Bacteriological Observations on the Ripening of Cheddar Cheese

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Bacteriological Observations on the Ripening of Cheddar Cheese

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THE PENNSYLVANIA STATE COLLEGE THE GRADUATE SCHOOL DEPARTMENT OF BACTERIOLOGY

BACTERIOLOGICAL OBSERVATIONS ON THE RIPENING OF CHEDDAR CHEESE

A Thesis by DARRELL DWIGHT DEANE

Submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August, 1942

APPROVED:

...... Head of Department of Bacteriology

In Charge of Major Work

DATE,

TABLE OF CONTENTS Page INTRODUCTION

..... ..................................

REVIEW OF LITERATURE EXPERIMENTAL METHODS

......... ...................... ........

.......... Manufacture of the Cheese Curing the Cheese ....... Preparation of Starters Media Bnployed .... Bacteriological Investigations on theCheese. Taking the Sample ........... Plating the Sample ..... Isolating the Cultures Cultural Studies ................ Classification .................. Direct Microscopic Examination of Cheese .... Experiments with Bacterial Enzyme Preparations Reagents used .... Media used ............ Selection of culture .... Experimental procedure .... EXPERIMENTAL RESULTS

....

Effect of Certain Pure Cultures on the Flavor .... of the Cheese Effect of Temperature of Curing ............. Morphological Studies of the Cheese Flora ... Cultural Studies................. Production of Bacterial Bi^ymes Capable of Proteolytic A c t i o n ..... Isolation of Acidoproteolytic Coccus ........ DISCUSSION

.......................................

1 5 17 17 18 18 19 24 24 24 24 25 26 28 50 50 51 52 52 55

55 47 62 65 75 75 78

CONCLUSIONS........................

82

LITERATURE C I T E D ..................

86

APPENDIX............................

i

LIST OF TABLES Page Table 1.

Table 2*

Table S.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

Table 10.

Table 11.

Source of the Pure Cultures and Starter Used in the Preparation ofthe Cheese ......

19

Type of "Starter" and Quantity Used in Prepara­ tion of the C h e e s e . ..............

20

Bacterial Plate Counts of Milk Used to Prepare the Cheese Studied in this Investi­ gation .....

50

Bacterial Plate Counts of Cheese Prepared with Commercial Starter (Control) and with the Same Commercial Starter Supplemented with ..... L. bulgaricus (Experimental)

52

Bacterial Plate Counts of Cheese Prepared with Commercial Starter (Control) and with the Same Commercial Starter Supplemented with Str. paracitrovorus (Experimental) ........

53

Bacterial Plate Counts of Cheese Prepared with Commercial Starter (Control) and with the Same Commercial Starter Supplemented with Str. citrovorus (Experimental) .........

54

Bacterial Plate Counts of Cheese Prepared with Commercial Starter as Compared to Cheese Prepared with Str. lactis # 1 .....

55

Bacterial Plate Counts of Cheese Prepared with Str. lactis #1 (Control) and with Str. lactis #1 Supplemented with Str. paracitrovorus (Experimental) .........

56

Bacterial Plate Counts of Cheese Prepared with Str. lactis #1 (Control) and with Str. lactis #1 Supplemented with Str. citrovorus (Experimental)

57

Bacterial Plate Counts of Cheese Prepared with Commercial Starter Supplemented with an Acidoproteolytic Coccus(Experimental) .....

58

Bacterial Plate Counts of Cheese Prepared with Commercial Starter (Control) and with Commer­ cial Starter Supplemented with an Acidopro­ teolytic Coccus (Experimental) ............

59 l

ESge Table 12.

Table 13.

Table 14.

Table 15..,

Table 16.

Table 17.

Bacterial Plate Counts of Cheese Prepared with Str. lactis #2 (Control) and with Str. lactis #2 Supplemented with an Acidoproteolytic Coccus (Experimental) ........ .

60

Per Cent of the Morphological Types Isolated from Cheese while Ripening under Different Temperature Conditions •••••....... .

62a

Morphological and Physiological Characteristics of Certain Groups of Acid Producing Strepto­ cocci Isolated from Cheese Prepared in this Stuty .....

66

Morphological and Physiological Characteristics of Certain Acid Producing Cocci KVhich Have Been ...... Isolated fromDairyProducts

67

Per Cent of the Different Groups of Strepto­ cocci Isolated from Cheese Prepared in this Stucfy................................. .

72

The Relationship between the Bacterial Counts of the Original Milk, the Flavor Score of the Cheese and the Predominant Groups of Strepto­ cocci IsolatedduringCuring .......

74

I

LIST OF FIGURES Page Figure 1*

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Comparison of Flavor Scores of Cheese made with Commercial Starter and Supplemented with L. bulgaricus and Cured under Different Temperature Conditions .......................

36

Comparison of Flavor Scores of Cheese made with Commercial Starter Alone and Supplemented with Str. paracitrovorus (Hammer) and Cured under Different Temperature Conditions .......

37

Comparison of Flavor Scores of Cheese made with Commercial Starter Alone and Supplemented with Str. citrovorus (Hammer) and Cured under Different Temperature Conditions

38

Elavor Score of Cheese made with Commercial Starter as Compared to that of Cheese made with Str. lactis and Cured under Different ..... Temperature Conditions ;-s

39

Comparison of Flavor Scores of Cheese made with Str. lactis Alone and Supplemented with Str. paracitrovorus (Hammer) and Cured under Different Temperature Conditions ......

40

Comparison of Flavor Scores of Cheese made with Str. lactis Alone and Supplemented with Str. citrovorus (Hammer) and Cured under Dif­ ferent Temperature Conditions .....

41

Comparison of Flavor Scores of Cheese made with Commercial Starter Alone and Supplemented with an Acidoproteolytic Coccus and Cured under Dif­ ferent Temperature Conditions ...... .

42

Comparison of Flavor Scores of Cheese.made with Commercial Starter Alone and Supplemented with an Acidoproteolytic Coccus and Cured under Dif­ ferent Temperature Conditions

43

Comparison of Flavor Scores of Cheese made with Commercial Starter from Different Lots of Milk and Cured at 43°F.......

48

Page Figure 10. Comparison of Flavor Scores of Cheese made with Commercial Starter from Different Lots of Milk and Cured at 6S°F. for 4 weeks and then at 45°F...............................

49

Figure 11. Comparison of Bacterial Plate Counts of Cheese made with Str. lactis Alone and Supple­ mented with Str. citrovorus (Hammer) and Cured tinder Different Temperature Conditions

61a

INTRODUCTION

Since the investigations of Freudenreich and his associates, bacteria, and especially the lactic acid bacteria, have been considered important agents in producing the flavors of cheese and also in break­ ing down the insoluble nitrogenous compounds. ing the ripening process*

These changes occur dur­

Attempts to explain the phenomena by a

study of the flora of cheese, however, have met with difficulty since the organisms which grow best in milk and its products do not flourish upon many artificial media.

In morphology many of the cultures are so

nearly alike that a separation into the accepted coccus or bacillus is difficult, while their physiological characteristics may vary suf­ ficiently from those of established species to make it difficult to assign a species name to them*

Also, in studying physiological

characteristics recourse must be taken to various culture media* The difficulty here encountered is that such reactions may be determined by

the treatment to which the organism had been previously exposed.

The ripening of cheese has been of interest also from an economic standpoint*

It requires from six to nine months, depending

on

the type of market for which it is intended, for a cheddar cheese

to

ripen under normal conditions of manufacture and storage*

This

storage period contributes a great deal to the cost of manufacture of the cheese.

If it were possible then to shorten the ripening period,

2. It would eliminate some of the expense of manufacture*

Attempts have

been made to reduce this ripening time through the addition of chemicals, enzymes, the use of various microorganisms, and altering the methods of manufacture as well as the conditions under which the cheese is stored during ripening*

It is important from the standpoint of ripening to obtain the desired flavor in the cheese at the time it is placed in the hands of the consumer and this point must be considered when attempting to shorten the ripening period.

Even if the insoluble casein fraction

can be converted to soluble products more quickly, unless the desired flavor is attained in the same length of time, the cheese will not possess its potential value.

This is also true if the desired flavor

is attained and at the same time the boty and texture of the cheese indicates that a sufficient quantity of the insoluble casein fraction has not as yet been broken down into its soluble fractions.

The purpose of this investigation was to study the influence of certain pure cultures of bacteria and temperature conditions during storage on the flavor of Cheddar cheese.

It was also the purpose of

this investigation to study some of the bacteriological phases of cheddar cheese during the curing period.

This bacteriological investi­

gation was to be conducted by making cultural as well as direct micro­ scopic studies of the flora of the cheese at intervals as it was being ripened in the curing rooms.

It was hoped that in this manner more

information might be obtained concerning the role of the lactic acid bacteria in the ripening of cheddar cheese.

5. REVIEW OF LITERATURE

It is not within the scope of this investigation to review all the early work which led to the discovery that microorganisms played an important role in the ripening and in the development of the flavor of cheese*

The early work published by various investigators

has been reviewed by Hucker (1).

Ho consecutive bacteriological examinations had been made of a ripening cheddar cheese before 1896.

The first detailed work on

this variety of cheese as far as the writer could find was that of Russell (2) who made a quantitative examination of the cheese during ripening by means of gelatin plates and reported that acid forming organisms make up 99 per cent and over of the banteria thus determined* In 1901 Harding began his work on the biological phases of the rela­ tion of the bacterial flora of cheddar cheese to ripening and in a report with Van Slyke and Hart (5) discussed the action of enzymes in cheese*

At this time the enzymes in cheese were considered to be

of three kinds, namely:

(a-) bacterial, (b) pepsin from the rennet

extract, and (c) natural ferments of the milk discovered by Babcock and Russell (4)*

By subjecting the. cheese to. the action of chloroform

or other antiseptics to check the bacterial action, Harding and Van Slyke (5) found that -typical flavors failed to develop, while the insoluble casein was split into compounds similar to those found in ripening.

Finding that the action of bacteria played an important

part in the development of flavors, Harding and Prucha in 1908 (6)

made an extensive stucty of the flora of cheddar cheese*

By studying

the flora of nine good quality cheeses through all stages of curing (ripening) they found that Bacterium lactis acidi was the organism which was alweys found, and that it made up more than 99 per cent of the total count*

This substantiated, in part, Harding's conclusion in

1903 (7) that the lactic organisms were the principle types in cheese and that they were important in ripening, especially in the early stages, since they suppressed other forms and facilitated the action of the rennet by breaking down the sugars into acids*

It was found by Hastings, Evans and Hart (8) that while Bact. lactis acidi was consistently present in great numbers; in the early ripening stages it was replaced, after the sugars had been con­ verted into acids, by the Bacterium ca3ei group which utilized other sources of carbon.

Cocci were also found in large numbers.

Hart and

co-workers (9) studied the physiological activities of the organisms isolated from cheddar cheese and found that the cocci when grown in milk were able to produce large quantities of volatile fatty acids, "probably from citric acid, lactose, or protein as the medium was practically free from fatn*

It was concluded that as the cocci are

present in large numbers in cheese they produce much of the volatile fatty acids which arise during the ripening process.

They also showed

that the Bact. casei group produced large amounts of acetic and also of lactic acid (both active and racemic) and could decompose citric acid when available*

5. Evans et* al. (10) again studied the flora of cheddar cheese and Isolated several hundred cultures from a number of cheeses made under various conditions.

These she arranged in the following groups:

(a) Bact. casei group, (b) cocci, (c) streptococci, and (d) Bact. lactis acidi.

She again found that the Bact. lactis acidi group is

replaced in the later stages of ripening by the Bact. casei group and a large number of cocci and a few streptococci.

Her results tended to

show that the Bact. casei group was largely responsible for the develop­ ment of the pungent flavor and that Bact. lactis acidi contributed to the characteristic flavor of cheddar cheese.

Of the cocci isolated,

the streptococci were thought to contribute to the flavor produced if present in large numbers, while the micrococcus group imparted a distinct "bitter" taste.

In a check on her results she was successful

in producing a cheese with a characteristic flavor using a mixed cul­ ture of Bact. lactis acidi with a streptococcus.

(Bact. casei group

always developed in cheese and was not added as a starter.)

However, in this work up to this time (1920) no correlation had been made between the quality of the poorer grade of cheese, and the flora present, to determine which are this undesirable bacterial types to be avoided in the production of cheese from milk of satis­ factory quality.

Hucker (11) examined 37 sampler of cheddar cheese

from 25 factories and found that the better grades of cheese had a distinctly different flora than that of the poorer grades. better types

In the

' Streptococcus lactis and the lactobacilli predominated

6. while in the poorer grades spore formers and Gram-negative rods were present in the largest numbers.

Allen (12) found in 1950 that cheese

made from "market milk" sold for cheese making undergoes a quicker and more extensive process of ripening (as evidenced by protein decomposi­ tion) than cheese made from clean milk of a low bacterial count.

He

reported that cheese made from milk with a very low bacterial count showed a retarded rate of ripening.

"Market milk" cheese ripened more

quickly but was more open and more pungent in flavor than "clean milk" cheese.

Allen and Knowles (15) studied the bacterial flora of cheese

made from milk of a very low bacterial count and reported that the predominant flora for the first 19-20 weeks was lactic acid strepto­ cocci.

They found that if the milk was produced under strict condi­

tions of cleanliness either lactobacilli might be absent entirely or the few species present might be of a type unable to contribute towards the ripening process; therefore, one of the factors regarded as essential to produce full flavor in the cheese was absent.

Their results

are not in complete accordance with those of Davis (14) who found that, in cheese made from clean milk (having a bacterial count of about 1000 per gram) while the lactic acid streptococci are most abundant during the first month after that the rod shaped organisms start to pre­ dominate.

Hammer and Bailey (15) in 1919 studied the volatile acid production of starters and of organisms which they isolated from them. They concluded that there was more than one organism in a number of

very satisfactory starters since cultures of Str. lactis did not pro­ duce as much volatile acid in pure culture as the starter it was isolated from.

These research workers isolated two organisms from

starter which in combination gave a volatile acidity equal to that of the original starter but did not identify the organisms (besides Str. lactis) which they found.

They concluded that high volatile acid pro­

duction was not due to Str. lactis alone.

In 1920 Hammer (16) found

two organisms associated with Str. lactis in starter and proposed the names Streptococcus citrovorus

and Streptococcus paracitrovorus.

He

reported that satisfactory starters could be made from Str. lactis plus these two organisms but not from Str. lactis alone.

Hucker and Marquardt (17) reported that Str. paracitrovorus (Hammer), when added to pasteurized milk in addition to the usual lactic culture, improved the flavor of the cheese made from the milk, while Str. citrovorus (Hammer) produced a cheese with a slightly bitter flavor.

Cheese made with Str. lactis was found to be similar to that

made with cohmercial cheese cultures.

Similar researches by Hansen,

Bendixon, and Theophilus (18) indicate that cheese made from raw or pasteurized milk with Str. citrovorus (Hammer) or Str. paracitrovorus (Hammer) alone as the culture becomes bitter and has a poor body, while cheese made with Str. lactis as the culture is almost equal in flavor and superior in body and texture to that of the cheese made with the commercial starter.

8. It was reported by Hacker and Marquardt (17) that most com­ mercial starters contain Streptococcus cremoris in addition to Str. lactis*

Kelly (19, 20) found very little difference in comparing the

protein breakdown in cheddar cheese made with Str. lactis as compared to that of cheese made with Str* cremoris.

The cheese made with Str*

cremoris was better in flavor during the early stages of ripening but there was little or no difference between the two when ready for con­ sumption*

In studying the effect of certain bacteria on the ripening of cheddar cheese, Lane (21) found that four out of seven strains of Lactobacillus casei when added individually to pasteurized milk appeared to improve the flavor and hasten the ripening of the resulting cheese* Two of the strains consistently produced a buttery flavor which was distinctly pleasing and desirable.

The addition of small numbers of

Streptococcus liquefaclens produced a well ripened cheese in a compara­ tively short time but a relatively large number produced a bitter flavor and soft bodied cheese.

Str. paracitrovorus (Hammer) appeared to

slightly improve the flavor and hasten protein breakdown of the pasteuri­ zed milk also.

Lane and Hammer (22) studied the effect of I»* casei

on the nitrogenous decomposition and flavor of pasteurized milk cheese and reported that six of the eight strains of the organisms studied appeared to bring about more rapid and extensive decomposition of the protein in the cheese when inoculated into the milk as compared to cheese made with milk not inoculated with J». easel*

The two remaining

strains had little effect*

This was essentially in agreement with the

findings of Davis, et. al. (23), who reported that the addition of lactobacilli to the starter used in making the cheese appeared to accelerate the proteolytic process in the early stage of ripening*

Comparisons of the nitrogenous decomposition in cheese made from raw and pasteurized milk and from a mixture of the two (ratio 1 to 9) were made by Lane and Hammer (24)*

They found a more rapid and

extensive protein breakdown in the raw than in the pasteurized milk cheese and also that the protein breakdown in the cheese made from the mixture was more nearly comparable to that of the raw milk than to the pasteurized milk cheese.

From these results Lane and Hammer con­

cluded that the addition of the raw milk contributed various types of desirable bacteria, and suggest that pasteurization destroys certain I

bacteria or enzymes important from the standpoint of cheese ripening.

Sherwood (25) found that cheese made from a "natural mixed culture" prepared from mature cheeses, whether good or bad, had in each case an unclean fermented flavor.

However, this flavor appeared

more quickly and was stronger when the mixed culture was taken from poor cheese.

Sherwood then added pure cultures of lactobacilli and

found that cheese in each case had a characteristic flavor (found cheeses with both good and bad flavor).

Mixed strains of pure cultures

also produced a characteristic "individual" flavor.

This same investi­

gator (26) also found that small inoculations of lactobacilli produced the best cheese.

Sherwood also reported (27) that lactobacilli or betar-

10. cocci (Leuconostoc) capable of producing carbon dioxide relatively rapidly could be isolated from "open” cheese.

The addition of such organisms

resulted in the development of slit openness in the cheese.

Harris and Hammer (28) studied the effect of various micrococci isolated from cheddar cheese on the flavor of cheese made from pasteurized milk.

Of the 34 strains which they used, 7 had an undesir­

able effect, producing bitter or unnatural flavors.

Fourteen had no

definite effect but 15 of the strains of micrococci had desirable effectj producing a flavor more or less characteristic of well ripened cheddar cheese.

From these results they concluded that if micrococci were used

in making cheese they should be selected on a "strain" basis.

Research work has also been carried out to study the cocci found in milk showing proteolytic tendencies.

Barthel and Haglund

(29) discounted the possibility of using strong casein-digesting strains of "lactococci" for hastening the ripening process.

After conducting

experiments with strains of lactococci having varying casein-digesting abilities they reported negative results and stated that "pure cul­ tures of lactococci are always inferior to ordinary starters".

They

also showed that a given strain of organism might lose its proteolytic properties, even with daily transfers to sterile milk.

In studying

the strains of Str. lactis having varied ability for splitting casein, Haglund, et. al. (30) found that differences in the cultures did not seem to affect the rapidity of ripening.

Anderegg and Hammer (51)

11. found that while certain cultures of Str. lactis showed a definite pro­ teolytic activity in milk, others did not.

When CaCOj was added to

the milk culture to prevent the accumulation of acid, simulating in this respect the conditions found in cheese, the proteolysis was more pronounced.

Str. citrovorus and Str. paracitrovorus did not cause

proteolysis.

It was reported by Harriman and Hammer (32) that certain pure cultures of Str. lactis could be split (by pouring plates and picking colonies) into rapidly and slowly coagulating strains and that in general the former proteolyzed and the latter did not.

Kelly (53)

compared proteolytic strains of Str. lactis and Str. cremoris to a com­ mercial starter and found that Str. cremoris and the starter produced approximately the same amount of amino nitrogen (Van Slyke method). The amount of amino nitrogen found in the Str. lactis cultures was less than that produced either by Str. cremoris or the "starter”.

Gorini reported (34) that the liquefying cocci, which he termed acido proteolytes, are the most important organisms from the standpoint of cheese ripening.

The acido proteolytes include organisms

which attack both lactose and casein, and which are capable of proteolyzing in an acid medium.

Gorini (35) gave the origin of these acido­

proteolytic organisms as, the udder, the rennet extract and the intestine. Orla-Jensen (36) reported the results of a study of the proteolytic activity of several lactic organisms by inoculating milk plus chalk and holding at 55°C for one and one-half months.

He found that rennet

12. alone could attack the milk pritein and this peptonizing action was greatly increased by the addition of Str. lactis.

However, the mixture

did not produce as much soluble nitrogen as Str. liquefaciens by itself. Wojtkiewiez and Inikkoff (57) have reported that the addition of 0.4 per cent of selected proteolytic, strongly acidifying, cultures of Str. lactis. Lactobacillus bulgaricus or either of two strains of Lactobacillus casei to pasteurized milk produced a degree of ripeness in the cheese at one month equal to that normally obtained in two months.

Ripening temperature Considerable research work has been done since 1895 on the ripening temperature for cheddar cheese.

Before this time the curing

rooms had very little or no provision against changes in temperature; thus the ripening temperature fluctuated considerably, depending some­ what upon the season of the year.

The first experimental work to deal with the ripening of cheese at lower than room temperature was initiated in 1895 by Babcock and Russell of the Wisconsin Experiment Station (58).

In this experi­

ment cheese was cured at three temperatures, 50°, 60° to 65°, and 85°F*

It was found that the cheese cured at 50°F., though requiring

a much longer time than the cheese cured at the higher temperatures, broke down fully as well.

It was considered to have approximately the

same quality and value as the cheese cured at temperatures of from 60°F. to 65°F., while the cheese cured at 85°F. was very strong and almost unfit for use.

15. Smith (59) reported an investigation in 1900 in which cheeses were ripened at 55°, 60?, 65° to 70°, 75° and 80°P.

He

stated that the chteese cured at 55° and 65°F. scored an average of 6 points higher than cheese cured at the higher temperature.

In 1901

McKay (40) found that cheese cured at 60°F. scored higher than that cured at 65°F. and shove. .

Babcock, etal. (41, 42, 45) published a series of papers reporting on experiments dealing with the use of much lower ripening temperatures such as 15°, 55°, 40°, and 50°F. as well as 60°F.

In

o storing cheese at these different temperatures it was found that 40 F. and 50°F. gave the best results when considered by the market standards of that time.

In noting the effect of holding for a long time, these

o

investigators found that cheese held at 55

o

to 40 F. was of fine

quality after 2 years, while that held at 50°F. was on the decline after 16 months.

Since this cold cured cheese never acquired the

sharp-biting flavor of old cheese which was cured at 60°F. an attempt was made to ”correct” this. then placed at 60°F.

Cheeses were cold cured several months,

After 5 months at 15°, 40° and even 50°F. the

flavor of the cheese was very mild and clean.

Placing at 60°F. at

this time brought about a change in degree of flavor, not quality, toward a more desirable cheese flavor, rather than toward the sharp-biting tang of old cheese ripened at high temperature.

If left for consider­

able length of time at 60°F., however, flavor soon deteriorated.

In

summing up the work done by the Wisconsin Experiment Station, Babcock

14. and Russell (44) called attention to the fact that cheese cured in cold storage was much more uniform than that cured under the old conditions. Similar results were reported by Dean, et al. (45) who found that cheese cured at 40°F., or as close to this temperature as possible, was'much more uniform in quality than cheese cured at room temperature. Lane (46) after studying the effect of low temperatures on the curing of cheese reported that 40°F. cheese scored slightly higher than 28° and 34°F. cheese from the second to the eighth month.

Doane (47)

found, however, that cheese placed directly into 52°F. curing rooms had a slightly higher score than those cheeses placed directly at 40°F.

Van Slyke, Smith and Hart (48) ripened cheese at 40°, 50® and 60°F.

The average scores at the end of 20 weeks were;

94.2, and 91.7 respectively.

95.7,

Van Slyke and Hart (49) studied the

effect of ripening temperature on the amount of soluble nitrogenous 0

Q

compounds in the cheese, and found that between 32 F. and 70 F. one degree increase in temperature caused a 0.5 per cent increase in sol­ uble nitrogen.

They found that the greater portion of this increase

was contributed by amino acids and ammonia.

Rodger (50) after curing

cheese at approximately 50°F. and 75°F., reported that in all cases the cheeses held at the higher temperature ripened rapidly and soon developed a strong overripe flavor while the cheese cured at the lower temperature ripened slowly and retained an agreeable flavor for a long time.

To ripen cheese for early consumption (51) Sammis recommended a temperature of 60°to 70°F. in the curing room*

Atkinson (52) found

that the most suitable temperature for ripening cheese ranged from o o 40 to 60 F. according to the time of the year and the purpose for which the cheese was intended.

He reported that higher temperatures

favored -the growth of undesirable organisms, while very low tempera­ tures induced a somewhat bitter flavor.

Wilson, et al. (55) more recently reported that cheese made from good quality milk by proper manufacturing procedures could be cured at 50°F. with reasonable certainty that clean and characteristic flavors would develop.

If poor milk was used, then the cheese had to

be stored at 54°F. to retard the development of defects as much as possible.

,

Reichart (54) in experiments conducted with cheese canned I in a specially constructed 12 ounce vented can found that a ripening period of 30 to 40 days at 60° to 70°F. was sufficient to produce a satisfactory ripened cheese for ordinary trade demands.

He also

reported that cheese made from good quality milk and cured in this °c? manner scored materially higher after a considerably shorter ripening period than cheese put up as daisies or five pound prints and cured "at the usual temperature"•

HcCubbin and Reichart (55) ripened 12

ounce prints of cheddar cheese in valve—vented cans at 40°F. or com­ binations of these two temperatures and scored than at various intervals up to 3ix months of age.

They reported that cheese ripened at 40°F.

16. continuously was superior in both body and flavor.

Cheese exposed to

the 70°F. temperature for any appreciable length of time became very gas^y and developed a fruity fermented flavor.

Wojtkiewicz and Inikoff (56) have expressed the opinion that the use of higher ripening temperatures for producing a quick ripened cheese is. limited, since it has an unfavorable effect on the physical properties of the cheese.

They offer no experimental evidence to

support their contention.

Freeman (57) believes that the ripenin g period could be shortened by holding the cheese at a temperature of 60-65°F. for one month and then transferring the cheese to a storage room having a temperature of 40-45°F.

He also mentioned that if the cheese must be

kept in storage for a long period of time, a more desirable method might be to store the cheese at the lower temperature, then transfer it to the higher temperature two to three weeks before it is sold.

17 EXPERIMENTAL METHODS

Manufacture of the Cheese All cheeses were made from raw milk obtained from the college creamery and represented a typical commercial supply for cheese making. The milk fat content desired mas 5.5 to 5.6 per cent and in the few cases that standardization was necessary pasteurized skim milk was used.

In each trial 80 gallons of milk was divided into two equal

portions, and each portion was placed in separate 100 gallon stainless steel cheese vats. pounds of milk.

Color was added at the rate of 1/2 ounce per 1000

One vat of milk was used to prepare the control

cheese; the other, to prepare the "experimental" cheese.

The milk

used to prepare the "experimental" cheese was inoculated with the same kind and amount of "starter" as was used to prepare the control cheese plus a definite amount of a pure culture of the test organism to be studied.

The amount and type of starter used in each instance is noted

in a subsequent portion of this thesis.

After ripening to about 2j

spaces on the Marshall tester, the milk was set at 86°F. using three ounces of rennet per 1000 pounds of milk.

The curd was cut with half

inch curd knives when it had reached the proper degree of firmness. No heat was applied during the first 15 to 20 minutes of agitation. The curd was then slowly heated to 99 to 102°F. where it was held until the desired firmness was obtained; the acidity of the whey at this point was usually 0.16 per cent.

The curd was then dipped and cheddared

until the acidity in the whey was 0.50 to 0.70 per cent.

After milling

18. the curd was stirred for 5 to 10 minutes and 2 per cent salt added. After all the salt was dissolved the curd was hooped and pressed 45 minutes in specially constructed hoops 17 inches long and 4 inches in diameter. night.

Each cheese was then dressed and placed in the press over­

After pressing 18 to 20 hours the cheese was removed and cut

into ten 12-ounce portions.

Each portion was wrapped in cellophane and

then sealed in the specially constructed valve vented cans developed by Rodgers (58) and manufactured by the Continental Can Company.

This

valve permits the escape of gasses formed in the ripening of cheese without allowing the entrance of air.

Curing the Cheese After canning, each bath of cheese, which consisted of approximately 50 cans, was divided into two lots, one lot was placed at 45-45°F. while the other lot was held at 65-65°F. for four weeks and then placed at 45-45°F.

The cheese was scored for flavor at inter­

vals during ripening by members of the Department of Dairy Husbandry.

Preparation of Starters All starters were prepared by inoculating flasks of sterile milk.

These with one exception were incubated at room temperature

for 18 to 24 hours.

The milk inoculated with the acidoproteolytic

organism was held at room temperature for 48 hours before being used. The source of the pure found in Table 1.

cultures and the coimercial starter may be

19. TABLE 1.

Source of the Pure Cultures and Starter Used in the Pre­ paration of the Cheese

Starter or Pure Culture

Source of Isolation

Commercial Starter

Flav-O-lac Flakes (Dairy Labora­ tories, Philadelphia, Pa.)

Streotococcus lactis #1

Milk

Penn State

Streptococcus lactis #2

Cheese

Penn State

Streptococcus paracitrovorus (Hammer)

*

Iowa State

Streptococcus citrovoms (Hammer)

*

Iowa State

Lactobacillus bulearicus

*

Penn State

Acidoproteolytic coccus

Cheese

Penn State

•^Specific daily product not known

The type of starter and the amount of each which was used in the various trials may be found in Table 2.

Media Employed

Tomato Juice Broth (59)

Filtered tomato juice Peptonized milk (Difco) Peptone (Cudahy) Yeast Extract (Difco) Distilled water

200 ml. 10 grams 5 grams 5 grams 800 ml.

The medium was adjusted to pH 8.0 with IN NaOH.

Tomato juice agar

was prepared by adding 2 per cent agar to this broth.

The broth was

tubed in 10 ml. quantities and the agar prepared in 250 ml. amounts in Erlenmeyer flasks and sterilized by autoclaving at 15 pounds pressure for 20 minutes.

TABLE 2.

Type of "Starter" and Quantity Used in Preparation of the Cheese

Cheese Series |Trial ! • •

Control •

AA Experimental Control

: B

Experimental

* Type and quantity of "starter" •

0.75% commercial starter 0.75 commercial starter + 1% L. bulgaricus 0.75% commercial starter 0.75% commercial starter + 1% Str. paracitrovorus

I Control •

n o Experimental

0.75% commercial starter 0.75% commercial starter + 1% Str. citrovorus

Q

Control *

Experimental Control

V



0.75% commercial starter

T u| 1.0% S. lactis #1 1.0% S. lactis #1

T ? Hi Experimental

1.0% S. lactis #1 + Str. paracitrovorus

fK)

11 Control •

Experimental Control :

1.0% S. lactis #1 + Str. citrovorus 0.75% commercial starter

G Experimental

Control III

1.0% S. lactis #1

1? r

0.75% commercial starter + 0.05% acidoproteolytic coccus 0.75% commercial starter

: H Experimental *-

0.75% commercial starter + 0.05% acidoproteolytic coccus

4--------

Control

1.0$ Str. lactis #2

I Experimental

1.0% Str. lactis #2 + 0.05% acidoproteolytic coccus

Litmus Milk

The litmus milk was prepared by adding a sufficient amount of saturated litmus solution to fresh skim blue

color.

milk togive thedesired

The medium was then tubed and heated bysteaming in an

Arnold sterilizer for five minute3 before it was sterilized in the autoclave at 12 pounds pressure for 15 minutes.

Carbohydrate Broth

The basic medium was nutrient broth to which 0.5 per cent of the various carbohydrates was added according to the following formulas

Peptone (Cudahy) 5 Beef Extract (Difco) 5 Distilled water 1000 Brom thymol blue sol.(0.04$) 20 Carbohydrate (C.P.) 5

grams grams ml. ml. grams

The reaction was adjusted to pH 7.2 with IN NaOH and tubed in 10 ml. quantities.

A gas detection vial was placed in each tub6.

With the

exception of maltose»sterilization was carried out in the autoclave at 15 pounds pressure for 15 minutes.

The maltose was sterilized at

10 pounds pressure for 12 minutes after pre-heating in the Arnold sterilizer for five minutes.

Methylene Blue (60)

The medium was prepared to contain concentrations of 0.01 per cent and 0.1 per cent methylene blue in skim milk as follows.

22. The skim milk was sterilized in 250 ml. and 225 ml. portions in 500 ml. flasks by preheating for five minutes in an Arnold sterilizer and then holding in an autoclave for 15 minutes at 12 pounds pressure. An aqueous dye solution containing 1 per cent .methylene blue was sterilized separately in the autoclave.

By adding, sseptically, 2.5

ml. and 25 ml. of the dye solution to flasks containing 250 ml. and 225 ml. sterile skim milk respectively, a concentration of 0.01 per cent and 0.1 per cent was obtained.

This medium was then dispensed

aseptically into previously sterilized test tubes and allowed to stand 3 days before being used.

Sodium Chloride Tolerance Medium (61)

A cabbage infusion was made by disintegrating 100 grams of coarsly chopped cabbage in 200 ml. of distilled water for 2 minutes in a TChiz-Mix* beater. pulp.

This treatment reduced the cabbage to a fine

This mixture was added to 600 ml. of distilled water, heated

10 minutes

in an Arnold sterilizer, filtered through absorbent cot­

ton to remove coarse material and then through a Buchner funnel. The volume was adjusted to 800 ml. with distilled water.

The follow­

ing formula was used: Cabbage infusion Tryptone (Difco) Dibasic sodium phosphate (NagHPO^ Dextrose (C.P.) Agar

800 ml. 10 grams 2.5 grams 1*0 grams 20.0 grams

*Made by the Bersted Manufacturing Company, Fostoria, Ohio

25. This medium was heated in the Arnold to dissolve the agar and dispensed in 200 ml. portions in 500 ml. Erlenmeyer flasks. at 15 pounds pressure for 20 minutes.

It was sterilised

YRien the medium was to be used

it was melted in the Arnold, cooled to approximately 45°C. and 50 ml. of a sterile NaCl solution added.

Sufficient saline was added to

produce the desired final concentration of 2.0, 4.0 and 6.5 per cent NaCl in the agar.

Peptone Medium for Production of Ammonia (61)

Infusion from 100 grams of cabbage Peptone (Difco)

1000 ml. 40 grams

This medium was adjusted to pH 7.5 with IN NaOH, tubed and sterilized at 15 pounds pressure for 20 minutes.

Medium for Determining Tolerance to pH of 9.2 and 9.6 (61)

Nutrient broth was prepared and the pH adjusted to 9.5 and 9.9 with IN NaOH with a Coleman potentiometer.

The broth was then

dispensed in 250 ml. portions into 500 ml. flasks and sterilized at 15 pounds pressure for 20 minutes.

After sterilization 10 ml. of a

sterile 12.5 per cent glucose solution was added to each flask. medium was tubed aseptically in 8 ml. portions.

This

The pH of each medium

was checked potentiometrically and found to be 9.2 and 9.6 respec­ tively.

24. Bacteriological Investigations on the Cheese

Taking the Samples

Samples of cheese were taken for bac­

teriological analysis at definite intervals during the ripening process; a separate can of each cheese was opened each time.

A

plug was withdrawn from one to two inches from the perimeter of each cheese with a carefully flamed and cooled trier.

One gram of

cheese from the center of the plug was transferred to a mortar which had been previously sterilized by moistening with alcohol and then flaming.

The cheese was emulsified by grinding in the mortar with

a small amount of sterile quartz sand and approximately 2 ml. of sterile water from a 9 ml. water blank.

During the grinding process

the remainder of the water was added, to make a final 1 to 10 dilu­ tion of the cheese from which further dilutions for plating were made.

Plating the Samples; the dilutions in triplicate.

Tomato juice agar was used to plate The plates were counted after four

days incubation at room temperature with the aid of a Quebec plate counter.

Sufficient dilutions were prepared to assure securing a

"countable" plates (50 to 500 colonies) and from the average number of colonies on these plates and the dilution factor, the bacterial content of the cheese on the day of sampling was determined.

Isolating culture8s

It was realized that the method used

in isolating cultures should show the representative flora and at the same time the comparative number of each type of bacteria present in the cheese mass.

In the first method the Petri dishes were recounted

25. after the initial total count to find the per cent of each type of colonies present.

A total of ten colonies representative of all types

on the plate were then picked. of the flora.

Each colony represented 10 per cent

For example, if the countable plate was found to con­

tain three types of organisms in the relationship of 10,20,70 per cent, one, two and seven colonies respectively of each representative type would be picked for further study.

It was thought, however, that this manner of selecting colonies did not always give a true picture of the representative flora of the cheese since colonies of the same species of organism had a noticeably different appearance depending on where they were located in the agar.

The organisms, however, were identical.

ferent method of picking colonies was therefore adopted.

A dif­

All the

colonies from a definite area on the Petri dish were picked.

This

area varied in size up to l/Z of the Petri dish and contained from 15 to 25 colonies.

In all cases, however, only those colonies were

picked where there were two or more of one type present. I

Cultural studies:

The selected colonies were transferred

to tomato juice broth and after 24 hours incubation at room tempera­ ture, Gram stains were made and litmus milk tubes were inoculated. The litmus milk tubes were examined over a period of 10 days to two weeks.

Cultures were selected as most representative of the various

types of Streptococci found according to the Gram stains and litmus . milk reaction and transferred to tomato juice agar slants for further

26. study in an attempt to classify them.

Classification:

The cultures were subjected to the follow­

ing tests: 1. Reaction produced in various carbohydrate media. 2. Tolerance to 0.01 and 0.1 per cent methylene blue in skim milk. S. Tolerance to 2, 4 and 6.5 per cent NaCl. 4. Ability to produce ammonia from peptone. 5. Growth in glucose broth with a pH of 9.2 and 9.6.

The various carbohydrate broths prepared as previously des­ cribed, were inoculated from a 24 hour tomato juice broth culture and were incubated at room temperature for 10 days to two weeks.

The method of determining the tolerance of the cultures to various concentrations of methylene blue was very similar to that described by Avery (60).

The methylene blue skim milk medium was

inoculated with a 24 hour tomato juice broth culture, and incubated at room temperature for two weeks.

The medium used to determine the tolerance to various con­ centrations of sodium chloride was that of Yawger and Sherman (61) modified by the addition of two per cent agar.

This modification was

made necessary because of the formation of a precipitation in the broth on sterilization.

Pour plates were inoculated with a loopful

of a 1-1000 dilution of a 24 hour tomato juice broth culture.

The

27. Petri dishes were incubated 5 days at room temperature and then examined for the presence of colonies.

A cabbage infusion broth as described by Tawger and Sherman (61) was used to determine the ability of the cultures to produce ammonia from peptone.

The medium was inoculated with a loopful of

a 24 hour culture and the presence of ammonia determined after four days incubation at room temperature according to the following method*

Reagents*

(l) A 5 per cent solution of phenol in water. (2) A solution of NaOCl containing approximately 1 per cent available chlorine.

Tests

0.2 ml. of medium were added to 8 ml. of ammonia free distilled water; 1 ml. of the phenol reagent and 1 ml* of NaOCl wa' added.

These were well mixed and

allowed to stand thirty minutes.

The development

of a distinct blue green indicated the presence of ammonia.

In order to check the tolerance of these organisms to dex­ trose broth with a pH of 9.2 and 9.6 the method employed by Tawger and Sherman (61) was followed.

The tubes of broth, prepared as pre­

viously described in this thesis, were inoculated and incubated at room temperature for one week.

At this time the tubes were examined

for signs of growth and the pH of each tube was measured by the potentiometer.

28. Direct Microscopic Examination of Cheese

In preliminary triale in this investigation the use of frozen sections was not found applicable.

More success was had by

fixing the cheese and imbedding it in paraffin previous to section­ ing.

After experimentation with various fixing agents and dyes the i

following procedure was found to work fairly satisfactorily although it does have some disadvantages.

Duplicate pieces of cheese which measured approximately 3/8 x 3/8 x 1/8 inches were cut from the sample of cheese after the plug had been removed and the cheese plated.

These blocks of cheese were

placed first in 80 per cent ethyl alcohol for 36 to 48 hours; then into 95 per cent ethyl alcohol for 2 hours and finally into absolute ethyl alcohol for 1-1/2 hours.

This dehydrating process caused the

pieces of curd to shrink slightly and express small globules of milk fat.

After removing from the absolute alcohol the pieces of cheese

were placed in cedar wood oil for 36 hours in an attempt to "clear" them.

The oil did not permeate the entire piece of cheese even when

a small hole was punched through the center with a glass rod.

After dehydrating and attempted clearing the cheese was then placed in beakers of paraffin with a melting point of 50-52°C. and held in an oven at 52-55°C. for 6 hours.

At the end of this time the

cheese was imbedded in 56—58°C. paraffin in metal boats.

After har­

dening, the paraffin was removed from the boat and cut into approxi—

29. mately 3/4 inch cubes, each cube containing a single piece of cheese. From these blocks sections were cut seven microns in thickness with a microtome.

The sections were affixed to clean glass slides with the

aid of egg albumin, allowed to dry for 24 hours at room temperature, and then placed in the 52-55°C. oven for one hour before staining +• according to the following procedure.

The slides were placed in the following solutions in the order indicated and for the length of time stipulateds

1. Xylol

4 minutes

2. Xylol

4 minutes

3. 95

percent ethyl alcohol

3 minutes

4. 70

percent ethyl alcohol

2 minutes

5. 50

percent ethyl alcohol

2 minutes

6. 30

percent ethyl alcohol

2 minutes

7. Loefflers alkaline methylene blue

24 hours

8. Rinsed in tap water 9. Decolorized in 500 ml. of water containing 20 ml. of 70$ acid alcohol

10 seconds

10. Rinsed in tap water

.picric acid

11 Counterstained in saturated aqueous 30 to 60 seconds

.

12 Rinsed in tap water 13. 30 per cent ethyl alcohol

30 to 60 seconds

14. 50 per cent ethyl alcohol

30 to 60 seconds

15. 70 per cent ethyl alcohol

30 to 60 seconds

30. 16. 95 per cent ethyl alcohol

30 to 60 seconds

17. Absolute ethyl alcohol

2 to 3 minutes

18. Carbol xylol

30 seconds

19. Xylol

4 minutes

20. Xylol

4 minutes

The slides were removed from the last jar of xylol, a drop of Balsam (dissolved in cedar oil) added to the stained section and a coverslip placed over it to make a permanent mount.

After drying

a few days the slides were examined under the oil immersion lens.

Experiment with Bacterial Enzyme Preparations

Because of the interest shown in bacterial enzymes an attempt was made to determine if enzymes showing proteolytic activity could be extracted from certain selected organisms.

Reagents used; 1. M/15 NagHPC>4

9.47 grams of NaHP04 (anhydrous)

(Merck) dissolved in 100 ml. of freshly boiled distilled water (62) 2. M/15 KHgP0 4 ---- 9.08 grams of KHgP04 dissolved in 1000 ml. of freshly boiled distilled water (65) 3. Phosphate buffers

pH 7.4 -----

80.4ml. of

M/15 Na 2HP04 added to 19.6 of M/l5 KH 2PO4 to make 100 ml. of buffer.

31. 4. Phosphate buffer*

pH 5.6

5 ml. of M/15

NagHP04 added to 95 ml. of M/15 KH2P04 to make 100 ml. of buffer. 5. Acid alcohol solution

made by diluting 15 ml.

of glacial acetic acid to final volume of 100 ml. with 45 per cent ethyl alcohol. 6. Thymol solution ----- 10 grams of thymol dissolved in sufficient 95 per cent ethyl alcohol to make 100 ml. of solution.

Media used;

Gelatin (63)

Commercial gelatin to make a 5 per cent solution was dissolved by heating in a water bath at 60°C. in phosphate buffer (pH of 7.4 or 5.6) and 10/S thymol added to make a concentration of 0.1 per cent thymol*

The gelatin was then tubed and plugged in 5 ml. lots.

Casein Stock Solutions (64)

Merck casein to make 1 per cent solution was dissolved in phosphate buffer, pH 7.4, containing 0.9 per cent NaCl and 10$ alcoholic thymol solution added to make concentration of 0.1 per cent thymols

Casein

The stock solution of casein was dilu$®4

with the phosphate

buffer (pH 7.4 or 5.6) containing 0.9 per cent NaCl and 0.1 per cent

32. thymol until a solution was obtained which, when further diluted to allow for maximum amount of culture filtrate added, became opalescent when acidified with 0.2 ml. of the acid alcohol solution.

The media

was then tubed and sterilized.

Selection of Cultures

The cultures studied were selected from the isolations made from cheese prepared in this investigation.

Experimental Procedure

Sterile centrifuge flasks (250 ml. capaeily) containing the 100 ml. of tomato juice broth were inoculated with the organism to be studied and incubated 48 hours at 37°C.

After incubation the

flasks were centrifuged for one hour, the supernatant decanted and filtered through a sterile Berkfeld candle.

The sterile filtrate was

then transferred to a sterile flask and enough 10 per cent thymol solu­ tion added to give a final concentration of thymol of 0.1 per cent. The precipitated cells from the centrifuge flask were washed with saline by resuspending and recentrifuging and finally triturated by grinding in a small mortar.

The tubes of casein and gelatin medium were inoculated with one ml. of the filtrate, several tubes being inoculated with filtrate from each culture studied.

The casein medium was also inoculated

with triturated cells; several tubes being inoculated with a loopful each of the treated cells.

After inoculation, the cotton plugs were

53. cut off even with the topes of the tubes and .the tubes were then sealed with "parafilm" or gelatin caps.

The tubes of casein medium were

incubated at 37°C. while one set of gelatin tubes was held at 37°C. and a duplicate set incubated at 20°C.

The tubes of casein medium were checked for evidence of enzymatic activity by adding 0.2 ml. of the acid alcohol solution. If the medium became opalescent it was concluded that no activity had taken place.

To determine whether or not enzymatic activity had

o occurred in the gelatin tubes, those held at 20 C. were examined for o evidence of liquefaction while those held at 37 C. were held in icewater bath for 20 minutes.

In the latter case if the gelatin failed

to solidify it was considered evidence that enzyme action had occurred.

In addition to the above cultures an experiment was conducted with an aged cheddar cheese.

Thirty-five grams of a 12 ounce canned

cheddar cheese 4 years old were weighed into a nWhiz Mix" cup, 100 ml. of phosphate buffer (pH 7.4) added and t he contents agitated by the machine for four minutes.

The contents of the cup were then

poured into a 250 ml. centrifuging flask and centrifuged at 2000 rpm for 1 hour.

The supernatant was then filtered first through absorbent

cotton and then through a sterile Eerkfeld candle.

The sterile fil­

trate was then divided into 2 equal portions; to one half sufficient 10 per cent thymol solution was added to give a concentration of thymol of 0.1 per cent; to the other half nothing was added.

The un­

34. treated filtrate and the filtrate containing thymol was then added in amounts varying from one to 3 ml. to individual tubes of litmus milk and buffered casein medium prepared as before.

Incubation of dupli­

cate sets of tubes was carried out at room temperature and 57°C. Control tubes of totoato juice broth were also inoculated.

It appeared from the results which are presented elsewhere in this thesis that there was a proteolytic enzyme present in this aged cheddar cheese.

To determine whether or not the enzyme was pro­

duced by an organism still viable in the cheese, the cheese was plated in an effort to isolate the microorganism responsible.

Dilutions were made and plated with tomato juice agar. After incubation at room temperature for four days some 30 colonies, including all types of colonies present, were picked into tomato juice broth.

After 24 hours incubation at room temperature, transfers were

made to litmus milk.

After incubating several days at room tempera­

ture one culture . showed evidence of proteolytic activity.

Subsequent

plating of this culture yielded but one type of organism, an "acidproteolytic" coccus form.

This organism will be referred to as an

"acidoproteolytic" organism throughout the writing of this thesis. Its characteristics are discussed elsewhere*

35. EXPERIMENTAL RESULTS

Effect of Certain Pture Cultures on the Flavor of the Cheese The data with respect to the effect of certain pure cultures on the flavor of the cheese are presented in Figures l to 8, inclusive. The actual scores received by the cheese and the criticisms noted are shown in Tables I to Till in the appendix.

The trials A, B, C and D commercial starter was used to prepare the control cheeses.

The two temperature conditions 43°F, during the entire

curing period and 63°F. for four weeks previous to placing at 4 S°F., were used.

In trial A the commercial starter supplemented with

bulearicus

produced a cheese with a lower flavor score than the control under the same conditions of curing.

The control cheese cured at 45°F. entirely was found

to maintain its maximum flavor score for 11 weeks.

ThiR was not found to be

true with the other cheeses prepared in this trial.

When Str. paracltrovorus (Hammer) was used to supplement the commercial starter (Trial B) it was found as shown in Figure 2, that this cheese received a slightly higher flavor score than the control and maintained that flavor score at a fairly high level when cured at 43°F. entirely.

In

comparing the cheese partially cured at the higher temperature (65°F.) before placing at 45°F. the difference was not too significant. Str. citrovorus (Hammer) was also used to supplement commercial starter in trial C and, as shown in Figure 3 was found to be tinsatisfactory

LEGEND COMMERCIAL. 5 T A R T R R COMMERCIAL

□--— a

STARTER' 1 CUR ED A T 63"K F O R ■+ L BULGARICUSl WEEKS T H E N A T 4A?

36 A GE IN

C O M P A R IS O N O F C O M M E R C IA L

A N D CURED

W EEKS

V/ O R AND

OF L B U L G A R /C U S

COMMERCIAL STARTER

CURED A T 4 3*F . t 3TR R A R A C ITR O VO R U S COM M ERCIAL S T A R T E R CURED A T 6 3 * FOR, 4 + STR PARA C /TRQ VQRUS j WEEKS TH EN AT 4 3 * E

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IS

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- C O M P A R IS O N O R FL A VO R SCORES O F C H EESE

M A D E W I T H CO^M ERCIAL^ST^RTE'R'

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COMMERCIAL COMMERCIAL

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'COM PARISON O F ELAVOR SCORES O F CHEESE MADE~WITH~-C014HERC1AL STAR TER ALONE A N D SUPPLEM ENTED V *!T H -STR.—CITROVORUS (HAMMER) A N D CURED

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CUR E D A T 6 3 *F. FI * A C ID O P R O T E O L Y T IC COCCUS) 4 W E E K G T H E N A

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A G E I N WEEKS COMPARISON O F FLA VOR SCORES ~ OF CHEESE M A D E W IT R r C O M M ER C IA - ALONE A N D S U P P L E M E N T E D i Y IIT R A N r A C ID O P R O T E O LY TiC COC CU&: ^ C U R E D xMNDER- DIFFERENT.: TEMRERATU R E ± C O N a i T J p N S

----

43.

pf

5

44.

since the flavor score was

not improved, but caused a bitter flavor in the

cheese.

In trial D.. cheese made with commercial starter was compared to that aade with a pure culture of Str. lactis ffl. The data obtained indicated as shown in Figure 4 that the cheese aade with Str. lactis received a higher flavor score than the commercial starter

cheese when cured at45°F.

However, there was no significant difference found when the cheeses were cured at 6S°F. for

4 Weeks before placing at 43°F.

In the second series of trials a pure culture of Str.lactls ffl was used to prepare the control cheese.

When this organism was supplemented

by for, paradtrovorus (Haamer) to prepare the experimental cheese (Trial and the chesses were cured at 43°F. entirely £he maximum flavor scores attained were the saae, however, the flavor of the control cheese remained i

at a slightly higher level than the experimental cheese as the curing continued.

In comparing the cheese partially cured at the higher temperature i

the maximum flavor received by the control cheese was equal to that received by the experimental cheese although it required a longer curing period to be reached.

Soon after the

mnw flavor score had been reached it started

to decrease in both cheeses.

In trial F the experimental cheese was made with fog*, citrovorus (Hammer) as the supplementary organism.

In comparing the flavor score of

this cheese to that of the control made with Str.. lactis. ^

it was found as

shown in Figure 6 that the control cheese1scored higher as the ripening period continued beyond 14 weeks.

In trial G. the control cheese was made from commercial starter and t.



45.

the experimental by supplementing this with an acldoproteolytic coccus that had been isolated from a 4 year old Cheddar cheese.

From the data

presented in Figure 7 it may be seen that under the same storage conditions the experimental cheese had a somewhat higher flavor score.

In this trial,

however, the flavor score decreased after the maximum had been reached, due to the development of a slight bitter flavor In both the control and experimental cheeses.

This experiment was repeated (Trial H) and, as shown in Figure 8 the cheese containing the acldoproteolytic coccus received a higher flavor score than the control.

It is interesting to note, however, that in this

trial the flavor scores of the cheede cured at the higher temperature remained at their maximum for a period of four weeks (when thelast observations were made).

In trial

I

This did not occur in Trial G.

Str. lactla ^2 was used to prepare the control cheese

and this organism was supplemented with the acldoproteolytic coccus to prepare the experimented, cheese.

However, the cheese made inthe trial had not been

examined to determine the flavor score at the time ofwriting.

The results obtained by using the pure cultures were somewhat similar to those reported by other investigators.

Wojtkiewics and

Inikkoff^57) found that by using 0.4 per cent of a selected proteolytic, strongly acidifying strain of L,. bulgaricua in pasteurised milk a degree of ripeness was produced in the cheese at one month equal to that normally obtained in two months.

Other lactobacilli have been used (22) (21) (25)

with varied results depending upon the species and even the strain of lactobacillus used.

46. It had previously been reported by Hucker (17) that Str. pgracitrovorus (Hammer) when added to pasteurized milk, had a pronounced effect upon the cheese flavor in that a decided "cheese" flavor developed more quickly than when commercial starter was used.

Hansen (16) however*

reported that when Str. paraeitrovorus was used bitter flavors were found in both raw and pasteurized milk cheese*

Lane (21) found that this

organism apparently improved the flavor of the cheese slightly.

With respect to Str* citrovorus (Hammer), the results of this study agree closely with those reported by Hucker (17) and Hansen (18) in that the organism produced a bitter flavored cheese*

The use of acid proteolytic organisms has been of interest since they were reported by Gorini (65) mho believed them to be micrococci* Hucker (17) reported that cheese prepared with an acid proteolytic coccus always had a bitter flavor.

Hansen (66) however, reported that an un­

identified micrococcus improved the flavor of the cheese as did Streptococcus liouefaciens which proteolizes under acid conditions.

More recently Harris

and Hammer (28) reported the results of a study of the influence of some 34 micrococci upon the flavor of Cheddar cheese.

Of these, 14 had no definite

effect, seven had an undesirable effect while 15 cultures generally improved the flavor.

They concluded that if micrococci was used they should be

selected on a strain basis*

Wojtkiewicz and Inikkoff (37) found that welected proteolytic strongly acidifying cultures of Str* lactls used with pasteurized milk produced a degree of ripeness in the cheese at one month equal to that normally obtained in two months*

ifffact of Temperature of Curing By examining Figures 1 to 8 inclusirely it nay be s e n that the effect of temperature was sooewhat Inconsistent.

In nost Instances

the maximum score of the cheese held at 43°F. entirely was equal to or greater and maintained longer than that of the cheese partially cured at 65°F.

The nost notable exception to this was found in the cheese prepared

In trials G and H*

A maximum flavor score was usually reached more quickly when cheese was cured at 65°F. for four weeks and then placed at 43°F.

In most

instances, however, the score of this cheese was not maintained as high as that of the cheese cured entirely at 43°F.

It can also be seen from Figures 1 to 8 inclusively that the age at which the maximum flavor score was attained could not always be correlated with the temperature of curing or the type of starter used. show this more clearly Figure 9 and 10 are presented*

To

From these date it

is evident that the original milk supply might have some influence upon the fluctuation in flavor score*

The bacterial plate counts, on tomato juice agar, of the original supply of the cheese are presented in Table 3*

By comparing the data

it may be seen that mere counts are not very significant since there is no definite trend shown between the flavor and the original bacterial plate count*

It has also been reported by Hucker (67) that there was no striking

relationship between the bacteria cound of the milk and the score received by the cheese*

48.

* ® VJ

Q vs

L

0

T

*

5fr h t

w

.

a/oJiy

49.

I

I

50

Table 5,

Bacterial Plate Counts of Milk Used to Prepare the Cheese Studied in this Investigation J.

Series

Trial

I

A B C D

152,000 117,000 296,000 154,000

II

E F

160,000 57,000

G H I

53,000 151,500 185,500

III

Bacteria per ml* of milk

It must be remembered, however, in attempting to explain the fluctuations in flavor, or for that matter any biological phenomena, that it is almost impossible to vary but one factor*

It should not be assumed that

the variation in the behavior of a biological system resulting from the alteration of one condition is due entirely and directly to that one condition alone since the behavior of a system is the result of its component parts acting upon and Influencing tme another*

Bacterial Counts The first detailed work concerning the numbers of organisms found in ripening cheddar cheese was reported by Bussell (68) in 1896* This investigator reported that, following an initial decrease in the number of organisms during the first five days, the count Increased approximately ten times to reach a maximum of from 100 to 150 million

51.

bacteria per graa of cheese in 12 to 20 days.

Harrison and Connell (69)

reported that they usually found the highest count ehen the cheese eas one day old although occasionally the m m H m w cheese was three to five days old. 635 million per graa.

count occurred when the

The highest number they found was

Later it was reported by other research workers

(6) (19) that the highest counts were obtained within the first ten days to two weeks.

Hansen (70) more recently has found that the highest

count occurred when the cheese was two weeks of age.

In every instance

these investigators reported that there was an immediate decrease in the number of microorganisms after the maximum count had been obtained.

In Tables 4 to 12 inclusive are presented the bacterial counts of the cheese studied in this investigation.

It can be seen from these

data that in 24 Instances the highest counts were found when the cheese was first sampled at one day of age; in 7 cases when the cheese was one week old, in three instances when two weeks old and twice not until the cheese had been stored for a period of three weeks.

These results were

in d ose agreement with what has been previously reported by Harrison and Connell (69).

The

number of organisms obtained per gram of cheese

ranged from 200 million to approximately 4 billion with an average of 600 million.

Such a variation in the count of the cheese is difficult to

explain.

The method of manufacture might Influence the count yet this

procedure was kept constant.

A variation in starter could also have some

Influence but •frh'M can not be used to explain it entirely since in any tae trial where the same milk is used with different starterB and different

Table 4.

Age in weeks

Bacterial Plate Cotints of Cheese Prepared with Commercial Starter (Control) and with the Same Commercial Starter Supplemented with L. bulgaricus (Experimental)* Commercial Starter Cured at 63°F. for Cured at 43UF. 4 weeks-then at 43°F.

Commercial Starter + L. bulgaricus Cured at 43d'F. Cured at 63°F. for 4 weeks-then at 43°F.

0

598.011**

598.OM

158.5M

158.5M

1

587.5M

455.OM

565.OM

414.OM

2

280.QM

224.OM

415.OM

550.611

4

153.011

165.OM

373.OM

312.0M

8

105.0M

130.0M

96.CM

82.0M

16

335.5M

129.OM

457.OM

426.OM

27

11.OM

12.4M

103.OM

80.0M

* Counts are averaged from triplicate plates. ** M = Million.

Table 5.

Age in weeks

Bacterial Plate Counts of Cheese Prepared with Commercial Starter (Control) and with the Same Commercial Starter Supplemented with Str. paracitrovorus (Experimental)* Commercial Starter. Cured at 43°F. Cured at 65WF. for 4 weeks-then at 43°F.

Commercial Starter + Str. paracitrovorui Cured at 43UF. Cured at 65°F. for 4 weeks-then at 43°F.

0

419.OM**

419.OM

299.(81

299.OM

1

116.OM

156.OM

15.OM

67.5M

2

18.OM

18.OM

25.5M

16.51!

3

47. OM

33.5M

32.0M

50. OM

4

18.7M

98. OM

50.0M

51. 5M

6

2.0M

43.0M

17.01!

6.5M

10

5.5M

19.6M

6.3M

6.3M

15

11.OM

11.8M

28

10.1M

8.6M

* Counts are averaged from triplicate plates. ** M = Million. *** Plates lost.

8.5M 7.4M

5.6M

Table 6.

Age in weeks

Bacterial Plate Counts of Cheese Prepared with Commercial Starter (Control) and with the Same Commercial Starter Supplemented with Str. citrovorus (Experimental)* Commercial Starter Cured at 45°F. Cured at 65°F. for 4 weeks-then at 45°F.

Commercial Starter + Str. citrovorus Cured at 45WF. Cured at 65°F. for 4 weeks-then at 45°F.

419.01***

419.0M

654.OM

654.0?*

2

577.01*

552.0M

285.0M

556.01*

5

474.0M

462.OM

782.OM

546.OM

4

550.OM

205.0M

450.OM

66 .OM

6

55.0M

57 .OM

105.OM

51. 6M

10

100.OM

45. OM

4.8M

37. OM

18

59. a*

80.0M

88.OM

65.0H

29

4.7M

9.7M

6.9M

7.9M

0 1

* Counts are averaged from triplicate plates. ** M * Million. *** Plates lost.

Table 7.

Age in weeks

Bacterial Plate Counts of Cheese Prepared with Commercial Starter as Compared to Cheese Prepared with Str. lactis #1* Commercial Starter Cured at 45°F. Cured at 65°F. for 4 weeks-then at 45°F.

0

200.08S**

1

Str. lactis #1 Cured at 43°F. Cured at 65°F. for 4 weeks-then at 43°F.

200.OM

55. CM

55.OM

104.GM

95.5M

556-OM

310.5M

2

70.022

42. 5M

63.5M

50.011

3

250.5H

24.OM

147.5M

88.0M

4

28 .OM

52.0M

51.5M

39. OM

6

2.9M

1.722

4.5M

14.0H

10

3.9M

20.821

8.4M

15

5.0M

16.9M

4.952

10. 3M

28

5.8M

6.2M

6.7M

5.1M

* Counts are averaged from triplicate plates. ** M = Million.

'

15.OM

Table 8.

Age in weeks

Bacterial Plate Counts of Cheese Prepared with Str. lactis #1 (Control) and with Str. lactis #1 Supplemented with Str. paracitrovorus (Experimental)* Str. lactis #1 _ Cured at 43°F. Cured at 65°F. for 4 weeks-then at 43°F.

Str. lactis #1 + Str. paracitrovorus Cured at 43°F. Cured at 63°F. for 4 weeks-then at 43°F.

0

550.0M**

550.OM

850.OM

850.(8!

1

315.OM

295.OM

265.(81

296.OM

2

262.OM

250.OM

245.OM

280.0M

3

290.OM

440.0M

308.OM

278.0M

4

250.OM

285.OM

257.OM

190.OM

6

191.0M

265.OM

200.01!

145.01!

10

176.OM

212.OM

163.OM

125.(8!

14

185.OM

175.OM

155.0M

86 .OM

19

212.OM

186.OM

170.QM

110.OM

23

255.OM

74.5M

81. OM

60.5M

28

272.OM

79.OM

52. OM

38.0M

35

219.OM

85.OM

41.OM

36.0M

37

175.QM

117.OM

12. 3M

13.3M

* Counts are averaged from triplicate plates. ** U « Million.

Table 9.

Age in weeks

Bacterial Plate Counts of Cheese Prepared with Str. lactis #1 (Control) and with Str. lactis #1 Supplemented with Str. citrovorus (Experimental)* Str. lactis #1 Cured at 65°F. for Cured at 45°F. 4 weeks-then at 43°F.

Str. lactis #1 + Str. citrovorus Cured at 43F. Cured at 63°F. for 4 weeks-then at 43°F.

0

945.OM**

945.OM

1,010.OM

1,010.0M

1

282.01!

267.0M

370.0M

525.OM

2

256.0M

550. CM

405.0M

360.0M

5

560.OM

14S.5M

370.OM

156.5M

4

415.0M

126.5M

505.OM

97.5M

5

550.OM

133.0M

405.OM

118.OM

7

183.5M

115.5M

122.0M

80.0M

9

85.OM

109.OM

81. OM

98*0M

14

23.CM

40.OM

30.OM

56. OM

18

9.8H

19.2M

16. 8M

22. 3M

* Counts are averaged from triplicate plates. ** M = Million.

Table 10.

Age in weeks

Bacterial Plate Counts of Cheese Prepared with Commercial Starter (Control) and with Commercial Starter Supplemented with an Acidoproteolytic Coccus (Experimental)*

Commercial Starter Cured at 43°F. Cured at 65°F. for 4 weeks-then at 43°F.

Commercial Starter + Acidoproteolytic Coccus Cured at 43°F. Cured at 63°F. for 4 weeks-then at 43°F.

0

340.0M**

340.OM

505.OM

305.OM

1

276. m

136. CM

152.OM

93.5M

2

65.OM

23.8M

47. OM

34. OM

3

51. OM

21. 9M

47. OM

28.7M

4

37 .OM

45.5M

33.0M

25.9M

6

52.1M

27.4M

32. 5M

56.OM

8

10.7M

36.0M

22.2M

51. OM

12

2.7M

5.3M

2.5M

9.3M

16

lO.Offi

3.4M

13. 4M

12. 9M

20

8.1M

7.1M

14. 6M

12. 4M

* Counts are averaged from triplicate plates. ** M = Million.

Table 11*

Age in weeks

Bacterial Plate Counts of Cheese Prepared with Commercial Starter (Control) and with Commercial Starter Supplemented with an Acldoproteolytic Coccus (Experimental)*

Commercial Starter Cured at 63°F. for Cured at 43°F. 4 weeks-then at 43°F.

Commercial Starter + Acldoproteolytic Coccus Cured at 63°F. for Cured at 43°F. 4 weeks-then at 43°F.

0

258.OM**

258.OM

260.OM

260.OU

1

150.0M

117*5M

163.5M

217.5M

2

69.OM

54. OM

55.OM

42. OM

3

46.OM

48.CM

45.0M

41. OM

4

30.7M

46.0M

24.1M

26 .OM

6

35.OM

B7.3M

19.8M

26.4M

8

31.0M

28. 8M

19. 9M

28.4M

12

9.6M

28.2M

Z5.SH

32.OM

* Counts are averaged from triplicate plates. « Million.

** M

Table 12.

Age in weeks

Bacterial Plate Covints of Cheese Prepared with Str. lactis #2 (Control) and with Str. lactis #2 Supplemented with an Acldoproteolytic Coccus (Experimental)* Str. lactis #?. Cured at 43°F. Cured at 63°F. for 4 weeks-then at 43°F.

Str. lactis #2 + Acidoproteolvtic Coccui Cured at 43°F. Cured at 63^F. for 4 weeks-then at 43°F.

0

2,900.0M#*

2,900.DM

5,490.0M

3,490.0M

1

5,900.0m

2,940.OM

3,620.era

3,930.0M

3

3,160.era

1,230.0M

2,900.CW

301.OM

4

3,300.0M

249.5M

3,loo.era

380.OM

6

1,515.0M

175. m

2 ,200.era

272.OM

O

* Counts are averaged from triplicate plates. ** M = Million. *** Plates lost.

61.

ripening temperatures the counts* while not being identical, do not show such a variation.

This fact would also tend to show that it is not due

to a variation in the curing temperature.

Kelly (19) has reported that

the bacterial counts of cheese made the same day were more nearly alike than the counts of cheese made on different days with the same species of organism or starter. original milk,

In Table 5 are presented the bacteria counts of the

in examination of the date shows that there is no very

significant correlations between the initial plate counts of the cheese or the maximum number of organisms found and the plate count of the original milk.

It could not be ascertained from these date whether or not

the type of flora of the original milk; could contribute to the variation in count but it must

be recognized that such a possibility exists.

During the ripening period the number of organisms found in the cheese tended to decrease after the maximum count had been reached, however, this decrease in numbers was not constant.

The "growth curves "of the

cheese made in trial F are presented in Figure 11 and show the general trend found with respect to the decrease in numbers during the curing period. Semi-log paper was used to plot., the data in an effort to show the results more dearly.

It will be noted that in this instance the type of. starter used did not seem to be a significant factor but that the temperature conditions of storage did influence the counts.

It can be seen that the number of

bacteria in the cheese held at the higher temperate** first decreased sharply from the

maximum count, during the first four weeks.

This

was followed by a slow decrease in count during the next five weeks and

61a

62.

then the rate of diminution increased again.

On the other hand when the

cheese was cured entirely at 45®F* the decrease In the nuniber of organises which occurred in two weeks ,was less than with the cheese partially cured at 65°F.

This decrease was followed by a noticeable Increase in number for

two weeks at which tine a decrease In number again took place.

Russell and Hastings (71) had reported that the bacterial count of cheese cured at 60°F. Underwent in three weeks a greater diminution in numbers than that of cheese cured at 40°F.

I hare found, however, that

while In sane instances the decrease In bacterial count was found to be grea est In the cheese held first at 65°F. before placing at 45° In other trials the reverse was true.

The data that was obtained in this study seen

to indicate that the number of bacteria and the fluctuation of those numbers is dependant upon several interrelated factors rather than a single one.

ManM lsfllsal

.Am

It was possible to roughly classify the organisms isolated from cheese into streptococci, lactobacilli and miscellaneous types by means of the gram stain and the reaction produced in litmus milk.

The per cent of these various morphological types found under the two temperature conditions of storage are presented In Table IS.

As these data show the streptococci were found to be the pre­ dominant morphological type during the entire curing period that the cheese was examined. weeks

Lactobacilli were found in significant numbers only after 20

at no time were they found to make up more than 15 per cent of the

Table 15.

Age in weeks

Per Cent of the Morphological Types Isolated from Cheese while Ripening under Different Temperature Conditions Cured at 65°F,. for 4 weeks-then at 45°F. Per Cent Per Cent Per Cent Streptococci Lactobacilli Miscellaneous

Cured at 45°F. Per Cent Lactobacilli

Per Cent Miscellaneous

0

99.2

0.0

0.8



----

-----

1

99.1

0.0

0.9

100.0

0.0

0.0

2

100.0

0.0

0.0

99.1

0.0

0.9

5

99.1

0.0

0.9

99.1

0.0

0.9

4

98.0

0.0

2.0

99.0

0.0

1.0

6

100.0

0.0

0.0

97.0

0.0

5.0

8

97.6

1.2

1.2

94.0

5.5

2.5

12

100.0

0.0

0.0

99.0

0.0

1.0

100.0

0.0

0.0

98.6

1.4

0.0

20

89.7

9.0

1.5

97.0

1.5

1.5

24

95.0

5.0

0.0

95.0

5.0

0.0

28

85.0

15.0

0.0

85.0

15.0

0.0

55

85.0

15.0

0.0

85.0

15.0

0.0

57

90.0

10.0

0.0

85.0

15.0

0.0

16

62a.

Per Cent Streptococci

65.

flora*

The miscellaneous types consisted mainly of cocci other than

streptococci and occasionally spore formers were isolated.

These results

are not entirely in accordance with those reported by ail investigators* Hastings, Brans and Hart. (72) found that in the initial ripening period of 60 days almost all the organisms were Bact. lactla acldl.

The

lactobacilli then started to appear and increased steadily,

up

approximately 10 per cent of the flora during the third to fourth month of storage and as much as 50% by the fifth to sixth month*

At the end of 7

months there were very few organisms other than lactobacilli found*

Davis

and Hammer(14) have reported more recently that the Str. lactis types of organisms predominate over the lactobacilli only in the early ripening period*

Freeman, on the other hand (57) found very few lactobacilli in

raw milk cheese which had been canned and cured by the same method as the cheese prepared in this study*

He reported that, "the preponderance

of diplococci and streptococci throughout the ripening period is noticeable". If the lactobacilli were present in small numbers then it would be difficult to make isolations because of their infrequency in the high dilution required to plate the cheese accurately*

As another means of determining whether or not lactobacilli were present, histological techniques were used to prepare permanent slides of the cheese which could be stained and then examined directly.

The amouht of work published concerning methods of examining cheese directly as a means of studying the flora present has not been very great. Haralson (75) was one of the first to publish a procedure of imbedding,

64. sectioning and staining cheese according to histological techniques.

This

investigator dehydrated the cheese in alcohol and imbedded it in paraffin. The sections were cut, affixed to slides and stained by various methods. He obtained the best results by staining *ith the "ordinary method o f . Gram- and counter staining with eosin".

Percival and Mason (74) studied the

distribution of organisms in Stilton cheese by cutting thin slices of the cheese with a razor and staining with eosin methylene blue or thionine. These investigators published no details of the procedure used.

Hucker (75)

made a microscopic study of bacteria in cheese but gave no details as to the "fixing" and imbedding of the cheese other than that "the usual histological technics were employed". with methylene-blue.

The sections were cut at 5 microns and stained

Templeton (76) studied the structure of processed

cheese by making frozen sections which

were cut 20 microns thick.

It was found that by using the method as outlined in this thesis fairly satisfactorily slides could be prepared.

The organisms were not

too difficult to distinguish from the cheese mass if care was used in decolorizing and counterstaining the sections.

In the sections prepared from cheese during the early stage of ripening the bacteria were more widely scattered and appeared mainly in pairs.

As the ripening period increased, however, the bacteria were

apparently less numerous and were found more in clusters or clumps.

The

bacterial flora of cheese up to 20 weeks of age was examined in this manner and no organisms comparable to lactobacilli could be seen in any of the sections.

In the cheese that was 20 weeks of age a few rod shaped, non-

sporeforming organisms were seen which were presumed to be lactobacilli.

65.

Cultural Studies The reactions produced In litmus milk by the organisms isolated from the cheese prepared In the third series of trials (G, H, I) were followed more closely than had been done with the previous series.

From the G, H, and I. trials, 1556 cultures which, on the basis of their gram morphology were assumed to be streptococci, were classified into ten groups or types on the basis of their reaction in litmus milk.

Sixty-

two cultures were then selected from the ten groups for thorough study. Sherman's (77) review supplied most of the tests used and this paper as well as Bergey's manual (78) was followed in attempting to classify these organisms.

The characteristics of these groups and the number of cultures

classified under each may be seen in Table 14.

They were all gram positive

and in none of the litmus milk cultures was definite protrolysis observed. It was noted in fact that from none of the cheese made in the third series of trials could the original acldoproteolytic coccus used in preparing the cheese be isolated.

This might be explained by the fact that a very small

amount of this culture (0.05$) was added to the milk.

In Table 15 are

presented some of the characteristics of the various organisms which have been isolated from dairy products.

Group 1 Of the 1556 cultures Isolated 807 could be classified in group 1. The cultures of this group were those which completely reduced litmus milk prior to coagulation which was brought about in 24 hours.

The selection of

cultures on this basi3 would not allow differentiating Str. lactis from Stjf*,. cyynrn^ «T Separation was not considered of great importance, however, since no great difference has been found between these two types as far as effect

Morphological and Physiological Characteristics of Certain Groups of Acid Producing Streptococci Isolated from Cheese Prepared in this Study

© g

Group No.

Morphology (All Gram +)

Litmus Milk (Incubated at room temperature) in Days 1

1

2 5 4 5 6 7

8 9 10

SI. elongated Str. in Pairs + short chains SI. elongated cells; mostly in pairs and 4’s SI. elongated cells in pairs; no chains SI. elongated cells in pairs: few short chains SI. elongated cells in pairs: few short chains SI. elongated cells in pairs and short chains SI. elongated cells in pairs; few chains Small si. elongated cells in pairs Coccoid cells in pairs + medium long chains Coccoid cells in pairs and short chains

* Curd torn Litmus milk reactions* Carbohydrate reactions*

5

7

14

-S P. ©

'O Q) •H n> & 3

• • • • • •

Growth in

• iH PQ O . aS i S3 to E. « . >1? © •b'< 05 05 rH (O H 85 uj O '* • X X % to . a . S B & $ P< n

5

Carbohydrate Reaction

• •

• • E tA H . o

*© *8 *-p *8 ,R

Lactose

Table 14.

$ .H -0p a

+

St

1 s R

v?

+

+

+

o ©

ACRo

+

+

+

+

+

+

+

ACR-1/6

+

+ '+

+

+

+

+

+

ACR°

ACR-l/2

+

+

+

+ SI. +

+

+

AR-l/2

ACR-l/2

+

+

+ SI.

+

+

ACR-7/8

ACR-3/4

ACR-l/2

+

+

+

+

+

+

+

+

+

+

A

AR-l/2

ACR-2/3

ACR-l/2

+

+ SI.

+

+

+

+

+

+

+

a

ACR-l/2

ACR°*

ACR°*

+ SI.

+

+

A

A

+

+

+

+

+

+

+

+

+

+

SI.

+

+

+

+

+

+

ACR-7/8

ACR-2/3

a

A

a

AsC

a

A

AR-1/2

ACR-1/3 A

+ +

a

a

AR-l/2

AR-l/4

AR-l/2

ACR-l/2

SI.

A

ACR-3/4

ACR-2/3

ACR-l/2

SI.

a = slight acid;

A - definite acid;

+ = acid; — — no acid; SI. = slight acid





C — coagulated;

SI.

*

+

+

R° = no reduction;

may or may not produce acid;

+

+ SI. SI. + +

+ SI. +

+

SI.

+

R-l/2 = l/2 reduced

Morphological and Physiological Characteristics of Certain Acid Producing Cocci TShich Have Been Isolated from Dairy Products (77, 78) ©

ta

Organism

Str. lactis

Str. cremoris

Str. fecalls

Str. liauefaciens Str. bovis Str. paraci trovorus (Hammer) Str. citrovorus (Hammer)

Morphology of Organism (All Gram +)

Litmus Milk Reaction

Many cells elongated in direction of chain; mostly pairs and short chains Cells often larger than Str. lactis; long chains in milk Usually in pairs; vary in size; sometimes fairlv large Usually in pairs; sometimes short chains Spheres; in chains and pairs Spheres; in pairs and short chains Spheres; in pairs and short chains

* These reactions were not reported. Carbohydrate reactions: + = acid; U

-

Complete reduction before coagulation

T3 0) •H ft

o •p © ft d) & ft a o rH u a P (o ft -s a to rH w •6253

8

+



ft

rH

o

0}

W

• w. ffi LO •

03

to ft

+



+

= no acid;

as usually fermented

ro ©

to

(D

O



05

©

W

©

©

■a O +

+

+

+

+

+

+

+

+

R



H

© o CQ -P

£ -3 g 3

H O

Complete reduction before coagulation; milk may become slimy Acid; usually reduced before coagulation Acid; curdled and peptonized; reduced before acidulation Acid; curdled in 55 days; followed by reduction of litmus Acid; usually si. soft coagulation; si.reduc­ tion at base of tube Slight acid; partial reduction of litmus milk

Carbohydrate Reaction

Growth in

8

3 § 3 1 ^ a s a

a

+ +

a a * 5 8 -a ft CO

±

+

R

-

almosl always *

Table 15.

U +

+

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

*

+

_

*

+

+

+

+

t

*

+

+

+

-

R SI.

_

+

+

*

*

*

*

*

*

*

*

+

+

*

*

*

*

*

*

*

*

+

+ GN GN GN GN GN

. - * may or may not produce acid; R * rarely fermented

GN

SI. = slight acid

generally not fermented

upon the flavor of cheese is concerned.

The 10 cultures selected from

this group gave the reactions shown in Table 14.

There was a slight

variation In the fermentating ability for sucrose, other than that the characteristics were quite constant.

This type of organism was considered

to be Str. lactis since it agreed very closely with the organism as described by Sherman (77).

Group 2 The cultures in this group produced a d d in litmus milk but no coagulation until after seven days incubation at room temperature.

There

was only slight reduction of the litmus milk at any time after coagulation. Eight cultures were isolated from four of the twelve cheeses. these cultures were selected for study.

Three of

It was interesting to note that

these cultures did not show evidence of growth in either of the concentrations of methylene blue until after two weeks.

It was not possible to definitely

assign a species name to this organism and since it occurred infrequently it is entirely possible that it may be a strain variation.

Group 5 The cultures representative of this group showed a characteristic reaction in litmus milk.

After an initial acid development the milk was

coagulated in three to seven days but showed no reduction of the litmus until the second week.

Twelve cultures which showed this type of litmus

milk reaction were isolated from six of the twelve cheeses.

Four of these

cultures were selected to be studied.

Group 4 Litmus «