ICROBIOLOGICAL STUDIES OF INCIDENTS OF EARLY GAS PRODUCTION IN COMMERCIAL CHEESE PLANTS

The results of initial microbial studies (Table 2) at one plant (factory X) revealed that with the exception of catalase-negative citrate-utilisers, 'total count' on milk agar and 'counts' on Rogosa agar, microbial levels in blown and 'normal' cheeses were similar. Because bacterial numbers on Rogosa agar were relatively low <1 10⁶ cfu/g of cheese, these bacteria were not studied further at this stage but in later studies, blown cheeses were found to have 10-100 fold higher counts on Rogosa agar compared with normal cheese at factory X.

Notes:

All cheeses were obtained from the same commercial plant and from the same production run. Cheeses had been held at 7°C for 8 weeks after manufacture before sampling. Moisture, pH and salt levels were within acceptable limits and similar in all samples.
(1)Determined using differential agar media^13,14.
(2) Determined using Rogosa agar¹⁵

The scheme described by Billie et al.³ was used to identify presumptive lactic acid bacteria to the genus level and the API-CHL computer-assisted identification system to classify isolates to the species level. Detailed investigation of some of the catalase-negative citrate-fermenting isolates revealed that they resembled Lc. lactis ssp. lactis biovar. diacetylactis in their general properties. The data obtained were consistent with the Lc. lactis ssp. lactis biovar. diacetylactis isolates originating from the mixed strain starter cultures used.

These observations prompted us to study citrate levels in milk to determine if there was a relationship between citrate concentration and the incidence of gas production in factory X (Fig. 1). Although lowest citrate levels occurred during April, May and June, the incidence of gas production in this period was not markedly different than at other times during the period of study.

Pilot-scale cheese-making experiments were used to define the conditions required for gas production by Lc. lactis ssp. lactis biovar. diacetylactis in experimental cheese of satisfactory chemical composition. Sufficient gas to give overt "blowing" of barrier bags was only produced when mixed-strain cultures containing high levels of Lc. lactis ssp. lactis biovar. diacetylactis were used and the citrate level in cheese ex-press was 0.07% (w/w) or higher. To obtain this level of citrate, the cheese milk always had to be supplemented with additional citrate.

Attempts were made to correlate citrate levels in cheese, ex-press, with the development of gas in cheese at factory X. Some of the data obtained are shown in Table 3. These results show that gas production occurred in cheeses containing low and high levels of residual citrate and suggest that early gas production at factory X was not simply due to the activity of Lc. lactis biovar. diacetylactis. These findings also indicated that gas was being produced from substrates other than citrate.

During this study an opportunity arose to investigate the "blowing" of 4-kg wheels of Leicester cheese at another factory. The Leicester wheels had "blown" after 3 weeks storage at 7ºC.

Analyses revealed that:

i. salt/moisture (S/M) levels were satisfactory (ca. 5%)
ii. pH values were acceptable (ca. 5.1)
iii. levels of gas-producing non-lactic acid bacteria and yeasts were within acceptable limits.
iv. cheese samples gave counts >l0⁸ cfu/g on Rogosa agar
v. the major gas present was mainly carbon dioxide with small concentrations of nitrogen and oxygen.

After discussions with production staff, it became apparent that a new starter had been introduced into the starter rotation sequence and that it was this culture which had been used to make the defective cheese.

A can of unopened starter of the same code and batch number as that used to make the bulk starter used in the manufacture of the blown cheeses was shown to contain organisms capable of inducing high levels of gas in two experiments:

i. when reconstituted skim milk (RSM) was inoculated with culture, with or without added citrate, and the contents sealed with wax and agar, gas sufficient to displace the seals in control and citrated- milk was produced; and
ii. gas was produced when the culture was used as a starter in the manufacture of experimental Cheddar cheese.

NOTE! the use of simple milk based media as in i above cannot be relied upon to confirm the gas producing potential of isolates in cheese.

Detailed examination of the starter revealed the presence of high levels of strains (>50%) that grew on Rogosa agar. Some characteristics of the Rogosa isolates are given in Table 4. The relative salt insensitivity of the isolates and the fact that they grew at 6°C were of interest. The characteristics of these isolates were consistent with those of Leuconostoc mesenteroides ssp. dextranicum.

In controlled cheese-making experiments, gas production was induced using a 0.00l% inoculum of Leucon. mesenteroides ssp. dextranicum (isolate HM8/10). A section of experimental cheese showing the open texture of cheese produced using HM8/10 is shown on the left.

In view of these results, 13 of the mixed strain starters at factory X were screened for the presence of organisms capable of growing on Rogosa agar. Seven of the starters contained strains which grew on Rogosa agar. Unlike the situation described with the starter used at factory Y, starters at factory X generally contained <0.0000l% i.e. 10 ²-10³ cfu/ml of heterofermentative strains. Most Rogosa agar-isolates were capable of growth in 6% (w/w) NaCl and at 6°C.

Further study of cheese from factory X generally revealed that counts on Rogosa agar for "gassy" cheese differed from normal cheese by a factor of 10-100. Levels of non-starter lactic acid bacteria (NSLAB) generally increased with age of cheese. The Rogosa agar isolates had properties consistent with leuconostocs or heterofermentative lactobacilli. Most were also salt tolerant and capable of growth at 6°C.

An examination of water, rennet, pasteurised whole milk and annato at several plants for NSLAB (detection limit 1 cfu/ml) was unsuccessful. Low levels of NSLAB generally <100 cfu/ml were found in raw milk. Variable levels <10-10³ cfu/ml were found in tanker skim-milk used to standardise milk for cheese manufacture. Characterisation of these bacteria revealed properties consistent with L. fermenti. These isolates were also capable of growth at 6ºC and at S/M levels of 5.5%.