SOME ASPECTS OF THE MICROBIOLOGY OF FROZEN PEAS

WHIl%, A. 8r WHITE. H. It. (1962). J. appl. Hact. 25 ( I ) . ti2-il.

SOME ASPECTS OF THE MICROBIOLOGY OF FROZEN PEAS

BY ANNE WHITE AND HELEN R. WHITE Depurtment of Microbiobgy , Queen Elizabeth College, University of London

SUMMARY: The number of bacteria present in commercially frozen peas was found to be usually between lo' and 10e/g. Most of the organisms were of two species, Leuconostoc wtesenteroides and Streptococcus lartis. After thawing the leuconostocs multiplied more rapidly than the streptococci and in 48 hr the former constituted almost the entire population.

Growth studies of four icrolatee of each of the above species, both in laboratory media and on blanched peas, showed no difference in growth rates before and after freezing. When some organisms, including potential pathogens, were inoculated on to peas rapid multiplication took place even in the presence of leuconostocs or streptococci.

IT IS A COMMON belief that frozen produce when thawed is more perishable than fresh produce. If this is so there are at least two possible reasom: fist, that plant or animal cells which have been frozen and thawed are more suitable for baoterial growth owing to breakdown of cell structure and release of free fluid and nutrients; second, that freezing has a direct effect on the growth rate of bacteria.

The published results relating to this subject are somewhat contradictory. Kallert (1928) studied the bacteriology of minced meat and concluded that frozen meat was not more perishable than fresh meat, whereas Gressel & Griife (1929) who continued this work found that it was. Hastsell (1951) studied the growth of four bacterial species in frozen whole eggs and found that these bacteria multiplied more rapidly after freezing and thawing. He suggested that this was due to a physiological stimulation of bacterial cells which was brought about by freezing. Sulzbacher (1962) inoculated it psychrophilic pseudomonad on to meat before freezing it, and found that the lag phase on frozen meat after thawing was longer than on the fresh meat and also that growth was slower on thawed frozen meat.

Squires & Hartsell (1955) investigated the survival of Escherichia wli in different frozen storage menstrua and found that after such storage the generation times during subsequent growth varied according to the medium. Kitohell& Ingram (1956) investigated the growth of bacteria on meat, but found no evidence that the liberation of free fluid in thawed tissue favoured bacterial multiplication or that bacteria built up a 'growth potential' in the frozen state. They concluded that frozen meat was neither more nor less perishable than the corresponding fresh meat. Further work (Kitchell & Ingram, 1959) supported this conclusion.

The present work is confined to certain aspects of the bacteriology of frozen peas.

EXPERIMENTAL METHODS The numbers of bacteria present in commercial frozen peas. In all? 107 packets of

on0 brand of commercial frozen peas were examined. Each sample, which consisted

Microbiology of frozen peas 63

of approximately 1 g of peas, was macerated for 2 min in an M.S.E. macerator (Measuring and Scientific Equipment Ltd., 14-28, Spenser St., London, S.W.l) run at maximum speed, before being plated for colony counts. Several plating media were tried : glucose-Yeastrel agar, nutrient agar, tomato juice agar, malt extract agar, Rogosa's medium (Rogosa, Mitchell & Wiseman, 1951), V8 vegetable juice (Campbells Soups Ltd., Kings Lynn, Norfolk) medium and a pea extract medium. Gludose-Yeastrel agar (GYA) (peptone, 1 g; Yeastrel, 0.3 g; glucose, 1 g; NaC1,O-25 g ; agar, 1 g; distilled water, 100 ml; pH 7.6) was chosen as the most suitable general growth medium. It gave higher counts than all the other media used and was readily available in the laboratory; other authors have reported this medium to be most suitable for counting bacteria in thawed food products (Goresline, 1946; Hartsell, 1951). It was found that 30" was the most suitable temperature for incubation and this was used throughout.

The microjlora of commercial frozen peas and the effect of storage at high temperatures. Statistical picks were carried out on the colonies obtained on GYA from three packets of a single brand of commercial frozen peas. The packets were sampled while frozen and forty colonies were picked by the disc method described by Harrison (1938) from the plates obtained from each sample. Each of these cultures was then identified.

The types of organisms present in forty packets of frozen peas were determined by another method. Counts were obtained using the Miles & Misra (1938) counting technique modified by the use of platinum tipped pipettes (Davis & Bell, 1958, 1969). Two media were employed; these were GYA and a medium containing sucrose (Garvie, pers. comm.) (tryptone, 1 g; Yeastrel, 0.6 g; K,HPO,, 0.6 g; sucrose, 6 g; ammonium citrate, 0.2 g; agar, 1 g; pH 6.8). Slime forming colonies could be counted on the sucrose medium and in the statistical picks described above it had been found that all slime formers present were leuconostocs. Colonies which did not form slime on this medium were taken to be streptococci if they were of the typical colonial morphology and size, since more detailed examination of a sample of colonies indicated this to be a fair assumption. The results obtained on GYA were used to obtain the total count.

In order to determine which organisms multiplied during the storage of peas a h r removal from the deep freeze and were responsible for spoilage, further statistical picks were carried out on plates prepared from single packets before and after incubation a t 30" and 22" for 18 and 48 hr respectively. (Incubation for 18 hr was chosen as it was usually the time a t which spoilage first became apparent a t 30"; after 48 hr spoilage was much more advanced).

Growth studies with laboratory media and peas. From one of the statistical picks just described, 8 isolates were selected as representative of the species present. Four of the isolates were identified as Leuconostoc mesenteroides and four as Streptococcus Iactis, although two of the streptococci failed to ferment lactose. Experiments were done on these 8 isolates to observe the effect of freezing on growth rates in laboratory media and on fresh and thawed frozen peas (both blanched and unblanched). Counts were carried out by the Miles & Misra technique modified as before. All material was frozen by placing in a deep freeze a t -23".

64 Anne White and Helen R. White

Growth rate studies in laboratory media were done in triplicate in the broth equivalent of GYA. The inocula used were: first, 0.01 ml of an 8 hr culture in 10 ml of broth; and second, 0.01 ml of a similar 8 hr culture which had been kept frozen for 14 days, then thawed in a water bath at 37" for a few minutes before inoculation into broth.

The peas were shelled in the laboratory under aseptic conditions. They were blanched by immersing in boiling water for 1 min and then simultaneously cooled and inoculated by transference to a litre of cold water to which the inoculum, 5 ml of an 8 hr culture, had been added, In this the peas were left for 3 min, and they thus acquired an infection of 103-104 organismslg. The peas were then placed in sterile wax cartons. Three of these were incubated at 30" and the growth of the bacteria in the peas was followed. The remainder were frozen. One was removed from the freezer after 6, 12, 18 and 24 weeks, respectively, and taken directly to an incubator at 30" for the determination of growth rates. This process was repeated with fresh unblanched peas.

The growth of organisms other than the normal jlora. It is often thought that the presence of the so-called 'normal' flora of frozen produce is a safeguard against public health hazards and that initially these organisms are likely to outnumber any pathogenic organisms which might be introduced in the factory or elsewhere and prevent their increase to a dangerous level. It was thought of interest, therefore, to investigate the ability of some organisms not normally found on the peas to multiply on thawed frozen peas, and to establish their growth rates. The multiplication of these organisms on thawed frozen peas in the presence of streptococci or leuconostocs was also investigated.

Five organisms were selected for these studies: two from the National Collection of Type Cultures-Staphghcoccus aurew (NCTC 7447) and E. coli (NCTC 86); and three from the stock culture collection of this laboratory-Salmonella typhi-murium, Bacillus subtilis and Strep. faecalis. Each organism was inoculated on to peas which had been previously prepared, blanched and frozen in the laboratory. The inoculations were carried out as before, to give concentrations of 103-104 organismslg.

Mixed cultures were also used as inocula. Five experiments were carried out using the following combinations of the above stock cultures and strains of leuconostoc and streptococci isolated from frozen peas: Staph. aureus and Leuc. mesenteroides (strain 101) ; Salm. typhi-murium and L e w . mesenteroides; B. subtilis and Leuc. mesenteroides; E. wli and Leuc. mesenteroides; E. wl i and Strep. lactis (strain 118). In each case approximately equal numbers of the two organisms were inoculated and the development of both organisms was followed for 20 hr.

RESULTS The microfira of commercial frozen p e m a d the effect of storage

at high temperatures The numbers and types of bacteria present. The bacterial counts on packets of frozen

peas varied from 103 to 1071g. Out of the 107 packets examined, 58 had counts between lo4 and 105/g and a further 23 had counts between lo5 and 106/g.


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$ 40 Y 20

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The'statistical picks of the colonies obtained by platifig frozen peas showed that the main types present were catalase negative Gram-positive cocci. The commonly found organisms were studied in more detail. They fell into two groups, one of which consisted of heterofermentative, catalase negative, Gram-positive cocci which had no action on litmus milk and generally produced slime on sucrose media; these organisms were provisionally identified as leuconostocs. The strains included in the second group were homofermentative, catalase negative, Gram-positive cocci, which reduced litmus, produced ammonia from arginine and did not produce slime on sucrose media. These were identified as streptococci.

In 27 of the 40 packets examined leuconostocs constituted between 40% and 600,; of the population; in the remaining 13 packets the leuconostocs formed as few as 10% or as many as 90% of the total organisms. The rest of the population consisted of streptococci, except for a small percentage of other organisms; this was usually less than 10% of the total popuIation and consisted of Gram-negative rods and some sporeformers.

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a t 30"

a t 2 2 O

Fig. 1 . The development of the flora of commercial frozen peas on incubation at two temperatures. Cross-hatched areas, streptococci; white areas, leuconostocs; hatched areas, other organisms.

The effect of storage temperature after removal from the deep freeze. The results of the statistical studies are shown in Fig. 1. The packet of frozen peas which was incubated a t 30" initially contained approxiniately lo4 organismslg, comprising 33% of streptococci and 57% of leuconostocs. After 18 hr the total count was approximately 108/g, and the proportions then were 15% of streptococci and 85% of leuconostocs. After 48 hr the total count was still about 108/g, but 97% of these were now leuconostocs.


The second packet, which was incubated a t 22", gave similar results, although initially 43% of streptococci and 40% of leuconostocs were present; after 48 hr only 10% were streptococci, the remaining 90% being leuconostocs.

Accompanying the multiplication of streptococci and leuconostocs there was always a drop in pH from about 7 to about 4. The colour change from green to yellow normally associated with spoilage of frozen peas occurred at pH 4.6.

Growth studies with laboratory media and peas Growth in broth. The results obtained in broth are shown in Figs. 2 and 3. The

results shown for Leuc. mesenteroides and Strep. luctis are typical. A variance analysis carried out on the logarithmic phase of the graphs obtained for seven isolates showed no significant difference between the gradients at the 95% level. Results were not obtained for the eighth isolate. A longer lag (2-9 hr) was shown by the cultures which were frozen and thawed before subculturing compared with those which were not frozen.

, I I I I I J 0 I 2 3 4 5 6 7 8 9 10

Time (hr)

Fig. 2. The development of Leuc. mesenterodes (strain 101) in broth with prior freezing (full circles) and without prior freezing (open circles). Tripli- cate tests were done on each occasion.

Growth on peas. Figs. 4 and 5 show the results obtained for the same two cultures grown on blanched peas. These are typical of the results obtained for eight isolates. A variance analysis was carried out on the gradients of each set of graphs for eight isolates and there was no significant difference between gradients at the 96% level.


An apparent lag occurred due to the time taken for the peas to thaw before bacterial multiplication could begin. A period of approximately 2 hr was required for peas to reach 30" after they were placed in the incubator; thus with an apparent lag of 24 hr, the true lag would probably be less than 1 hr. The apparent increase in lag shown in Figs. 4 and 5 is between 2 and 3 hr. This increase is very much less than that which occurred when freezing took place in broth.

Time (hr)

Fig. 3. The development of Strep. Zactis (strain 118) in brothwith prior freezing (full circles) and without prior freezing (open circles). Triplicate tes ts were done on each occasion.

The growth rate determinations carried out on unblanched peas, both fresh and frozen, indicated that no change in growth rates had been brought about by freezing, but the curves were too irregular for any calculations to be made.

The growth on peas of organiw other than the normal $om. Each of the five organisms studied grew well as pure cultures inoculated on to frozen peas, each reaching a population of approximately 1081g after incubation for 1P16 hr.

The results obtained when mixed cultures were inoculated on to peas showed that E. COG, Staph. aureus, Salm. typhi-murium and 3. subtilis were all capable of multiplying on peas and reaching populations of about 108/g after incubation for 1P16 hr even in the presence of Leuc. mesenteroides.

A similar result was obtained for E. coli when inoculated on to peas with Strep. lactis.

68 Anne White and Heren R. White

Time (hr)

Fig. 4. The development of Leuc. mesenteroides (strain 101) on peas with prior freezing (full circles) and without prior freezing (open circles). Triplicate tests were done before freezing and single tests at 6-weekly intervals after freezing.

DISCUSSION The viable counts of frozen peas which we obtained on glucose-Yeastrel agar are of the same order as those reported by Pederson (1947), who found that counts of 104-106/g on tryptone-yeast extract-glucose agar were to be expected in good quality frozen vegetables. Pederson suggested that counts of 106/g or more were indicative of poor handling, and it is of interest to note that very few of our counts reached this level. Many of the bacterial counts reported elsewhere in the literature are not strictly comparable with our figures since the authors either gave no indication of the culture medium used or used a less rich medium on which fewer organisms would be able to grow. For example Tressler (1938) stated that counts of below SO,OOO/g in frozen vegetables were satisfactory; but he did not specify the medium used. Our evidence suggests that the dominant flora of frozen peas consists of Strep.

lactis and Leuc. rnesenteroides and that the spoilage of thawed frozen peas results from their multiplication. This is partly supported by the literature. Several workers, Lochhead t Jones (1938), Perry et al. (1948) and Hucker (1954) have


recorded the presence of Gram-positive cocci on frozen vegetables but have not identified them further. However, Sanderson (1941) and Fitzgerald (1947) reported that Strep.. faecalis occurred in frozen vegetables and attributed spoilage to the acid produced by this organism. Fitzgerald had also reported the isolation of similar strains of Strep. faecalis from the seed coats of fresh peas. Stark & Sherman (1935) found Strep. luctis on fresh peas. Only one reference has been found to the presence of leuconostocs on frozen vegetables (Borgstrom, 1955) and no references at all to their association with spoilage of these foods. This is surprising as the principal sugar present in peas is sucrose and, on incubation of commercial frozen peas, the production of slime by the leuconostocs becomes obvious after 24-36 hr.

Time (hr)

The development of Strep. h t i s (strain 118) on peas with prior freezing (full circles) and without prior freezing (open circles). Triplicate tests were done before freezing and single tests at 6-weekly intervals after freezing.

Fig. 5.

Our studies indicate that there is no change in the growth rate of cultures of Strep. lactis and Leuc. mesenteroides after freezing and thawing either in broth or on peas. However, the lag phase of the thawed frozen culture is prolonged to a considerable extent in cultures frozen in broth and slightly so in cultures frozen on peas. Thus freezing, particularIy in broth culture, appears to exert a physiological effect on leuconostoc and streptococcus cells, affecting their ability to initiate growth on thawing but not affecting their subsequent rate of growth. Observations reported


in the literature concerning the effect of freezing on growth rates and on length of lag phase seem to be conflicting, although it should be borne in mind that different strains, and usually different genera of bacteria, have been used by the various authors. Gram-positive cocci are noticeably more resistant to freezing than other organisms (Jones & Lochhead, 1939) and for this reason might possibly be expected to be less affected by freezing and thawing. However, among the Gram-negative organisms consistent behaviour has not been recorded. Hartsell (1951) showed that the growth of E. coli was stimulated in thawed frozen eggs, Squires & Hartsell (1955) found that the growth rates of E. coli cultures were altered after freezing and thawing in yeast extract-veal infusion broth and that the generation times changed with the length of time of freezing storage, and according to Sulzbacher (1952) the growth rate of a pseudomonad was slower and the lag period prolonged after freezing and thawing on meat. However, Kitchell & Ingram (1956, 1959), working with a pseudomonad frozen in laboratory media and in meat, obtained results similar to ours, finding no change in the generation time but an increase in length of lag after freezing. It seems that organisms in food are partially protected from the effects of freezing. This was shown in our experiments where the lag phase was extended more markedly after freezing the organisms in broth than after freezing them in peas. A similar observation was made by Kitchell & Ingram (1959), who demon- strated that when unheated aqueous extracts of meat were added to liquid laboratory media pseudomonad cells were protected, the effects of freezing being reduced.

Our results with mixed cultures, in which we showed that E. coli, Staph. aurew, Salm. typhi-murium and B. subtilis were all capable of multiplying in the presence of the normal flora of frozen peas, demonstrate the importance of factory hygiene in the production of frozen vegetables. The multiplication of the normal flora cannot be relied upon to prevent the growth of potential pathogens, a t least if the latter are initially present in high numbers.

The authors wish to express their thanks to Birds Eye Foods, Ltd. for supplying samples of frozen peas, and to Dr. P. M. Scholes of this Department for her valuable suggestions throughout the work and especially for her help in preparing the manu- script. They are also grateful to Miss P. Reed for her excellent technical assistance.

This work was underteken while one of us (A.W.) ww holding a postgraduate scholarship awarded by Queen Elizabeth College and later, a research studentship awarded by the Department of Scientific and Industrial Research. She would like to thank the authorities concerned.

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(Received 23 August, 1961)

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SOME ASPECTS OF THE MICROBIOLOGY OF FROZEN PEAS