No. 2, Volume 13, of the Proceedings of the Society for .Applied Bacteriology was issued on 21 November, 1951

SEASONAL VARIATION I N CHEESE STARTER ACTIVITY

BY J. CZULAK AND L. J. MEANWELL

United Dairies Research Laboratories

SUMMARY : Depression of activity of certain single strain starter cultures in H.T.S.T. pasteurized milk during the October-April semon was observed, but the effect waa removed by momentary boiling. The addition of a small percentage of such milk to autoclaved milk reduced the activity of these cultures. These ' inhibited ' cultures were unable to grow in modified Niven's medium even when additional known growth factors were added. Using a whey agar medium, active ' uninhibited ' colonies may be picked and a ' failing culture ' revived.

An hypothesis postulating an inhibitor-growth factor antagonism is advanced, to explain the findings.

As a result of the increase in winter milk production during the past four years, there has arisen a problem of slowness in cheesemaking associated with winter milk. This problem became serious in the period January-March 1949, when one large creamery, manufacturing daily over 20,000 gallons of milk into cheese, found that their routine supply of single strain starter cultures which had worked satisfactorily during the conventional cheesemaking season from April to October no longer gave satisfactory acid production in the cheese vat and a period of slow cheesemaking was experienced. It was decided to investigate this phenomenon, and the results are described below. In this paper effects due to phage attack are not considered.

EXPERIMENTAL AND RESULTS

Determination of starter activity. The starters studied were cultures of Streptococcus lactis and Str. cremoris only. Activity was determined by inoculating 1% of each starter culture into milk, incubating a t 30" for 6 hr. and then titrating with N/9 NaOH. The total titration was expressed as lactic acid, and the activity expressed as yo lactic acid.

Preliminary experiments on starter activities

First, all the starter cultures in normal use were tested for activity in cheese vat (H.T.S.T. pasteurized) milk and in autoclaved milk, and i t was found that while the activity of most of the cultures in autoclaved milk was between 0.4 and 0.6%, some of the starter cultures examined were far more suited to growth in cheese vat milk than others. When those starters which were more active in winter cheese vat milk were used the trouble due to winter slowness was overcome.

In order to gain information on the seasonal variation of starter activity in the cheese vat, a series of tests was carried out over twelve months during 1949. Eighteen

2 J . Czulak and L. J . Meanwell

aingle strain starter cultures selected on account of their good activity in autoclaved milk were tested each week for activity in both autoclaved and in bulk H.T.S.T. pasteurized milk. The average activity in pasteurized milk in each month of the test is given in Table 1, and in Table 2 the average activity of five individual starters is given for the four seasons in both autoclaved and pasteurized milk. A marked increase in average activity in H.T.S.T. pasteurized milk was recorded in mid-April and a marked fall began in mid-October, the months in which many normal changes occur, such as marked rise or fall of night temperature, change of feed, etc.

Table 1. Seasonal variation in starter activity (6 hr. at 30"), in H.T.S.T. pasteurized milk, of 18 cultures

highly active in autoclaved milk Month Mean activity*

(yo lactic acid) January 0.294 February 0.299 March 0.279 April 0.360

0.388 0.365

May June

Month Mean activity (yo lactio acid)

July 0.435 August 0.426 September 0.428 October 0.403 November 0-309 December 0.299

* All 18 cultures were tested weekly.

Table 2. Seasonal variation in activity of individual starter cultures Starter Activity* in autoclaved milk Activity* in pasteurized milk

h A no. r 7 r 7 Jan.- Apr.- July- 0ct.- Jan.- Apr.- July- 0ct.- March June Sept. Dec. March June Sept. Dec.

496 0.534 0.541 0.500 0.490 0.206 0.277 0.315 0.220 497 0.506 0.511 0.564 0.470 0.172 0.175 0.239 0.160 573 0.504 0.499 0.551 0.470 0.470 0.576 0.570 0.530 723 0.428 0.465 0.544 0.430 0.380 0.601 0.615 0.440 738 0.395 0.423 0.544 0.390 0.399 0.598 0.572 0.520

* yo lactic acid after 6 hr. at 30".

As an example of the individual variation, Table 2 shows that in autoclaved milk starters 496 and 497 gave exceIlent activities a t all seasons of the year, while in pasteurized (cheese vat) milk they,had little or no activity, except starter 496 during the months of July-September. Thus these two starters would have been useless for cheesemaking purposes during the winter months. This property of winter pasteurized milk, which results in slow acid production by certain starter cultures, was found to be a general one for milk supplies collected from widely separated areas, and was shown to be equally intense for milks of both high and very low original bacterial content. Similar behaviour was noted when winter spray-dried milk powder was used, but not with the more severely heated roller-dried powder. The property was destroyed by momentary boiling and thus i t was clearly dis- tinguished from any action due to antibiotics such as penicillin, diplococcin and nisin which withstand boiling €or considerable periods.

Seasonal tiariation in starter activity 3

Even the addition of ly0 of winter bulk pasteurized milk to autoclaved milk reduced the activity of a sensitive starter from 0.70 to 0.38% lactic acid, while a 1006 addition still further reduced the activity to 0.280~.

From these preliminary observations it became evident that the fall in activity of certain starter cultures might be due to an inhibitory substance active in H.T.S.T. past,eurized milk, but inactive in autoclaved milk.

Nutritional expPrirneT& with starter cultures

It is known that cultures of Str. lactis and Str . cremoris are subject to mutation. e.g. Harriman & Hammer (1931) found that Str. lac& produced stable variants which formed acid slowly in milk. Thus, a t any given time a culture may contain a fast and a slow acid-producing element. Storrs & Anderson (1949) found that these slow acid-producing cells required a factor present in yeast extract or fish meal in order to form acid rapidly. The present authors have since found that starter cultures which produce acid rapidly in milk lose this power after prolonged growth in yeastrel broth. Similar results have been reported by Eagles, Okulitch & Campbell (1936). l h u s i t appeared a t this stage that the difference between a fast and a slow acid-producing culture might be related to their synthesizing ability, and i t was decided to determine the nutritional requirements of fast and slow acid- producing cultures.

Sixteen cultures were selected, eight of which were known to be inhibited in winter (H.T.S.T.) pasteurized milk, while the remainder were active (' uninhibited ') in this type of milk. Attempts were made to grow them in the synthetic medium proposed by Niven (1944) which, after some preliminary work, was modified a s follows :-(1) In the group of amino acids, considered important by Niven, L-proline and m-lysine were omitted, as were also all amino acids in the unimportant group. (2 ) Glucose was replaced by lactose. (3) Potassium dihydrogen phosphate (3 g./l.) was added. (4) The pH was finally adjusted to 6.8 instead of 7-4. Tubes containing 10 ml. of this modified medium were inoculated with a small loop from 24 hr. litmus milk cultures of the starters and incubated for 48 hr. a t 22". Where growth was observed it was confirmed by making further transfers with a needle. The results are summarized in Table 3.

With one exception (806) all the ' uninhibited ' cultures were able to grow satis- factorily, although not as rapidly as in yeastrel broth or autoclaved milk. The ' inhibited ' cultures, on the other hand, showed no growth even after a further 10 days incubation a t 30". In some cases limited growth could be seen in the first tube and this was probably caused by the growth-stimulating effect of the milk constituents carried over.

The experiment was then extended to determine whether the addition of known growth factors and amino acids, alone or in combination, to the modified Niven's medium would result in growth of the ' inhibited ' cultures. The substances tested were : ascorbic, folic, desoxyribonucleic and nucleic acids : adenosine, aneurin, creatine, inositol, nicotinic acid amide, pyridoxal acetal hydrochloride, pyridox-

4 J . Czulak and L. J . Meanwell

aminedihydrochloride and Tween 80, as well as the remaining amino acids originally used by Niven. There was no response.

Table 3. The growth of ' uninhibited ' and ' inhibited ' starter cultures* in modified Niven's medium and in a

selective whey agar medium Culture Behaviour in Growth? in

no. pasteurized modified

519 uninhibited + 573 + 712 ,. + 738 + 806 I .

8.52 + 87 1 + 874 + 459 inhibited - 496 - 497 - 760 - 771 - 880 ,, 801 - 803 -

winter milk Niven's medium

._

-

* All cultures were active in autoclaved milk. t Turbidity in liquid medium after 2 days at 22" plus 10 days at 30". $ Colonies on solid medium after 4 days at 22".

Growth: in selective whey agar medium

+ + + + + + +

-

While the modified Niven's medium gave a satisfactory differentiation between the ' uninhibited ' and ' inhibited ' cultures, i t was thought desirable to find a simpler solid medium which would be selective for the variants which produced acid rapidly. A medium was prepared, which in its vitamin content approached the modified Niven's medium and, owing to the presence of chalk and indicator, permitted the differentiation of the fast acid-producing colonies. I ts composition was : Difco whey agar, 4% ; Difco peptone, 0.75% ; sterile chalk, 3% ; bromcresol purple, 0.006%.

Serial dilutions of all the 16 cultures tested for growth in the modified Niven's medium were plated out, using 10 ml., quantities of this whey agar medium. As controls, the same cultures were plated out on tryptone yeastrel agar ; all plates were incubated for 4 days a t 22'. The counts on the control medium showed no significant differences between the two types of culture, while on the whey medium the ' uninhibited ' cultures (except 806) showed varying numbers of colonies and the ' inhibited ' ones showed no growth (Table 3).

The colonies that appeared on the whey medium were of two types, either with a well defined clear zone, or with a hazy zone. The clear zone colonies, when picked into litmus milk, gave in most cases ' uninhibited ' cultures, i.e. they were active in winter H.T.S.T. pasteurized milk. In every case the count on the whey medium was lower than that on the control, being sometimes only a fraction of 1% of the control count. It was then realised that the whey medium could be used for recovering a single strain culture which had declined in activity provided that it contained

Seasonal variation in starter activity 5

even a small number of ' uninhibited ' type organisms. A number of such cultures were plated, colonies with clear zones picked and new ' uninhibited ' cultures were thus obtained. However, the same procedure applied to a few commercial starters (mixtures of Str. lactis, Str . crenaoris, Str. citrovorus and Str. paracitrovorus) proved unsuccessful, the test plates being crowded with colonies of Rtr. citrouorus and Str. pa ra.citrovorus.

DISCUSSION

Cultures affected during the winter season by the postulated inhibitory substance in H.T.S.T. pasteurized milk were, in every case, unable to grow in a well defined synthetic medium even when additional known growth-promoting substances were added. They also failed to grow on a solid medium containing whey and peptone without enriching substances such as yeast extract. On the other hand, cultures active in winter H.T.S.T. pasteurized milk were able to grow in these media. It thus appears that organisms of the latter class possess the power to synthesize for themselves some growth-stimulating factor which the ' inhibited ' cultures are unable to make. If this is true, inhibition in winter pasteurized milk may be explained on the hypothesis that ' inhibited ' cultures have a reduced activity in winter because an unknown growth factor which they require is a t that season made unavailable by a heat labile inhibitory substance, This inhibitory substance may be supposed to be present in milk throughout the year, and that in summer when the cows are fed mainly on grass and green fodder, the concentration of the growth factor is sufficiently high to overcome the effect of the inhibitor. The ' inhibited ' cultures are thus able to grow readily in summer H.T.S.T. pasteurized milk and in milk a t all seasons after the heat labile inhibitor has been destroyed by autoclaving or momentmy boiling. On the other hand, the ' uninhibited ' cultures syiithesize their own growth factor in sufficient quantity to overcome the inhibitor a t all times, and it is presumed that synthesis of the same growth factor enables them to grow in the above synthetic medium.

The evidence in support of the . inhibitor-growth factor ' hypothesis is indirect. Proof can only be obtained if both the inhibitor and the growth factor are isolated. While this remains'to be investigated the results obtained so far in our experiments can find application in practice. Several transfers in the modified Niven's medium result in an increase of the ' uninhibited ' element. Cultures so treated can then be plated on the ' selective ' whey agar medium and the ' uninhibited ' colonies picked. Where the ' uninhibited ' organisms are still relatively numerous direct plating only may be necessary. So long as cheese is produced from winter milk the necessity for using cultures which are not affected by the inhibitory substance in H.T.S.T. pasteurized milk is evident. It is also clear that activity tests in autoclaved milk have little value in the case of starters to be used in the winter season.

While it is evident, therefore, that the fast element can be sub-divided into that which is inhibited in winter H.T.S.T. pasteurized milk and that which is not, a third type of cell remains to be discussed, for when numerous colonies are picked from tryptone yeastrel agar plates three different types of cultures may be obtained,

6 J . Czulak and L. J . Meanwell

namely (a) those which are slow in both autoclaved and winter pasteurized milk ; (b) those fast in autoclaved milk but slow in winter pasteurized milk ; and (c) those fast in both types of milk. Cells from cultures of type (a), which like those from cultures of type (b) fail to grow in either modified Niven’s or the selective whey agar medium, are assumed to require some further growth factor not present in milk but present in yeastrel. This is the factor referred to by Storrs & Anderson (1949). CuItures of types (b) and (c), on the other hand, are able to synthesize this factor, and accordingly to produce acid rapidly in autoclaved milk. It is assumed, therefore, that all starter cultures tend to mutate and that the behaviour as far as acid production is concerned of any particular starter in the cheese vat will depend upon the relative proportions of these three types of cells.

REFERENCES

EAGLES, B. A., OKULITCH, 0. & CAMPBELL, A. C. (1936). Cheese ripening studies. The influence

HARRIMAN, L. A. & HAMMER, B. W. (1931). Variations in the coagulation and proteolysis of

NIVEN, C. F. (Jr.) (1944). Nutrition of Streptococcus Zactis. J . Buct. 47, 343. STORRS, F. C. & ANDERSON, E. B. (1949). The ‘‘ activity ” of cheese starters. Proc. 12th int.

Dairy Congress, Stockholm, 2, 605.

of yeast extract on the types of streptococci found in starters. Canad. J . Res. (B) 14, 311.

milk by Streptococcus lactis. J . Dairy Sci. 14, 40.

(Received 23 .January, 1951)