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The Influence of Hydrogen Ion Concentration and Salt on the Surface Flora of Limburger Cheese1
T H E I N F L U E N C E OF HYDROGEN ION CONCENTRATION AND SALT ON T H E SURFACE FLORA OF LIMBURGER C H E E S E 1 C. D. K E L L Y AND J . C. M A R Q U A R D T
N e w York State Agricult~tral Experiment Station, Geneva, 2~. Y.
Two main types of microorganisms are found in the slime of limburger cheese and assumed to be responsible for the surface ripening (Kelly (1)). Budding yeast-like organisms appear first about the time the cheeses are put on the shelves and increasing rapidly in numbers make the surface of the cheese slimy. Later red pigment-producing, rod-shaped bacteria replace the yeasts and make the slime red and more like butter in consistency. In speculating as to the factors contributing to the development of the two types of microorganisms to the almost total exclusion of all other types, and the definite sequence with which.the rods followed the yeast forms it was thought that salt and hydrogen ion concentration, together with temperature might play a part. Accordingly, cultures of the organisms were isolated and examined as to the influence of these factors on their development. •acy and Erekson (2) studied the slime of a Roquefort type of cheese and of Port du Salut cheese. Large numbers of Streptococcus lactis were found on the surface of the Roquefort cheese before salting, but after salting they disappeared ahnost entirely and were replaced by yeast forms. As the yeasts increased the p H rose from 4.8 to 5.0. At 21 days the pI-I was 5.9 and the yeasts started to disappear and were replaced by rods. At 6 weeks the p H was 7.3, red color was found in the slime and torula, rods and cocci as well as Penicillium roqueforti were present. In the interior of the cheese the p i t was 4.8 when one day old and changed little during six weeks. The slime of well-developed Por t du S a h t cheese had a pH of 7.5 to 7.6, with rods predominating. Large numbers of organisms of the Bacterium linens type together with some degenerated forms of torula appeared in microscopic preparations. Examinations of the slime of Tilsiter and old Limburger cheeses showed characteristics similar to those of Port du Salut. INFLUENCE OF P n ~ SALT~ AND TEMPERATURE ON THE ORGANIS1E[S TOGETHER WITH THEIR ACTION ON CERTAIN CARBON AND NITROGEN COMPOUNDS
The cultures of the yeast forms were divided into two main groups, those represented by Cultures 65 (Table 1) growing more rapidly and producing Received for publication October 17, 1938. 1 Approved by the Director of the New York State A g r i c u l t u r a l Experiment Station f o r publication as J o u r n a l P a p e r No. 289, October 11, :[938. 309
TABLE T A B L E 11 The The influence influence of of temperature temperature and and hydrogen hydrogen ion ion concentration concentration on on the the growth of of yeasts from ,Limburger Limburger cheese cheese Culture C u l t u r e 65 65 pH pH
Reading days 11 22 3 7
-- -
3.5 3.5
-
4.5
11 22 3 7
5.5
11 22 3 7
t+ + +t t + +t t
+ +t t + +t+t t + +t+t t
-t+
11
-
-+t ++ + +t t ++ +t ++ +t
-
6.5 6.5
7.5
8.5 8.5
95 9.5
18° 18 ° CC..
I 25° 25 ° CC.. i
t+ + +t t + +t t
-
t+ + +t t + +t t
-
t+ + +t t + +t t
+ +t t + +t t + +t t
-
-
3 7
t +t + + +t t + + tt
t+ + +t t + +t+t t + +t+t t t+ + +t t + +t+t t + +t+t t
1
-
-
22
3 7
t +t + + +t t + +t t t
1
-
22
2 33 7 1 22 33 7
-+
t + +t t
-
-
= No N o growth. growth. - ::: + = First F i r s t sign s i g n of o f growth. growth. t::: = Good G o o d growth. growth. t+ t+ ::: = Heavy H e a v y growth. growth. t+ t+ t+ =
-
t+ + +t t
-
--
--
Culture C u l t u r e 67 67 [ 30° 30 o C 37 ° CC.. C.. I 37°
--
--
+ +t t
+ +t t + +t t
-
t+ t+ + +t t
-
--
-
-
-
-
-
-
-
-
-
I 45° 4.~;oqC.
-
pH pH
3.5
-
-
-
-
-
-
4.5
5.5
6.5
7.5
Reading i . days d 7s 1 2 3 7 1 2 3 7 1 2 3 7 1 2 3 7 1 2 3 7 1
8.5
9.5
2 3 7 1 2
3 7
I:i.:l
I-'
o
I
18° 18 ° C.
-
25° 25 ° C. C - - 30° 30 ° C.
-
-
-
-
-
--+ +t t
-
-
-
-
t+ + +t t
t4t ++ t+
-
-
t+
-
+
+ +t t
-
37° 37 ° C. C.
45° 45 ° C. C.
-
-
-
--
--
--
+÷ t+
-
-
-
-
-
--
-
-
t+ + +t t + +t t
-
-
t+ + +t t
t+ t+ + +t t
-
+ +t t
--
--
-
t+
+ +
-
-
-
-
-
--
-
-
-
t+ + +t t
-
-
+ +t t
--
+ +
-
-
-
-
-
-
-
-
-
-
-
-
-
-
pP Y ~
~ ~
I::l
~
P 9 ~
~
'§
§..,
311
SALT AND HYDROGEN ION CONCENTRATION
a heavier pellicle on liquid media than the others represented by Culture 67. In all other respects the cultures appeared the same and are probably variants of one species. As stated before (1) these yeast forms were found to reproduce by budding, showed no trace of sexuality, did not produce spores, formed a pelliele on liquid media from the beginning, and tolerated salt in concentrations as high as 18 and 20 per cent. They showed their best growth at a temperature of 25 ° C. and a p H about 6.5, though growth was observed from p H 3.5 to 8.5 (Table 1). Fructose, glucose, mannose, galactose, sucrose, maltose, lactose, raffmose, ethyl alcohol, and sodium and calcium lactate were utilized as carbon sources with the production of carbon dioxide. Both Bacto proteose peptone and Bacto peptone were readily utilized as a source of nitrogen without any other source of carbon. A proteose peptone (3), made by salting out with ammonium sulphate and dialyzing the precipitate to get rid of the sulphate, was able to support growth in the presence of a fermentable carbohydrate but not without. Ammonium sulphate, ammonium nitrate, ammonium phosphate, and asparagin could not be used as a sole source of nitrogen in the presence of a fermentable carbohydrate. TABLE
2
The influence of te~nperatgre and hydrogen ion concentration on the growth of Bacterium linens Cultures 4 and 5 6 a ~ B a c t e r i u m linens pH
Reading days
18 ° C.
25 ° C.
30 ° C.
37 ° C.
m
3.5 m
m
m
4.5 m
5.5 5.85
÷ ÷
6.0
÷ ÷
6.5
÷ +÷
÷ ÷÷
÷ ÷÷
7.5
÷ ÷÷
÷ ÷÷
÷ ÷
8.5
÷ ÷÷
÷ ÷÷
÷ ÷
9.5
÷ ÷÷
÷ ÷÷
÷ ÷
- - - N o growth. + - - S m a l l amount of growth. ÷ ÷ -- Good growth.
4 5 ° C.
312
C. D. KELLY AND J .
C. MARQUARDT
Gelatin was not liquefied, and a slight alkaline reaction was noted in litmus milk with reduction of the litmus at the bottom of the tube. At the present time no attempt is made to classify these yeast forms. The rod forms are represented by Culture 4, isolated in the present study, and Culture 56a (Table 2), obtained from Kiel. Both are typical Bacterium linens Weigmann. It is to be noted that Culture 4 and other cultures isolated in this study produced the typical orange-colored ring in milk in contrast to that previously reported (1). Bacto peptone was readily utilized both as a carbon and nitrogen source, but asparagin, ammonium sulphate, and ammonium nitrate could not be used. No apparent action was noted on any of the carbohydrates. The optimum temperature for growth was about 25 ° C. with no growth at 37 ° C. (Table 2). A hydrogen ion concentration of pH 6.5 proved most satisfactory with growth at pH 9.5 and as low as 5.85 but not lower (Table 2). Salt in concentrations as high as 18 and 20 per cent was tolerated in both solid and liquid media. In order to correlate the findings in the laboratory with conditions in factory cheeses, hydrogen ion determinations together with salt determinations were made on cheeses in the factory varying in age from the new cheese to those ready for market. I~ETHODS
The method previously employed of sampling factory cheeses, varying in age by a day, was largely employed in the present studies. Where possible the same batch of cheeses were sampled from day to day. In sampling the cheeses, the outer slimy surface was scraped off with a knife and labelled " A . " After this five-sixteenth of an inch of cheese was cut off the outside and called sample " B . " The next five-sixteenth of an inch was called " C " and the center " D . " Salt, hydrogen ion and in some cases moisture determinations were made of these portions. At the same time bacterial samples were taken of the surface by bringing a glass slide in contact with the surface slime (1). Hydrogen ion determinations were made with the quinhydrone electrode following the method of S~nke Knudsen as modified by Watson (4). Salt was determined in duplicate by the method recommended by Wilster et al. (5), for hard cheese. Though Limburger cheese is classed with the semisoft cheeses it is firm in the early stages and the method proved quite satisfactory. In all, six lots of cheese were examined for hydrogen ion concentration and four lots for salt, covering periods of May, July, and October.
SALT AND HYDROGEN ION CONCENTRATION
313
RESULTS Hydrogen Ion Changes During Ripening
The p H dropped from about 6.45 in the milk at time of renneting to below 5.0 in the cheese within a few days (Tables 3, 4, 5 and F i g u r e 1). TABLE 3 Changes in hydrogen ion concentration of Limburger cheese made in May Description of cheese
First color ............................ Good color .............................
Ready for storage .........
Age days
pH of surface
Microorganismsin surface smears
2 3 4 5 6 7 8 9 10 11
5.64 5.81 5.95 6.57 6.84 6.95 7.56 7.50 7.75 7.85
Few bacteria and yeasts Yeasts increasing Yeasts in masses, a few rods Yeasts in masses, a few rods Yeasts in masses, a few rods Yeasts in masses, a few rods Masses of yeasts and rods Masses of yeasts and rods Masses of rods, a few yeasts Masses of rods, a few yeasts
A f t e r the first or second d a y when the yeasts started to grow the p H of the surface slime was found to rise to around p H 7.0 or higher at about 7 to 12 days when the cheeses were r e a d y to go to storage. Because of lower temperatures in the ripening room, the p H of the October cheeses did not go above 6.7 before being t r a n s f e r r e d to storage. The recorded figures in some cases m a y be somewhat low as difficulty was encountered in scraping off the slime without getting some of the interior of the cheese. This factor together with the gas in two cases would no doubt explain the irregularities of the points of the curves designated surface and 1st 5/16th in. ( F i g u r e 1). Little f u r t h e r change was noted in the p H of the surface up to the time the cheese was ready for market. I n the interior of the cheese (Samples B, C, and D) the rise in p H was not as r a p i d and h a r d l y reached the neutral point at any time d u r i n g ripening, being a r o u n d p H 6.9 when r e a d y for market. The rods ( B a c t e r i u m l i ~ e n s ) did not appear until the p H of the slime was a r o u n d 5.85 and usually when it was higher. The red color appeared when the surface smear was somewhere about p H 6.8. ~Vhen Oospera were f o u n d in any numbers in the surface smear or the cheese was gassy the p H was invariably lower than normal (Table 4, F i g u r e 1). The desirability of this first drop in pH, due no doubt to the growth of lactic acid streptococci, was shown in some work carried out on cheeses made at this same time from milk pasteurized at 143 ° F. for 30 minutes. Two lots of cheese were made with the milk as received and the p H did not drop below p H 6.0 (Table 6) followed by a steady rise in pit. W h e n these cheeses were examined at a later date they were f o u n d to have a strong p u t r i d odor. Another lot was made from milk of the same supply but 2 ml.
Ci-' 50
"""
fI>.
TABLE T A B L E 44
Changes C h a n g e sin i n hhydrogen y d r o g e n iion o n c concentration o n e e n t r a t i o n oof f L iLimburger m b u r g e r e hcheese e e s e made m a d e iinn JJuly, u l y , 1937 1937 pH p H in in cheese eheese D e s c r i p t i o n of of cheese cheese Description
Age Age
W h i t e slime . White
G a s s y cheese Gassy r e d color ..... Good red G a s s y cheese Gassy R e a d y for f o r storage s t o r a g e .... Ready
R e a d y ffor or m a r k e t ............... Ready market
.S'" goo "',...., co .......
coQ
.S
~ en.
... ,...., ............ ~lQ
w.ot:>
4.95 5.05 4.95 6.05 6.45 6.45 5.70 7.00 5.75 7.25 7.35 7.40 7.35 7.35 7.35 7.25 7.45
4.90 4.90 4.85 5.20 5.45 5.00 5.70 5.20 5.20 5.35 5.60 5.35 6.00 6.25 7.00 6.25 6.25
4.80 4.75 4.65 4.70 4.75 4.50 4.80 4.75 4.75 4.85 4.80 4.85 5.45 5.55 6.05 6.05
~
day 11 day days 2 days 3 "c~ 4 "~c 6 "~ 7 "c~ 88 "cc 10 "c~ 11 "~c 12 "t~ 13 "" 4 weeks 55 "~c ct 66 " 7 " c~ 88 ~c "
~
'tloo
~
..<:l
'd
...co
Microorganisms M i c r o o r g a n i s m s in in surface s u r f a c e smear smear
~
..,o:l
~
Q)
0
4.65 4.80 4.70 4.70 4.60 4.70 4.70 4.70 4.70 4.70 4.65 4.70 4.90 5.10 5.55 5.55 5.80 5.80
~
A few f e w rods evenly scattered scattered A few f e w yeasts yeasts Fewer F e w e r rods, yeasts y e a s t s well distributed distributed Yeasts, Y e a s t s , some rods increasing increasing Yeasts Y e a s t s in masses, m a s s e s , rods fewer Yeasts Y e a s t s more numerous, n u m e r o u s , rods fewer fewer Yeasts Y e a s t s same, rods in masses masses Yeasts Y e a s t s fewer, rods fewer. Oospera Oospera present present Yeasts Y e a s t s few, rods in masses, a few f e w Oospera Yeasts Y e a s t s fewer f e w e r and a n d smaller, rods in in masses masses Yeasts Y e a s t s in masses m a s s e s but b u t small, rods rods in masses masses Yeasts small, rods in masses Yeasts rods masses rods in masses No yeasts, No y e a s t s , rods masses Yeast Y e a s t small s m a l l and a n d numerous, n u m e r o u s , rods rods in masses masses Yeast rods in in m masses Y e a s t small aand n d few, rods asses Yeasts rods in in m masses Y e a s t s small aand n d few, rods asses
>1 >
Z
t:I ~y
<-< ~
l:':: >
~
,c;
q
~
t:I >-3
TABLE T A B L E 55
Changes Changes in in hydrogen hydrogen ion ion concentration concentration of of Limburger Limburger cheese cheese made made in in October, October, 1937 1937
~
1 p i t in in cheese cheese1 pH
Description of of cheese cheese Description
Age Age
.g '""0;¢2
.......
"
F i r s t red color ......,....................... First
Ready o r storage Ready ffor storage
.......... , ..
3 4 66 7 88 99 10 11 12
"~
"tt "~t~t "tt
"tc
"ct "~t " "'~~
"
""
.S
Soc
"rl
"'--""
Microorganisms l~ieroorganisms in in surface surface smear smear
... 1:1'"
~
Z
1;:;1
I:Q
'"
t1
UJ.lf:l
4.37 4.30 4.97
5.65 5.65 5.33 4.37 3.95 4.75
4.30 3.95 4.80
4.25 4.25 3.95 4.80
Few F e w bacteria bacteria and and yeasts yeasts Yeasts Y e a s t s increasing Yeasts Yeasts the the same same
4.82 4.70 5.25 5.20 5.55 5.60 5.20 6.70 6.55
4.70 4.55 4.65 4.61 4.90 4.65 4.85 4.90 5.40 5.10
4.70 4.25 4.60 4.65 4.60 4.65 4.75 4.75 4.60
4.70 4.50 4.55 4.50 4.50 4.50 4.66 4.65 4.65 4.60 4.60
Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Y easts Yeasts Yeasts
hours hours day days
"0
~lf:l
UJ.
44 6 11 2 3
.S ~~ •.-<--...,
.g 0
§ in masses in in masses masses and and rods rods in in masses masses and and some some and and rods rods in in masses masses and and some some and rods in masses and a and rods in masses and a few few and and rods rods in in masses masses and and aa few few and rods rods in in masses masses and and aa few few and and rods rods in in masses masses and and rods rods in in masses masses and
~
Z (5 Z Q
o Oospera Oospera Oospera Oospera Oospera Oospera Oospera Oospera Oospera
Z
Q
~'-3
~ ~
The r o m cheeses made The first 6 samples were were ffrom made October October 4th. 4th. The The rremaining were ffrom made October October 1st. 1st. e m a i n i n g samples were r o m cheeses made 11 Cheese made r o m milk H 6.45 made ffrom milk with with ppH 6.45 at at the the addition addition of of rennet. rennet.
Co\:)
I-'
~
316
C.D. KELLY AND J. C. iVIARQUARDT I
I
I
•
•
e
'1
J
~-~
PH
e
efe
7
e -~
o
o
5
~--~=r~-~
4_
•
o o
e SURFACE o l S T . 5/161N. • 2 N D . 5/16 IN. <>CENTER
,.I ......
I ...... I
•
[ 2
I
i
r
I
I
r
3
4
5
6
7
8
WEEKS Ym. 1. Changes in hydrogen ion concentration of Limburger cheese made in July~ 1937.
of starter were added for each gallon of milk at the time of renneting. By the third day the p H had dropped to 5.0 and though this drop was not as rapid or as low as in many commercial cheeses a typical Limburger developed with no sign of the putrefaction found in other cheeses. This development of acid appears to be necessary to control putrefying bacteria in Limburger cheese as in the hard pressed varieties.
Concentration of Salt in the Cheese As would be expected and as shown by lYIrozek (6), Byers and Price (7), and others the salt in the cheese was found in greatest concentration in the surface layers and lowest in the inner portions soon after salting (Tables 7, 8, and 9 and Figure 2). On the first day the percentage of salt was between 3.13 and 3.30 in the surface layer (Sample A), 2.03 to 3.04 in the second layer (Sample B), 0.36 in the next layer (Sample C) and 0.06 to 0.31 in the center (Sample D). From this time on the salt tended to diffuse TABLE 6 Influence of added starter on hydrogen ion concentration and quality of Lin~burger cheese made f r o m rail# pasteurized at 143 ° F. )*or 30 minutes
Age in days 1 No starter added. Cheese became putrid No starter added. Cheese became putrid Starter added. Good cheese
2
3
6
7
pH
pH
pH
6.30
6.30
4
pH
pH
6.15
6.30
~5
6.15
6.65
6.15
5.55
6.0
pit
5.00
6.00
317
SALT AND HYDROGEN ION CONCENTRATION
t h r o u g h the cheese until an equilibrium was reached n e a r the tenth d a y when the percentage was about the same throughout the cheese. Considering t h a t the method of salting is b y rolling the cheese in the salt and rubbing with the hands it is surprising to find the salt content f r o m cheese to cheese as u n i f o r m as it is. Though moisture should have been taken into consideration in r e p o r t i n g the salt content it was difficult to obtain satisf a c t o r y samples. However when c o r r e c t i o n s w e r e made using the moisture determinations t h a t were made the variations in salt content were so small as not to change greatly the relationship of the amounts as given (Tables 7, 8, and 9 and F i g u r e 2). TABLE 7 Changes in salt content of L i m b u r g e r cheese made during July, I937 Age 1 day 2 days
....................................... .........................................
3
~
............................................
4
~
..........................................
6 " 7 "
............................................. .............................................
10 " 11 ~ 12 ~' 13 ~'
............................................. ............................................
............................................
.............................................
4 weeks
......................................
6
¢c
........................................
7
~
....................................
8
~
..........................................
Surface
First 5/16 inch
Second 5/16 inch
3.28 3.12 2.33 1.56 2.33 2.38 1.55 2.10 1.69 2.56 2.00
3.04 2.71 2.33 2.31 2.34 2.23 2.05 2.23 1.85 2.46 2.53
0.63 1.15 1.64 1.60 2.00 1.68 1.71 2.21 2.13 2.53 2.36
0.31 0.61 0.80 0.93 1.56 0.91 1.41 1.71 2.10 2.40 2.13
1.49 1.46 1.46 1.81 1.88
1.69 1.80 1.70 1.88 1.96
2.03 1.78 1.80 1.80 2.11
1.76 1.73 1.90 1.70 2.09
Center
TABLE 8 Changes in salt content of :Limburger cheese made during October, 1937 Age days 1 2 4 5 7 9 11 12
Surface
First 5/16 inch
3.13 2.15 2.03 1.76 1.66 1.64 1.46 1.66
2.03 1.20 1.90 1.73 1.96 1.68 1.43 1.56
l
Second 5/16 inch 0.90 0.88 1.40 1.73 1.86 1.48 1.53 1.66
Center 0.30 0.55 1.26 1.50 1.56 1.42 1.46 1.43
I n determining the influence of salt on the growth of bacteria in cheese several factors m u s t be taken into consideration in estimating the concent r a t i o n of salt in contact with the organisms. I t is to be expected t h a t most of the salt would be dissolved in the moisture of the cheese and because of this the percentage of salt as determined b y an analysis of the cheese does
C. D. KELLY AND J. C. MARQUARDT
318
TABLE 9 Changes in salt content o f Limburger cheese made during October, 1937
Per cent of salt Age days 1
............................................................
3
............................................................
3
...........................................................
4
...........................................................
5 6
........................................................... ............................................................
7
............................................................
8
............................................................
9
............................................................
10
............................................................
11
............................................................
12
..........................................................
Surface
First 5/16 inch
Second 5/16 inch
Center
3.30 2.26 2.30 2.13 1.80 1.60 1.53
2.86 2.13 2.23 1.82 1.76 1.46 1.56 1.76 1.76 1.73 1.76 1.76 1.76
0.36 1.50 1.86 1.44 1.50 1.70 1.54 1.60 1.60 1.60 1.63 1.60 1.63
0.06 0.36 0.76 1.02 1.03 1.06 1.15 1.50 1.46 1.53 1.46 1.56 1.53
n o t give a t r u e p i c t u r e of c o n d i t i o n s i n the cheese. T h e r e f o r e i n e s t i m a t i n g the salt c o n t e n t of the m e d i u m i n w h i c h t h e b a c t e r i a are g r o w i n g the moist u r e c o n t e n t of the cheese m u s t be t a k e n i n t o c o n s i d e r a t i o n . T h e m o i s t u r e c o n t e n t of the s u r f a c e l a y e r v a r i e d f r o m 40 to 42 p e r c e n t a n d f r o m 42.5 to 44 p e r c e n t i n the c e n t e r p o r t i o n . A s s u m i n g t h a t all t h e s a l t is i n solution i n the cheese moisture, this b r i n e i n the s u r f a c e l a y e r of a one-day-old cheese I
2.8
I
I
I
I
"~~
2.4 2.0_ o~ ~ e
o
1.6_
~ o
~ °°0. 8 I-
Z
"' U frO. 4 n
• / /
2.
0
o CENTER
l
I L 2 4 AGE IN D A Y S FIG.
•
: SURFACE IST 51161N • 2ND 5/16 iN
I /o g
~
o
I 6
I 8
I 10
I 12
Changesin concentrationof salt in Limburger cheese.
S A L T A N D H Y D R O G E N I O N CONCENTRATIOkNI
319
(Table 9) with 41 per cent moisture would have 8.0 instead of 3.3 per cent salt. At 2 days the per cent of salt would be 5.5 instead of 2.26 and at 5 days 3.9 instead of 1.6 per cent. lVIcDowall and Dolby (8), working with Cheddar cheese, came to the conclusion that a large part of the cheese moisture was not free moisture though they were unable to make conclusive tests. However they estimated that 20 to 30 per cent of the moisture in a young cheese was bound. Assuming that 30 per cent of the moisture is bound, the concentration of salt in the free water would be 11.4 per cent in the surface layer of a day-old cheese. The surface layer of the 2-day-old cheese would then have a salt content of 7.8 per cent and the cheese at 5 days, 5.5 per cent. These theoretical considerations establish that the salt concentration of the cheeses a few days after salting would in no way retard the growth of the organisms desired. A short time after the cheese is salted, on the evening of the day when made and the morning of the following day, the salt content of the brine in the surface layer must be near the saturation point as there is a considerable amount of undissolved salt present. Therefore for at least some hours after salting the cheese, only salt tolerant organisms would multiply. This is given support in the work of Macy and Erekson (2) where it was shown that Streptococcus lactis disappeared when the cheese was salted and yeasts took their place. DISCUSSION
During making and the early stages of ripening the rapid fall in p H is undoubtedly largely due to the action of lactic acid streptococci. The pH which falls lower than in normal Cheddar cheese is no doubt due to a higher moisture content in the Limburger cheese with more lactose to be converted to lactic acid. This lowering of the p H is desirable in making Limburger cheese in that it keeps pu~re±ymg . . . . . . . . . . . . . .oac~erJa . . . . . . from growing. The high concentration of salt on the surface suppresses the many miscellaneous organisms found on the new cheese and permits the development of the yeasts that can grow at a low ptI. The yeasts by acting on both the lactate and the protein soon raise the p t I above the point (pH 5.85) where Bacterium linens can start growth. Other organisms that might grow at this p i t are suppressed by the concentration of salt while Bacterium linens grows readily. At this point the yeasts have completed their work and soon die out, being overgrown by Bacterium linens. Acting on the protein Bacterium linens raises the p H still higher and is believed to complete the ripening of the cheese. The cheeses become inoculated with both organisms from the ripening shelves, which in the factory are heavily seeded with the organisms. Though no work as yet has been carried out on the internal flora of the cheese it does not seem probable that Bacterium linens would be a factor
320
C. D. KEI~Y AND J. C. lYIARQUARDT
because of its aerobic n a t u r e and its inability to grow at a p H lower than 5.85. This tends to strengthen the t h e o r y that the soft b u t t e r y texture and a p a r t of the flavor is produced by the action of enzymes of Bacterium linens rather than by direct action of the organism itself. The question whether the enzymes penetrate the cheese and digest proteins could best be established by a study of the enzymes. SUMMARY
The yeast forms first appearing on the surface of L i m b u r g e r cheeses will grow readily on artificial media between p H 3.5 and 8.5 and with the best growth at p H 6.5. Bacterium linens will not grow below p H 5.85 but will grow at p H 9.5 and grows best at p H 6.5. I n the cheese the p H of the surface falls to 5.0 or lower, a p H at which the yeast will grow but not Bacterium linens. The yeasts acting on both the protein and lactic acid, raise the p H above 5.85 at which point Bacterium linens can become established and overgrow the yeast. Only late in the period of ripening does the p H of the interior of the cheese rise above 5.85. This p H and the lack of oxygen would inhibit the growth of Bacterium linens in any p a r t of the cheese excepting the surface. The concentration of salt soon after salting is highest in the surface layer and low in the interior. There is a gradual penetration of salt into the cheese until an equilibrium is reached at about 10 days. With the lack of information on how much of the moisture of the cheese is available to form brine it is impossible to estimate accurately the concentration of salt in the medium in which the bacteria are growing. However, as the cheeses are salted by rubbing salt on the outside there is little doubt but that it is near the saturation point in the surface layer for some time. This high concentration of salt tends to inhibit the growth of organisms other t h a n the yeasts and Bacterium linens. REFERENCES (1) KELLY, C. D. The microbiological flora on the surface of Limburger cheese. J. DAIRYSC. 20: 239--246. 1937. (2) MACY, H.) AND EREKSON, J. A. Unpublished data supplied by the authors. St. Paul, Minn., November, 1937. (3) HUCKER, CA. J. Studies on the Coecaceae. III. The nitrogen metabolism of the micrococci. New York State Agr. Exp. Sta. Tech. Bull. No. 101. 1924. (4) WATSON, PAUL D. Use of quinhydrone electrode for following changes of pH in Swiss cheese. [ndust. Engin. Chem. 19: 1272-1274. 1927. (5) WILSTER,G. H., et al. Determination of fat, moisture, and salt in hard cheese. J. DAIRYSC. 20: 27-30. 1937. (6) MROZEK,O. Diffusion yon Kochsalz in K~se--Milchwirt. l%rsch. 4: 391. 1927. (7) BYERS, E. L., ANDPRICEW. ¥. Observations on the salting of brick cheese. J. DAIRY SC. 20: 307-318. ]937. (8) McDOWALL,F. H., AND DOLBY,R.M. Studies on the chemistry of Cheddar cheese making. J. Dairy Res. 7: 156-175. 1936.
N e w York State Agricult~tral Experiment Station, Geneva, 2~. Y.
Two main types of microorganisms are found in the slime of limburger cheese and assumed to be responsible for the surface ripening (Kelly (1)). Budding yeast-like organisms appear first about the time the cheeses are put on the shelves and increasing rapidly in numbers make the surface of the cheese slimy. Later red pigment-producing, rod-shaped bacteria replace the yeasts and make the slime red and more like butter in consistency. In speculating as to the factors contributing to the development of the two types of microorganisms to the almost total exclusion of all other types, and the definite sequence with which.the rods followed the yeast forms it was thought that salt and hydrogen ion concentration, together with temperature might play a part. Accordingly, cultures of the organisms were isolated and examined as to the influence of these factors on their development. •acy and Erekson (2) studied the slime of a Roquefort type of cheese and of Port du Salut cheese. Large numbers of Streptococcus lactis were found on the surface of the Roquefort cheese before salting, but after salting they disappeared ahnost entirely and were replaced by yeast forms. As the yeasts increased the p H rose from 4.8 to 5.0. At 21 days the pI-I was 5.9 and the yeasts started to disappear and were replaced by rods. At 6 weeks the p H was 7.3, red color was found in the slime and torula, rods and cocci as well as Penicillium roqueforti were present. In the interior of the cheese the p i t was 4.8 when one day old and changed little during six weeks. The slime of well-developed Por t du S a h t cheese had a pH of 7.5 to 7.6, with rods predominating. Large numbers of organisms of the Bacterium linens type together with some degenerated forms of torula appeared in microscopic preparations. Examinations of the slime of Tilsiter and old Limburger cheeses showed characteristics similar to those of Port du Salut. INFLUENCE OF P n ~ SALT~ AND TEMPERATURE ON THE ORGANIS1E[S TOGETHER WITH THEIR ACTION ON CERTAIN CARBON AND NITROGEN COMPOUNDS
The cultures of the yeast forms were divided into two main groups, those represented by Cultures 65 (Table 1) growing more rapidly and producing Received for publication October 17, 1938. 1 Approved by the Director of the New York State A g r i c u l t u r a l Experiment Station f o r publication as J o u r n a l P a p e r No. 289, October 11, :[938. 309
TABLE T A B L E 11 The The influence influence of of temperature temperature and and hydrogen hydrogen ion ion concentration concentration on on the the growth of of yeasts from ,Limburger Limburger cheese cheese Culture C u l t u r e 65 65 pH pH
Reading days 11 22 3 7
-- -
3.5 3.5
-
4.5
11 22 3 7
5.5
11 22 3 7
t+ + +t t + +t t
+ +t t + +t+t t + +t+t t
-t+
11
-
-+t ++ + +t t ++ +t ++ +t
-
6.5 6.5
7.5
8.5 8.5
95 9.5
18° 18 ° CC..
I 25° 25 ° CC.. i
t+ + +t t + +t t
-
t+ + +t t + +t t
-
t+ + +t t + +t t
+ +t t + +t t + +t t
-
-
3 7
t +t + + +t t + + tt
t+ + +t t + +t+t t + +t+t t t+ + +t t + +t+t t + +t+t t
1
-
-
22
3 7
t +t + + +t t + +t t t
1
-
22
2 33 7 1 22 33 7
-+
t + +t t
-
-
= No N o growth. growth. - ::: + = First F i r s t sign s i g n of o f growth. growth. t::: = Good G o o d growth. growth. t+ t+ ::: = Heavy H e a v y growth. growth. t+ t+ t+ =
-
t+ + +t t
-
--
--
Culture C u l t u r e 67 67 [ 30° 30 o C 37 ° CC.. C.. I 37°
--
--
+ +t t
+ +t t + +t t
-
t+ t+ + +t t
-
--
-
-
-
-
-
-
-
-
-
I 45° 4.~;oqC.
-
pH pH
3.5
-
-
-
-
-
-
4.5
5.5
6.5
7.5
Reading i . days d 7s 1 2 3 7 1 2 3 7 1 2 3 7 1 2 3 7 1 2 3 7 1
8.5
9.5
2 3 7 1 2
3 7
I:i.:l
I-'
o
I
18° 18 ° C.
-
25° 25 ° C. C - - 30° 30 ° C.
-
-
-
-
-
--+ +t t
-
-
-
-
t+ + +t t
t4t ++ t+
-
-
t+
-
+
+ +t t
-
37° 37 ° C. C.
45° 45 ° C. C.
-
-
-
--
--
--
+÷ t+
-
-
-
-
-
--
-
-
t+ + +t t + +t t
-
-
t+ + +t t
t+ t+ + +t t
-
+ +t t
--
--
-
t+
+ +
-
-
-
-
-
--
-
-
-
t+ + +t t
-
-
+ +t t
--
+ +
-
-
-
-
-
-
-
-
-
-
-
-
-
-
pP Y ~
~ ~
I::l
~
P 9 ~
~
'§
§..,
311
SALT AND HYDROGEN ION CONCENTRATION
a heavier pellicle on liquid media than the others represented by Culture 67. In all other respects the cultures appeared the same and are probably variants of one species. As stated before (1) these yeast forms were found to reproduce by budding, showed no trace of sexuality, did not produce spores, formed a pelliele on liquid media from the beginning, and tolerated salt in concentrations as high as 18 and 20 per cent. They showed their best growth at a temperature of 25 ° C. and a p H about 6.5, though growth was observed from p H 3.5 to 8.5 (Table 1). Fructose, glucose, mannose, galactose, sucrose, maltose, lactose, raffmose, ethyl alcohol, and sodium and calcium lactate were utilized as carbon sources with the production of carbon dioxide. Both Bacto proteose peptone and Bacto peptone were readily utilized as a source of nitrogen without any other source of carbon. A proteose peptone (3), made by salting out with ammonium sulphate and dialyzing the precipitate to get rid of the sulphate, was able to support growth in the presence of a fermentable carbohydrate but not without. Ammonium sulphate, ammonium nitrate, ammonium phosphate, and asparagin could not be used as a sole source of nitrogen in the presence of a fermentable carbohydrate. TABLE
2
The influence of te~nperatgre and hydrogen ion concentration on the growth of Bacterium linens Cultures 4 and 5 6 a ~ B a c t e r i u m linens pH
Reading days
18 ° C.
25 ° C.
30 ° C.
37 ° C.
m
3.5 m
m
m
4.5 m
5.5 5.85
÷ ÷
6.0
÷ ÷
6.5
÷ +÷
÷ ÷÷
÷ ÷÷
7.5
÷ ÷÷
÷ ÷÷
÷ ÷
8.5
÷ ÷÷
÷ ÷÷
÷ ÷
9.5
÷ ÷÷
÷ ÷÷
÷ ÷
- - - N o growth. + - - S m a l l amount of growth. ÷ ÷ -- Good growth.
4 5 ° C.
312
C. D. KELLY AND J .
C. MARQUARDT
Gelatin was not liquefied, and a slight alkaline reaction was noted in litmus milk with reduction of the litmus at the bottom of the tube. At the present time no attempt is made to classify these yeast forms. The rod forms are represented by Culture 4, isolated in the present study, and Culture 56a (Table 2), obtained from Kiel. Both are typical Bacterium linens Weigmann. It is to be noted that Culture 4 and other cultures isolated in this study produced the typical orange-colored ring in milk in contrast to that previously reported (1). Bacto peptone was readily utilized both as a carbon and nitrogen source, but asparagin, ammonium sulphate, and ammonium nitrate could not be used. No apparent action was noted on any of the carbohydrates. The optimum temperature for growth was about 25 ° C. with no growth at 37 ° C. (Table 2). A hydrogen ion concentration of pH 6.5 proved most satisfactory with growth at pH 9.5 and as low as 5.85 but not lower (Table 2). Salt in concentrations as high as 18 and 20 per cent was tolerated in both solid and liquid media. In order to correlate the findings in the laboratory with conditions in factory cheeses, hydrogen ion determinations together with salt determinations were made on cheeses in the factory varying in age from the new cheese to those ready for market. I~ETHODS
The method previously employed of sampling factory cheeses, varying in age by a day, was largely employed in the present studies. Where possible the same batch of cheeses were sampled from day to day. In sampling the cheeses, the outer slimy surface was scraped off with a knife and labelled " A . " After this five-sixteenth of an inch of cheese was cut off the outside and called sample " B . " The next five-sixteenth of an inch was called " C " and the center " D . " Salt, hydrogen ion and in some cases moisture determinations were made of these portions. At the same time bacterial samples were taken of the surface by bringing a glass slide in contact with the surface slime (1). Hydrogen ion determinations were made with the quinhydrone electrode following the method of S~nke Knudsen as modified by Watson (4). Salt was determined in duplicate by the method recommended by Wilster et al. (5), for hard cheese. Though Limburger cheese is classed with the semisoft cheeses it is firm in the early stages and the method proved quite satisfactory. In all, six lots of cheese were examined for hydrogen ion concentration and four lots for salt, covering periods of May, July, and October.
SALT AND HYDROGEN ION CONCENTRATION
313
RESULTS Hydrogen Ion Changes During Ripening
The p H dropped from about 6.45 in the milk at time of renneting to below 5.0 in the cheese within a few days (Tables 3, 4, 5 and F i g u r e 1). TABLE 3 Changes in hydrogen ion concentration of Limburger cheese made in May Description of cheese
First color ............................ Good color .............................
Ready for storage .........
Age days
pH of surface
Microorganismsin surface smears
2 3 4 5 6 7 8 9 10 11
5.64 5.81 5.95 6.57 6.84 6.95 7.56 7.50 7.75 7.85
Few bacteria and yeasts Yeasts increasing Yeasts in masses, a few rods Yeasts in masses, a few rods Yeasts in masses, a few rods Yeasts in masses, a few rods Masses of yeasts and rods Masses of yeasts and rods Masses of rods, a few yeasts Masses of rods, a few yeasts
A f t e r the first or second d a y when the yeasts started to grow the p H of the surface slime was found to rise to around p H 7.0 or higher at about 7 to 12 days when the cheeses were r e a d y to go to storage. Because of lower temperatures in the ripening room, the p H of the October cheeses did not go above 6.7 before being t r a n s f e r r e d to storage. The recorded figures in some cases m a y be somewhat low as difficulty was encountered in scraping off the slime without getting some of the interior of the cheese. This factor together with the gas in two cases would no doubt explain the irregularities of the points of the curves designated surface and 1st 5/16th in. ( F i g u r e 1). Little f u r t h e r change was noted in the p H of the surface up to the time the cheese was ready for market. I n the interior of the cheese (Samples B, C, and D) the rise in p H was not as r a p i d and h a r d l y reached the neutral point at any time d u r i n g ripening, being a r o u n d p H 6.9 when r e a d y for market. The rods ( B a c t e r i u m l i ~ e n s ) did not appear until the p H of the slime was a r o u n d 5.85 and usually when it was higher. The red color appeared when the surface smear was somewhere about p H 6.8. ~Vhen Oospera were f o u n d in any numbers in the surface smear or the cheese was gassy the p H was invariably lower than normal (Table 4, F i g u r e 1). The desirability of this first drop in pH, due no doubt to the growth of lactic acid streptococci, was shown in some work carried out on cheeses made at this same time from milk pasteurized at 143 ° F. for 30 minutes. Two lots of cheese were made with the milk as received and the p H did not drop below p H 6.0 (Table 6) followed by a steady rise in pit. W h e n these cheeses were examined at a later date they were f o u n d to have a strong p u t r i d odor. Another lot was made from milk of the same supply but 2 ml.
Ci-' 50
"""
fI>.
TABLE T A B L E 44
Changes C h a n g e sin i n hhydrogen y d r o g e n iion o n c concentration o n e e n t r a t i o n oof f L iLimburger m b u r g e r e hcheese e e s e made m a d e iinn JJuly, u l y , 1937 1937 pH p H in in cheese eheese D e s c r i p t i o n of of cheese cheese Description
Age Age
W h i t e slime . White
G a s s y cheese Gassy r e d color ..... Good red G a s s y cheese Gassy R e a d y for f o r storage s t o r a g e .... Ready
R e a d y ffor or m a r k e t ............... Ready market
.S'" goo "',...., co .......
coQ
.S
~ en.
... ,...., ............ ~lQ
w.ot:>
4.95 5.05 4.95 6.05 6.45 6.45 5.70 7.00 5.75 7.25 7.35 7.40 7.35 7.35 7.35 7.25 7.45
4.90 4.90 4.85 5.20 5.45 5.00 5.70 5.20 5.20 5.35 5.60 5.35 6.00 6.25 7.00 6.25 6.25
4.80 4.75 4.65 4.70 4.75 4.50 4.80 4.75 4.75 4.85 4.80 4.85 5.45 5.55 6.05 6.05
~
day 11 day days 2 days 3 "c~ 4 "~c 6 "~ 7 "c~ 88 "cc 10 "c~ 11 "~c 12 "t~ 13 "" 4 weeks 55 "~c ct 66 " 7 " c~ 88 ~c "
~
'tloo
~
..<:l
'd
...co
Microorganisms M i c r o o r g a n i s m s in in surface s u r f a c e smear smear
~
..,o:l
~
Q)
0
4.65 4.80 4.70 4.70 4.60 4.70 4.70 4.70 4.70 4.70 4.65 4.70 4.90 5.10 5.55 5.55 5.80 5.80
~
A few f e w rods evenly scattered scattered A few f e w yeasts yeasts Fewer F e w e r rods, yeasts y e a s t s well distributed distributed Yeasts, Y e a s t s , some rods increasing increasing Yeasts Y e a s t s in masses, m a s s e s , rods fewer Yeasts Y e a s t s more numerous, n u m e r o u s , rods fewer fewer Yeasts Y e a s t s same, rods in masses masses Yeasts Y e a s t s fewer, rods fewer. Oospera Oospera present present Yeasts Y e a s t s few, rods in masses, a few f e w Oospera Yeasts Y e a s t s fewer f e w e r and a n d smaller, rods in in masses masses Yeasts Y e a s t s in masses m a s s e s but b u t small, rods rods in masses masses Yeasts small, rods in masses Yeasts rods masses rods in masses No yeasts, No y e a s t s , rods masses Yeast Y e a s t small s m a l l and a n d numerous, n u m e r o u s , rods rods in masses masses Yeast rods in in m masses Y e a s t small aand n d few, rods asses Yeasts rods in in m masses Y e a s t s small aand n d few, rods asses
>1 >
Z
t:I ~y
<-< ~
l:':: >
~
,c;
q
~
t:I >-3
TABLE T A B L E 55
Changes Changes in in hydrogen hydrogen ion ion concentration concentration of of Limburger Limburger cheese cheese made made in in October, October, 1937 1937
~
1 p i t in in cheese cheese1 pH
Description of of cheese cheese Description
Age Age
.g '""0;¢2
.......
"
F i r s t red color ......,....................... First
Ready o r storage Ready ffor storage
.......... , ..
3 4 66 7 88 99 10 11 12
"~
"tt "~t~t "tt
"tc
"ct "~t " "'~~
"
""
.S
Soc
"rl
"'--""
Microorganisms l~ieroorganisms in in surface surface smear smear
... 1:1'"
~
Z
1;:;1
I:Q
'"
t1
UJ.lf:l
4.37 4.30 4.97
5.65 5.65 5.33 4.37 3.95 4.75
4.30 3.95 4.80
4.25 4.25 3.95 4.80
Few F e w bacteria bacteria and and yeasts yeasts Yeasts Y e a s t s increasing Yeasts Yeasts the the same same
4.82 4.70 5.25 5.20 5.55 5.60 5.20 6.70 6.55
4.70 4.55 4.65 4.61 4.90 4.65 4.85 4.90 5.40 5.10
4.70 4.25 4.60 4.65 4.60 4.65 4.75 4.75 4.60
4.70 4.50 4.55 4.50 4.50 4.50 4.66 4.65 4.65 4.60 4.60
Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Y easts Yeasts Yeasts
hours hours day days
"0
~lf:l
UJ.
44 6 11 2 3
.S ~~ •.-<--...,
.g 0
§ in masses in in masses masses and and rods rods in in masses masses and and some some and and rods rods in in masses masses and and some some and rods in masses and a and rods in masses and a few few and and rods rods in in masses masses and and aa few few and rods rods in in masses masses and and aa few few and and rods rods in in masses masses and and rods rods in in masses masses and
~
Z (5 Z Q
o Oospera Oospera Oospera Oospera Oospera Oospera Oospera Oospera Oospera
Z
Q
~'-3
~ ~
The r o m cheeses made The first 6 samples were were ffrom made October October 4th. 4th. The The rremaining were ffrom made October October 1st. 1st. e m a i n i n g samples were r o m cheeses made 11 Cheese made r o m milk H 6.45 made ffrom milk with with ppH 6.45 at at the the addition addition of of rennet. rennet.
Co\:)
I-'
~
316
C.D. KELLY AND J. C. iVIARQUARDT I
I
I
•
•
e
'1
J
~-~
PH
e
efe
7
e -~
o
o
5
~--~=r~-~
4_
•
o o
e SURFACE o l S T . 5/161N. • 2 N D . 5/16 IN. <>CENTER
,.I ......
I ...... I
•
[ 2
I
i
r
I
I
r
3
4
5
6
7
8
WEEKS Ym. 1. Changes in hydrogen ion concentration of Limburger cheese made in July~ 1937.
of starter were added for each gallon of milk at the time of renneting. By the third day the p H had dropped to 5.0 and though this drop was not as rapid or as low as in many commercial cheeses a typical Limburger developed with no sign of the putrefaction found in other cheeses. This development of acid appears to be necessary to control putrefying bacteria in Limburger cheese as in the hard pressed varieties.
Concentration of Salt in the Cheese As would be expected and as shown by lYIrozek (6), Byers and Price (7), and others the salt in the cheese was found in greatest concentration in the surface layers and lowest in the inner portions soon after salting (Tables 7, 8, and 9 and Figure 2). On the first day the percentage of salt was between 3.13 and 3.30 in the surface layer (Sample A), 2.03 to 3.04 in the second layer (Sample B), 0.36 in the next layer (Sample C) and 0.06 to 0.31 in the center (Sample D). From this time on the salt tended to diffuse TABLE 6 Influence of added starter on hydrogen ion concentration and quality of Lin~burger cheese made f r o m rail# pasteurized at 143 ° F. )*or 30 minutes
Age in days 1 No starter added. Cheese became putrid No starter added. Cheese became putrid Starter added. Good cheese
2
3
6
7
pH
pH
pH
6.30
6.30
4
pH
pH
6.15
6.30
~5
6.15
6.65
6.15
5.55
6.0
pit
5.00
6.00
317
SALT AND HYDROGEN ION CONCENTRATION
t h r o u g h the cheese until an equilibrium was reached n e a r the tenth d a y when the percentage was about the same throughout the cheese. Considering t h a t the method of salting is b y rolling the cheese in the salt and rubbing with the hands it is surprising to find the salt content f r o m cheese to cheese as u n i f o r m as it is. Though moisture should have been taken into consideration in r e p o r t i n g the salt content it was difficult to obtain satisf a c t o r y samples. However when c o r r e c t i o n s w e r e made using the moisture determinations t h a t were made the variations in salt content were so small as not to change greatly the relationship of the amounts as given (Tables 7, 8, and 9 and F i g u r e 2). TABLE 7 Changes in salt content of L i m b u r g e r cheese made during July, I937 Age 1 day 2 days
....................................... .........................................
3
~
............................................
4
~
..........................................
6 " 7 "
............................................. .............................................
10 " 11 ~ 12 ~' 13 ~'
............................................. ............................................
............................................
.............................................
4 weeks
......................................
6
¢c
........................................
7
~
....................................
8
~
..........................................
Surface
First 5/16 inch
Second 5/16 inch
3.28 3.12 2.33 1.56 2.33 2.38 1.55 2.10 1.69 2.56 2.00
3.04 2.71 2.33 2.31 2.34 2.23 2.05 2.23 1.85 2.46 2.53
0.63 1.15 1.64 1.60 2.00 1.68 1.71 2.21 2.13 2.53 2.36
0.31 0.61 0.80 0.93 1.56 0.91 1.41 1.71 2.10 2.40 2.13
1.49 1.46 1.46 1.81 1.88
1.69 1.80 1.70 1.88 1.96
2.03 1.78 1.80 1.80 2.11
1.76 1.73 1.90 1.70 2.09
Center
TABLE 8 Changes in salt content of :Limburger cheese made during October, 1937 Age days 1 2 4 5 7 9 11 12
Surface
First 5/16 inch
3.13 2.15 2.03 1.76 1.66 1.64 1.46 1.66
2.03 1.20 1.90 1.73 1.96 1.68 1.43 1.56
l
Second 5/16 inch 0.90 0.88 1.40 1.73 1.86 1.48 1.53 1.66
Center 0.30 0.55 1.26 1.50 1.56 1.42 1.46 1.43
I n determining the influence of salt on the growth of bacteria in cheese several factors m u s t be taken into consideration in estimating the concent r a t i o n of salt in contact with the organisms. I t is to be expected t h a t most of the salt would be dissolved in the moisture of the cheese and because of this the percentage of salt as determined b y an analysis of the cheese does
C. D. KELLY AND J. C. MARQUARDT
318
TABLE 9 Changes in salt content o f Limburger cheese made during October, 1937
Per cent of salt Age days 1
............................................................
3
............................................................
3
...........................................................
4
...........................................................
5 6
........................................................... ............................................................
7
............................................................
8
............................................................
9
............................................................
10
............................................................
11
............................................................
12
..........................................................
Surface
First 5/16 inch
Second 5/16 inch
Center
3.30 2.26 2.30 2.13 1.80 1.60 1.53
2.86 2.13 2.23 1.82 1.76 1.46 1.56 1.76 1.76 1.73 1.76 1.76 1.76
0.36 1.50 1.86 1.44 1.50 1.70 1.54 1.60 1.60 1.60 1.63 1.60 1.63
0.06 0.36 0.76 1.02 1.03 1.06 1.15 1.50 1.46 1.53 1.46 1.56 1.53
n o t give a t r u e p i c t u r e of c o n d i t i o n s i n the cheese. T h e r e f o r e i n e s t i m a t i n g the salt c o n t e n t of the m e d i u m i n w h i c h t h e b a c t e r i a are g r o w i n g the moist u r e c o n t e n t of the cheese m u s t be t a k e n i n t o c o n s i d e r a t i o n . T h e m o i s t u r e c o n t e n t of the s u r f a c e l a y e r v a r i e d f r o m 40 to 42 p e r c e n t a n d f r o m 42.5 to 44 p e r c e n t i n the c e n t e r p o r t i o n . A s s u m i n g t h a t all t h e s a l t is i n solution i n the cheese moisture, this b r i n e i n the s u r f a c e l a y e r of a one-day-old cheese I
2.8
I
I
I
I
"~~
2.4 2.0_ o~ ~ e
o
1.6_
~ o
~ °°0. 8 I-
Z
"' U frO. 4 n
• / /
2.
0
o CENTER
l
I L 2 4 AGE IN D A Y S FIG.
•
: SURFACE IST 51161N • 2ND 5/16 iN
I /o g
~
o
I 6
I 8
I 10
I 12
Changesin concentrationof salt in Limburger cheese.
S A L T A N D H Y D R O G E N I O N CONCENTRATIOkNI
319
(Table 9) with 41 per cent moisture would have 8.0 instead of 3.3 per cent salt. At 2 days the per cent of salt would be 5.5 instead of 2.26 and at 5 days 3.9 instead of 1.6 per cent. lVIcDowall and Dolby (8), working with Cheddar cheese, came to the conclusion that a large part of the cheese moisture was not free moisture though they were unable to make conclusive tests. However they estimated that 20 to 30 per cent of the moisture in a young cheese was bound. Assuming that 30 per cent of the moisture is bound, the concentration of salt in the free water would be 11.4 per cent in the surface layer of a day-old cheese. The surface layer of the 2-day-old cheese would then have a salt content of 7.8 per cent and the cheese at 5 days, 5.5 per cent. These theoretical considerations establish that the salt concentration of the cheeses a few days after salting would in no way retard the growth of the organisms desired. A short time after the cheese is salted, on the evening of the day when made and the morning of the following day, the salt content of the brine in the surface layer must be near the saturation point as there is a considerable amount of undissolved salt present. Therefore for at least some hours after salting the cheese, only salt tolerant organisms would multiply. This is given support in the work of Macy and Erekson (2) where it was shown that Streptococcus lactis disappeared when the cheese was salted and yeasts took their place. DISCUSSION
During making and the early stages of ripening the rapid fall in p H is undoubtedly largely due to the action of lactic acid streptococci. The pH which falls lower than in normal Cheddar cheese is no doubt due to a higher moisture content in the Limburger cheese with more lactose to be converted to lactic acid. This lowering of the p H is desirable in making Limburger cheese in that it keeps pu~re±ymg . . . . . . . . . . . . . .oac~erJa . . . . . . from growing. The high concentration of salt on the surface suppresses the many miscellaneous organisms found on the new cheese and permits the development of the yeasts that can grow at a low ptI. The yeasts by acting on both the lactate and the protein soon raise the p t I above the point (pH 5.85) where Bacterium linens can start growth. Other organisms that might grow at this p i t are suppressed by the concentration of salt while Bacterium linens grows readily. At this point the yeasts have completed their work and soon die out, being overgrown by Bacterium linens. Acting on the protein Bacterium linens raises the p H still higher and is believed to complete the ripening of the cheese. The cheeses become inoculated with both organisms from the ripening shelves, which in the factory are heavily seeded with the organisms. Though no work as yet has been carried out on the internal flora of the cheese it does not seem probable that Bacterium linens would be a factor
320
C. D. KEI~Y AND J. C. lYIARQUARDT
because of its aerobic n a t u r e and its inability to grow at a p H lower than 5.85. This tends to strengthen the t h e o r y that the soft b u t t e r y texture and a p a r t of the flavor is produced by the action of enzymes of Bacterium linens rather than by direct action of the organism itself. The question whether the enzymes penetrate the cheese and digest proteins could best be established by a study of the enzymes. SUMMARY
The yeast forms first appearing on the surface of L i m b u r g e r cheeses will grow readily on artificial media between p H 3.5 and 8.5 and with the best growth at p H 6.5. Bacterium linens will not grow below p H 5.85 but will grow at p H 9.5 and grows best at p H 6.5. I n the cheese the p H of the surface falls to 5.0 or lower, a p H at which the yeast will grow but not Bacterium linens. The yeasts acting on both the protein and lactic acid, raise the p H above 5.85 at which point Bacterium linens can become established and overgrow the yeast. Only late in the period of ripening does the p H of the interior of the cheese rise above 5.85. This p H and the lack of oxygen would inhibit the growth of Bacterium linens in any p a r t of the cheese excepting the surface. The concentration of salt soon after salting is highest in the surface layer and low in the interior. There is a gradual penetration of salt into the cheese until an equilibrium is reached at about 10 days. With the lack of information on how much of the moisture of the cheese is available to form brine it is impossible to estimate accurately the concentration of salt in the medium in which the bacteria are growing. However, as the cheeses are salted by rubbing salt on the outside there is little doubt but that it is near the saturation point in the surface layer for some time. This high concentration of salt tends to inhibit the growth of organisms other t h a n the yeasts and Bacterium linens. REFERENCES (1) KELLY, C. D. The microbiological flora on the surface of Limburger cheese. J. DAIRYSC. 20: 239--246. 1937. (2) MACY, H.) AND EREKSON, J. A. Unpublished data supplied by the authors. St. Paul, Minn., November, 1937. (3) HUCKER, CA. J. Studies on the Coecaceae. III. The nitrogen metabolism of the micrococci. New York State Agr. Exp. Sta. Tech. Bull. No. 101. 1924. (4) WATSON, PAUL D. Use of quinhydrone electrode for following changes of pH in Swiss cheese. [ndust. Engin. Chem. 19: 1272-1274. 1927. (5) WILSTER,G. H., et al. Determination of fat, moisture, and salt in hard cheese. J. DAIRYSC. 20: 27-30. 1937. (6) MROZEK,O. Diffusion yon Kochsalz in K~se--Milchwirt. l%rsch. 4: 391. 1927. (7) BYERS, E. L., ANDPRICEW. ¥. Observations on the salting of brick cheese. J. DAIRY SC. 20: 307-318. ]937. (8) McDOWALL,F. H., AND DOLBY,R.M. Studies on the chemistry of Cheddar cheese making. J. Dairy Res. 7: 156-175. 1936.