A study has been made of the ripening process of Limburger cheese, including microbiological investigations of the surface flora and chemical analyses of the cheese during the different stages of ripening. The microbial flora was studied both qualitatively and quantitatively. In addition to the morphological charac ters, the nutritional requirements of the main types of micro-organisms occurring on the cheese surface were studied. A survey dealing with the micro-organisms growing on the surface of Limburger cheese and present in the brine and on different shelves, used for cutting and holding the cheese for ripening, gave the following results:
1 . The main groups of micro-organisms to be found on the surface of commercial Limburger cheese included: arthrobacters, Br. linens,
'other coryneforms' and yeasts.
2. The percentage of colonies belonging to organisms of the Br. linens
type was always higher when counted on media supplemented with 4 % NaCl than on media without this amount of salt.
3. The brine flora consisted of the same series of micro-organisms as found on the cheese surface except for the presence of moulds which were almost absent on the cheese.
4. The organisms isolated from the scraped material of the shelves were mainly arthrobacters and yeasts with small numbers of Br. linens
and 'other coryneforms'.
In a morphological and nutritional study of 251 strains of cheese coryneform bacteria isolated from the surface of commercial Limburger cheese, the follow ing conclusions could be summarized:
1. All of the tested strains were found to be Gram-positive in both the coccus and the rod stages.
2. All of the strains were able to tolerate high salt concentrations. The greenishyellow arthrobacters and the Br. linens
organisms were able to tolerate higher concentrations of salt than were the cream + white, grey-white and red arthrobacters, and the 'other coryneforms'.
3. About 66 % of the greenish-yellow arthrobacters needed light to induce pigmentation.
4. Approximately 45 % of the Br. linens
strains required light for the development of the orange pigment.
5. No light effect was observed in the case of the cream + white-coloured, grey-white and red arthrobacters and in the strains of the 'other coryneforms'.
6. All the strains tested were able to utilize both glucose or lactate as the only carbon source, which was not true for lactose and to a larger extent for sucrose.
7. All the isolated strains were catalase-positive.
8. The greenish-yellow strains were very highly proteolytic while the grey arthrobacters and the Br. linens
strains were moderately proteolytic. The cream + white-coloured arthrobacters were almost entirely inactive, whereas about 45 % of the red arthrobacters were either moderately or slightly proteolytic.
9. The red and greenish-yellow arthrobacters, the Br. linens
strains and the 'other coryneforms' were largely unable to utilize inorganic nitrogen and required Casamino acids either with or without vitamins. About 50 % of the grey arthrobacters and 70 % of the cream + white-coloured ones were able to utilize ammonium sulphate in the presence or absence of vitamins.
10. Cell-form was found to be the most important differential character in these groups of cheese coryneforms. The organisms of the Arthrobacter
group were easily and quickly transformed from rod into coccus forms upon ageing or under poor nutritional conditions; this was a much retarded process in the group of Br. linens
and the 'other coryneforms'. The Arthrobacter
rods were mostly short and thick while those of the Br. linens
group were slender, relatively long and sometimes branched. However, all the isolated strains showed the morphological implications of the coryneform bacteria.
A chemical and microbiological analysis of Limburger cheese during ripening gave the following results.
(a) Throughout the ripening process of Limburger cheese the pH values gradually increased. The values of protein breakdown increased along with ripening. A low rate of proteolysis occurred during the first week of ripening, followed by a much higher rate until the age of 20 days, after which it slightly slowed down. At the age of 35 days, the cheese was heavily proteolysed and partly liquefied.
(b) The frequent changes in the composition of the surface flora in the course of ripening may be summarized as follows:
1. Fresh cheese contained only lactic acid bacteria.
2. Immediately after salting, the surface flora resembled that of the brine.
3. At the age of 5 days the surface flora consisted of yeasts only.
4. Four days later the yeasts had decreased and were replaced by arthrobacters which predominated until the end of the ripening period.
5. Strains of Br. linens
started to be a part of the surface flora of 14 days old cheese and increased upon ageing. They never reached numbers higher than 1/3 of the total of the surface flora, a value which was found on 35 days old cheese.
(c) The colour of the cheese surface ranged from white in fresh cheese to reddish-orange in 35 days old cheese. The following micro-organisms may be responsible for the colour of Limburger cheese:
1. Cream-coloured yeasts.
2. White, cream, grey, red and greenish-yellow arthrobacters.
3. Orange-coloured Br. linens.
Light effect on pigmentation was detected in the group of grey arthrobacters and the group of Br. linens
when grown on agar media as well as on the surface of Limburger cheese. A number of grey arthrobacters developed a greenish-yellow pigment when grown in light. The same light effect was essential for inducing the orange pigment in part of the Br. linens
strains. It may be stated that the colour of the cheese surface is a combination of the colour of the above-mentioned organisms together with that of the cheese curd.
(d) The highest percentage (of the total) of highly proteolytic organisms occurred in the period between 9-20 days of cheese age. The grey and greenish-yellow arthrobacters were the highest proteolytic organisms followed by strains of the Br. linens
type and the red arthrobacters. The majority of the white and cream arthrobacters had no proteolytic activity.
To study the effect of the micro-organisms of the surface of Limburger cheese on the ripening process, particularly on the transformation of protein and other nitrogenous compounds, a number of representative micro-organisms were separately grown on cheese slices placed in Petri dishes. Chemical properties of the cheese slices, including pH, degree of proteolysis, amino acid + ammonia content and growth features of the micro-organisms on the cheese slices, including colour and organoleptic ones, were recorded after different periods of incubation. For comparison, the same organisms were grown in Casamino acids-containing media, while some strains were cultivated in media with single amino acids. In all series of cultures free amino acids were estimated by paper chromatography. The results of these experiments may be summarized as follows:
a. During the first week of the incubation period, several Arthrobacter
strains brought about a drop in the pH of the cheese slices, which was followed by a rise during the second and third weeks, resulting in ultimate pH values being between 7 and 8. In the Casamino acids-containing media the pH changes, both into the acid and the alkaline directions, were much more pronounced than those of the more strongly buffered cheese slices. The organisms belonging to the Br. linens
group only caused an increase of pH, when growing on cheese slices as well as in Casamino acids-containing media.
b. After 3 weeks of incubation, the yeasts and most of the cream-coloured arthrobacters had not succeeded in liquefying the cheese slices. Owing to this fact, these organisms had caused only a slight increase of the soluble and amino acid nitrogen. Most of the grey, red and greenish-yellow arthrobacters and the strains of Br. linens,
after 3 weeks of incubation, showed a moderate and often a pronounced liquefaction of the cheese slices. They had hydrolysed about 70 and sometimes even more than 80 % of the protein, and in some cases had produced large amounts of free amino acids + ammonia.
c. The chromatograms obtained from the three weeks old cheese slices which had been inoculated with the weakly proteolytic cream-coloured Arthrobacter
strains contained relatively higher amounts of aspartic acid, glutamic acid, serine and glycine, when compared with the chromatograms of a diluted mixture of Casamino acids.
Cheese slices inoculated with the moderately proteolytic strains of the grey and greenish-yellow arthrobacters and of the Br. linens
strains gave chromatograms more or less resembling those derived from ripening Limburger cheese. In the case of the highly proteolytic grey, red and greenish-yellow arthrobacters and most of the Br. linens
strains, the chromatograms of the liquefied cheese slices resembled those of the Casamino acids mixture with the exception of the leucines and valine which occurred in higher concentrations and serine and threonine occurring in lower concentrations.
d. When the representative micro-organisms, used in the experiment with cheese slices, had grown in Casamino acids-containing media for 10 days, the chromatograms obtained with the weakly and highly proteolytic organisms were relatively similar to those of the uninoculated blank, indicating that there was no preference to decomposing particular amino acids. The chromatograms obtained with most of the moderately proteolytic strains were completely different from those of the other cultures. Many amino acids occurred in considerably lower concentrations than in the cultures of the strongly proteolytic strains, indicating a much higher degree of decomposition of the amino acids in the former cultures.
e. The results of the experiments with cheese slices and of those with Casamino acids-containing media, are in agreement with the experiment with single amino acids. A number of highly proteolytic organisms, also used in the previous experiments, had a much poorer ability to decompose various amino acids than this was true of a number of moderately proteolytic strains.
The quantitative analysis of the free amino acids of Limburger cheese, carried out at various stages of ripening, revealed pronounced differences between various amino acids as to their occurrence in the free state. This was demonstrated in two different ways, viz. (a) by calculating the different amino acids as percentage of the total weight of amino acids, and comparing the results with a similar set of values calculated from average figures recorded in the literature for acid- hydrolysed casein. (b) A more clear picture of the fate of the liberated amino acids during the ripening of Limburger cheese was obtained by comparing the free amino acids in the cheese sample (calculated as mg/15.65 g nitrogen) with the theoretical amounts of free amino acids to be found if a corresponding amount of casein would have been acid-hydrolysed. From this comparison the following conclusions may be drawn:
1. All the values found in the cheese samples were lower than their corres ponding values in casein hydrolysate, owing to the decomposition of the amino acids during ripening.
2. Of all the amino acids present in casein, alanine was recovered at the highest degree, viz. nearly 100%. Other amino acids recovered for more than 70 % were glycine, cystine, valine and leucine.
3. A low recovery value (below 30%) was found for: threonine, proline, glutamic acid and tyrosine, while aspartic acid and arginine were nearly absent from the cheese samples.
4. The remaining amino acids were recovered in amounts between 30 and 70 %.
The poor recovery of several amino acids was due to microbial decomposition, resulting in the liberation of most of the nitrogen as ammonia. A comparison of the total amount of ammoniacal nitrogen found in the cheese samples of different ages with the total amount of nitrogen calculated from the amounts of apparently decomposed amino acids during ripening, gave the following recovery values (%) for the different sampling dates: 9 days after salting, 55; 20 days, 67; 27 days, 66; and 35 days, 80. The amounts of nitrogen not accounted for in ammonia are partly present in those amino acids not recorded in casein hydrolysate (viz. glutamine + asparagine; y-aminobutyric acid and ornithine). These values accounted for another 6, 20, 13 and 12 % in 9, 20, 27 and 35 days old cheeses, respectively. The rest of the above-mentioned differences may have been due to losses in ammonia by evaporation, or to transformation of part of the amino acids to other nitrogenous compounds not detected in the analysis.