snapped over the tray. The finished kit is then frozen for storage but is thawed prior to use.

The BCP-milk-culture color is blue or blue-green. The bromcresol purple is a pH indicator dye which turns from blue color to yellow in response to acid development as the culture strain develops in the presence of the bacteriophage present in a whey sample collected before salting in current cheese production in the plant. The whey sample is filtered and then 0.03 cc (1 drop) is added to each test tube, preferably using a hypodermic syringe 18 as shown in FIG. 2 with respect to tube 12A to puncture the seal film 14 but other methods of inoculating can be used such as pipettes, etc. The tray is now placed in and supported by the foam plastic flotation collar 20 and then placed in an incubation water bath maintained at 30.degree. C. Alternatively the tray can be placed in a dry incubator at 30.degree. C. with the tray and collar supported on legs 22.

After 6 hours incubation (up to 16 hours) the tubes are inspected. The tubes which have formed a firm curd and have turned yellow contain culture strains which are not affected by the phage present and have proper acid production and growth. These strains are suitable for use in making a blend. One strain is selected from those indicated suitable in each row and the 3 strains are mixed. The cultures which remain blue or blue-green after incubation have not produced acid (or curd) properly by reason of the phage attacking the strain. Some tubes may turn yellow-green indicating partial susceptibility to phage attack and while they might be usable the cheesemaker preferably will select from the strains clearly unaffected by the phage. This assures virtually 100% results compared with 96% (at best) obtained with rotation. In a cheese plant processing 2,000,000 pounds of milk per day this means elimination of a 80,000 lb/day loss of milk (or a gain of 8,000 lbs. of cheese production each day).

Bromcresol purple is the preferred dye but other pH indicator dye are usable, such as methylene blue or resazurin dye. The color changes with BCP are deemed easier to work with. Less than full color change indicates various degrees of susceptibility and for safety sake it is easy to select from the cultures showing full change. The tray and tubes can be made as shown or as a unit and are preferably made of plastic to be a throw away item.

The method can be used in conjunction with freeze dried cultures. In that case the culture strain (blend) would be freeze dried with sterile non-fat dried milk power as in tube 12B in FIG. 2. The filtered whey would be added to a container of sterile water and BCP and the proper amount of that solution would then be added to tube 12B to reconstitute the dried milk and start the test. This approach may be more attractive to the cheesemaker since he does not have to meter out just one drop as in the "wet" approach. The film 14 is simply peeled back to allow addition of the proper amount of the solution of tube 12B and the film is then reclosed to maintain sterile conditions. A dispenser 24 for automatically metering the correct quantity to each tube can be used.

If this method is practiced in conjunction with the blends used by the cheesemaker in the past on a rotation basis, the method can prevent the 4% (or more) loss previously experienced. Thus the cheesemaker can test the next blend normally used in his rotation along with a few other blends. If the test shows his usual "next" to be susceptible to the current phage, he will select a blend which is clearly resistant and avoid the loss.

This method, therefore, can be used with rotation to raise the success rate to 100%. It can be used in conjunction with genetically distinct strains to permit the widest choice of blends and complete orchestration of the culture art. And it can be used to select a single strain with assured results for those preferring that approach.