- Email: [email protected]
Effect of Lactic-Streptococcal Agglutinins in Milk on Curd Formation and Manufacture of Cottage Cheese1
Effect of Lactic-Streptococcal Agglutinins in Milk on Curd Formation and Manufacture of Cottage Cheese 1 D. B. EMMONS, J. A. ELLIOTT, and D. C. BECKETT Food Research Institute, Research Branch, Canada Department of Agriculture, Ottawa Abstract
Shattered~ mealy curd, sediment on the bottom of the vat, an(t slow acid production were assoeiated with agglutinating strains of Cottage cheese cultures. Agglutinated bacteria formed centers of high acid production and resulted in (lense flecks of addprecipitated casein. These flecks either settled as a sediment or were trapped during coagulation and caused a fragile, mealy cm'd. The sediment was low in p H and high in protein and bacteria. Acid production in the supernatant skimmilk was retarded through displacement of agglutinated bacteria to the bottom of the vat, indicating that agglutinating antibodies in skimmilk are not inhibitory, per se. Prolonged agitation of skimmilk and starter promoted agglutination and, after agitation ceased, resulted in more sediment and reduced rates of aeid t)roduction. With agglutinating cultures, the p H of the A-C test sample, receiving less agitation, reduced more quickly than skhnmilk in the vat, thereby nullifying the test as a direct indicator of the time to eat the curd. tteating skimmilk (71 C/30 rain) or removing agglutinins by adsorption on heat-killed cells eliminated the defects. Pasteurization (72 C/16 see) reduced the agglutinating activity of skimmilk by 50-75%. Agglutinating strains differed markedly in the severity of defects produced; two strains settled so extensively that the ski,nmilk (lid not coagulate; whereas, defeets with some strains were evident only as small amounts of sediment. Auto-agglutination of certain strains also appeared to cause small amounts of sediment. Some commercial cultures contained agglutinating strains and produced sediment. Selection of agglutination-resistant strains for Cottage cheese cultures is reeommm~ded. A defect of sediment on the bottom of the vat has been observed during curd formation in Cottage cheese manufacture. Lucas (15) de-
scribed a. spongy sediment, 1/8 1/2 in. deep, and suggested it was due to the use of overheated nonfat dry milk. San(line et al. (20) reported that sediment formed occasionally when mixed-strain starters containing Streptococcw~ diacetilactis were used. This defect occurred more frequently when more than four mM of caleimn were added per liter of milk and when the milk was aged three to seven days. The defect was not observed when aetive, lowgas-producing, mixed-strain starters were used. They suggested that excess calcium ions~ either added or liberated by citrate fermentation, destabilized the casein which subsequently settled. Initial trials in our laboratory using two single-strain cultures ( I I P and E~) as starter resulted in sediment of very low p H on the bottom of the vat. Assodated with the sediment were undesirable shattered, mealy curd, a eoagulum that shattered excessively during cutting and cooking, and slow acid development. The low p H of the sediment indicated the presence of relatively larger numbers of starter bacteria. I n this conneetion, Wright and Tramer (24) observed that "slow (agglutinationsusceptible) starters, which give a better aetivity in separated and homogenized milk (than in whole milk), tend to form a deposit on the bottom of the test-tube after 6 hr at 30 C; whereas, fast (agglutlnation-resistant) starters do not, when grown in either kind of milk. Examination of this deposit reveals enormous numbers of starter organisms, presumably due to agglutination." The association of deposit formation in raw or pasteurized skimmilk with agglutination-suseeptible strains of starter was confirmed by Auelair (1) and Stadhouders (21). Gillies (9), Keogh (11), Portmann, Gate, and Auelair (18), Randolph (19), and Stadhouders (21) showed that acid production by agglutinating strains is retarded in unheated skimmilk. This paper 1)resents evidence linking the agglutination phenomenon to defeets that occurred during the manufacture of Cottage cheese with certain strains of starter ba<'teria (5). Experimental Procedures
Cultures. Sino'le strains were of New Zealand Received for publication June 30, 1966. and Australian origin. Commereial cultures • Contxibution no. 43 from tim Food Research Institute, Research Branch, (!amtda Del)art.me~lt of were donated by firms in the United States. Cultures were reactivated frequently i~rom Agriculture, Ottawa, Cmmda. 1357
1358
D.B.
EMMONS ET AL
freeze-dried or frozen (liquid n i t r o g e n ) stoeli: a n d p r o p a g a t e d usually less t h a n 2 wk. Cult u r e s were inoculated a t the r a t e of 1 % in autoclaved r e c o n s t i t u t e d n o n f a t d r y milk (10 l b / 15 r a i n ; 1 0 % ) a n d i n c u b a t e d 16 h r a t 22 C. Agglutinin titers. I n the a g g l u t i n a t i o n test (6)~ e i t h e r one-hal:f- or t h r e e - f o u r t h s - d i l u t i o n s of whey were used. N e g a t i v e t i t e r s ( < 1/2) represented no a g g l u t i n a t i o n in a n equal nfixture of cell s u s p e n s i o n a n d u n d i l u t e d whey. Reported values are log averages of duplicate determinations. Bacterial estimates. D u p l i c a t e p o u r e d plates of either H u n t e r ' s (10) or E l l i k e r ' s (4) media -were counted following i~mubation f o r 72 h r a t 22 C. ChaiJ~ lengths. Slides of cultures were prep a r e d from autoclaved (10 l b / 1 5 rain) skimnfilk a f t e r i n o c u l a t i o n w i t h 5ff{~ of culture a n d incuhation f o r 3 h r at 32 C. The Levowitz a n d W e b e r (14) s t a i n was used. E a c h s t r a i n was classified as diploeoeci, s h o r t ( 4 - 8 cells), medium ( 9 - 16) cells) or long ( > 1 6 cells) chains. (.',urd formation in ,vats. Coagulum was :formed a t 32 C f r o m 182 kg of p a s t e u r i z e d (72 C / 1 6 se(') skimmilk in 228- or 455-kg v a t s ; 5°//, s t a r t e r was added. Unless otherwise stated, r e n n e t was added ii h r a f t e r the s t a r t e r , at the r'tte o1'1 m l / 4 5 5 kg of s k i m m i l k ; the skimmilk was stirred mechanically f o r 15 lni~l a f t e r a d d i n g b o t h s t a r t e r a n d rennet. S a m p l e s f o r p l l a n d bacterial estimates were t a k e n a) 1 em u n d e r the sm'faee ( T ) , b) o n e - t h i r d of the distance from the b o t t o m of the v a t ( M ) , a n d c) tq'om a m i x t u r e of the b o t t o m 25 m m ( B ) . The bottom samph~ ( B ) was o b t a i n e d from 250 nil beal
over a p e r i o d of 130 rain, a n d washed with w a t e r at 16 a n d 4 C. Curd formation in beakers. C o a g u l m n was fomned at 32 C w i t h 5ff~ of s t a r t e r added to 550 ml of s k i m m i l k in 600dnl Berzelius b e a k e r s ; the d e p t h of s k i m m i l k was 13 era. The skin> milk was s t i r r e d f o r 15 rain a f t e r addb~g starter, a n d a g a i n ] h r later, with a mechanical s t i r r e r at a c o n s t a n t r a t e estimated to be the same as
that in the vats. Samples for pH were taken a) 1 em u n d e r the sm'face (T), b) 4 em f r o m the b o t t o m ( M ) , a n d c) iron1 a m i x t u r e of the b o t t o m 1 em of the b e a k e r ( B ) . F o r the bott o m sample ( B ) , the top was s i p h o n e d away u n t i l a d e p t h of 1 em remained. Results
Deseriptio.u o/' defects i~ curd for.marion. Defects r e s u l t i n g f r o m the a g g l u t i n a t i o n p h e n o m enon are i l l u s t r a t e d with S t r a i n H P . S t r a i n C~ was used f o r c o m p a r i s o n ; it did not agg l u t i n a t e in n o r m a l milk a n d p r o d u c e d curd free of these defects. S e d i m e n t always a p l , e a r c d on the b o t t o m of t h e v a t with S t r a i n I t P . The a m o u n t of sedim e n t increased as the p l [ el! the rest o£ the v a t decreased, first a p p e a r i n g between p I [ 6.2 to 5.6 and 3'eaehiljg the m a x i m u m j u s t before coagulation a t p l l 5.2. Usually the bottom 11.5 1 em of the sediment was a tan or cream color, whereas m,y a d d i t i . n a l sediment above it was white. The. sediment contained relatively large n u m b e r s of bacteria (Table 1 ) ; microscopic e x a m i n a t i o n sh~wed large masses of bacterial cells. The sediment also contained a b o u t twice as much nitrogen, i n d i c a t i n g t h a t casein was remow~d from. the skimmilk a n d c o n c e n t r a t e d in the sediment. This sediment was dense, tough, a n d difficult to cut a n d collected on the c u t t i n g k,,iw~s ( F i g u r e 1). D u r ing curd :formati,m iu a vat:, p l l values were relatively u n i f o r m ahow, the sediment. The p I l oJ' the s e d i m e n t was always h,w, apl,roach-
I AI L E 1 l)istribuiio~J of nitrogen, pit, and bacteria in pasteurized sldmmilk in a vat during Got.rage cheese curd formation using Strain H P ;ts starter
Point
of s'unpllng
Time of sampling
Nitrogen content of skimmi]k or curd 1
(hr :'miO Skimmi]k" and starter ] em below surface 1/3 distance from bottom Mixture of sediment (25 mm in depth)
2
Mean
pH
(rag N/g)
Standard plate count
(× 10~)
0 4:20 4:20
5.16 4.93 4.89
5.18 4.94 4.93
5.17 4.94 4.91
6.40 5.'25 5.25
4:20
10.24
8.57
9.40
4.49
1.6 1.9 58
" Agglutinin titers against l i p cells were 1,/14.2 and 1,/3.6 for the r'tw and pasteurized skbnmilks, respectively, J. DAII~Y SCIENCE VoL. 49, NO. 11
C U R D F O R ~ [ A T [ O N I N CI-~EESE
1359
tLr~m~zh thtid xi
I,'I(;. Ii ~,,!hmml th:ll c~dk.'l,'d ~)1~knhc~ d u r i ~ vIli~'ill~ ill: ('1}~;1~"' ViH',"~C,'[ll'd (~4~]'Hill IIl'). h ~ t h a t ~V the limiti~La pH bw lactic avi;l d c v c l o p l m ' . t ( F i g u r e 2): Just beli, le c,aguhltimL (h'ck.~ o£ curd were obtained , b y drawii~g- a screen thrm~g'h the skiullrliik (Fie'rive 3). Fi~:uve 4 shows a ~ , ~ of bacterial cells f r o m such :t fleck a f t e r dissolviu~' the c-t~ein in dih~te alMdi. The amouut of ag'it:ation of s k i m m i l k a f t e r a d d i n g s t a r t e r markedly affected the r a t e of acid deve]opw_ent (I~igure 5). A p H of 4.7 was reached lit 5 h r when the skimmiiI< was a g i t a t e d for only 10 rain a f t e r a d d i u g the s t a r t e r a n d rennet. P r o l o n g e d a ~ i t a t i o n delayed acid p r o d u c t i o n a~d resulted in more sedime~tt. Contiuuous a g i t a t h m f o r 3 b r resulted
~
m faihu'c ,I' lhc I,tl i - dl'~q~ beh~.~ .3~3 m the ttlq>el' liquid i.,rti,m ,,f the vat amI in a ,~edi.,e~a ,.o.,pvi~in~ .m.-thivd <~t~tlw vt~t c.nt~.ut~. ~,imilar agitati(m tveatment~ with ~trah~ tL. did ~,)t affect rate ~)~ acid (h'veh)l)ment , ran' did any sediment [))rut. At cutting, the em'd f o r m e d with S t r a i n l i p s~plit irvegutarly ( F i g u r e 6, A) a.~ c o m p a r e d to the clean split o[ curd £m'n~ed with S t r a i n (J~ ( P l g u r e 6, 1%). I t was ~requeut/y possible to see dense white flecks on the surface or' the cut curd p a r t M e s . Also, the sur£ace of the v a t of et~rd showed small raised b u m p s ; a % w millimeters of whey usually covered the surface. The dense white flecks on cut curd a n d
t~ 30 - -
4 HR
~
0 6.0
5.5
5 HR
5.0
8 HR
4.5
,-
4.0
~IO. 2. I)istribu~ion of pII at various times dur]ll~' elll'd forlil}t'Liot£ ill ~1 Vf[~ (StI'Hill H - P ) .
Fro. 4. Photomicrograph of mass of agglutinated cells in fleck that had bee~L placed on ~t slide in a small drop of N/10 NaOEI. J. DAIRY S(JIEXC!E VOL. 49, No
~I
1360
D.B.
EMMONS
5.7
pHso 4.5
TIME
- HR.
]?IG. 5. Effect of agitation of skimnfilk 11~ vat on rate of acid development in upper portion of skimmilk in vat, usiug Strain H P as starter. Treatments were: ( I ) agitated 10 rain, starter and rennet added together; ( ] I ) agitated 15 rain, rennet added 1 hr after starter and agitated 15 nfin; ( I I I ) agitated conthmouslv for 75 rain, adding rennet 60 nlin after starter; ( I V ) agitated continuously for 3 hi', addhig re,met 60 nlin after starter. the b u m p s on the surface were p r o b a b l y caused by the flecks observed on the screen j u s t p r i o r to coagulation. The curd formed with S t r a i n H P s h a t t e r e d excessively during; cutti~g a n d cooking, as evidenced by the Cornell g r i t test (13), a n d was
E T AL
very mealy in body a n d t e x t u r e (Table 2). 2fealiness was not due solely to the s h a t t e r e d c u r d p a r t i c l e s ; large i n t a c t c u r d particles, obtained with the 1A-in. sieve in the wash water, were j u d g e d to be as mealy as the bulked curd. Reduction of a g i t a t i o n to a m i n i m u m reduced the s e t t i n g time to a n acceptable value, b u t did not reduce the s h a t t e r i n g a n d mealiness in the finished curd. I n 20 cheese-making trials with S t r a i n H P , the em'd was always very. shattered a n d very mealy. A d s o r p t i o n o]' antibodies from..~t,:i:t~,,milk on heat-killed cells. I t was p o s t u l a t e d t h a t agg l u t i n a t i o n of s t a r t e r b a c t e r i a b y antibodies o c c u r r i n g n a t u r a l l y in the skimmilk caused the above defects. A n t i b o d i e s in skimmi]k were adsorbed on heat-killed H P cells a n d removed b y e e n t r i f u g a t i o n as follows: Each of two 450-ml q u a n t i t i e s of u n h e a t e d skinmdlk was t r e a t e d three times with saline-washed heatkilh,d (63 C / 3 0 rain) cells from 15(1 nfl of incubated ('2'2 C/16 h r ) , n e u t r a l i z e d ( p H 6.5) t t u n t e r ' s b r o t h . Then H P s t a r t e r was added to 550-ml q u a n t i t i e s of t r e a t e d a n d u n t r e a t e d skimmilk in beakers. The a d s o r p t i o n t r e a t m e n t increased the r a t e of acid dew~lopment a n d reduced the a m o u n t
A B VI~. 6. A. ]~agged split in curd formed with Strain t I P at time of cutting. Bumps may be seen on surface of vat of curd. B. Clean split in curd formed with Strain C._, at time of cutti~g. J . DAIRY SCIENCE XC:OL. 49, NO. 11 A
C U E D FOI%MATION ]IN C H E E S E
1361
TABLE 2 Quality and particle size distribution of curd made with st,'aius resistant (C~) and susceptible ( H P ) to agglutination Curd particle size distribution '~ Time
Rep.
(In.)
t~
cutting
> 1/2
1/2 7/4
( hr : rain)
1/4-1/8
1/8-1/16
Curd quality
(%)
Strain C.~--agitatcd for 15 rain; rennet added 1 hr after starter, and agitated 15 rain I 4 : 35 0.8 73.9 23.6 1.7 Cleau cut ; satisfactory curd I[ 4 : 42 0.5 72.3 24.6 2.5 Clean cut ; satisfactory curd Strain H P - - a g i t a t e d for 10 rain only; and starter and rennet added together I 4 : 38 2.1 57.1 33.7 7.1 Ragged cut ; shattered and very mealy cm'd lI 4 : 40 2.1 52.3 36.5 9.1 Ragged cut ; shattered and very mealy curd Strain H P - - a g i t a t e d for 15 rain; rennet added 1 hr after starter and agitated 15 rain l 7:09 1.2 54.6 35.0 9.2 g~gged c a t ; shattered and very mealy curd II 7 : ll0 0.9 54.6 32.9 11.6 Ragged cut ; shattered and very mealy curd Kosikowski's Corne]l Grit Test (13). I n a n o t h e r trial, the a d d i t i o n of heat-killed cells, w i t h o u t removal, a c c e n t u a t e d the defects of settling a n d slow acid development. Si,l~gle-.~train c u l t u r e s . W i d e differences were observed a m o n g 20 s t r a i n s in the thickness of the sediment, r a t e of acid development, distribution of p H values a n d of b a c t e r i a in the skimmilk, a g g l u t i n i n titers of whey f r o m the skimnfilk before a n d a f t e r p a s t e u r i z a t i o n , a n d l e n g t h of b a c t e r i a l chains ( T a b l e 4). I n general, the observations s u p p o r t the h y p o t h e s i s t h a t agglut i n a t i o n of s t a r t e r b a c t e r i a by antibodies caused the observed defects. F i v e strains, C,, C~, C~, C~, a n d C2, p r o d u c e d no a p p r e c i a b l e s e d i m e n t a n d were n e g a t i v e f o r a g g l u t i n a t i o n in whey f r o m both r a w a n d p a s t e u r i z e d s k i m m i l k ; p H values a n d bacterial d i s t r i b u t i o n were u n i f o r m t h r o u g h o u t the skimmilk in the vat. The 15 o t h e r strains, except C~o which autoa g g l u t i n a t e d , were positive f o r a g g l u t i n a t i o n , b u t differed widely in the seriousness of de-
feets produced. Cells of S t r a i n s K a n d ML~ settled so extensively t h a t acid p r o d u c t i o n ill the v a t p r a c t i c a l l y s t o p p e d at p H 6.0 a n d 5.5, respectively. Some a g g l u t i n a t i n g strains, e.g., C~ a n d C,, yielded only small a m o u n t s of sedi m e n t a n d p r o d u c e d acid quickly. T h e r e was no absolute r e l a t i o n s h i p between the a g g l u t i n i n titers of the whey a n d the seriousness of bacterial settling in s k i m m i l k ; f o r example, S t r a i n K yielded the lowest titers b u t settled the most extensively. C h a i n l e n g t h m a y h a v e some b e a r ing on this, because a g g l u t i n a t i n g s t r a i n s growing in longer chains t e n d e d to show more serious defects t h a n those g r o w i n g as p a i r s or s h o r t chains. S t r a i n C,o showed a u t o - a g g l u t i n a t i o n in the a g g l u t i n a t i o n test. I n the b e a k e r test, this s t r a i n p r o d u c e d a b o u t h a l f as much sediment in skimmilk h e a t e d (80 C / 3 0 rain) to destroy the a g g l u t i n i n s as in p a s t e u r i z e d s k i m m i l k ; both sediments c o n t a i n e d h i g h e r n u m b e r s o f h a c t e r i a
TABLE 3 :Formation of Cottage cheese coagulmn with Strain H P iu unheated skimmilk before and after adsorption with heat-killed cells Distribution of pH in skimmilk in beaker Time of observation
T"
M '~
B ~'
( hr : ,mi~ )
Sediment ('##~m )
Untreated skimmilk with an agglutinin titer of 1/10.5 in rennet whey 4 : 25 5.10 5.10 4.42 Treated skimmilk with an agglutinln titer of ~ 1/2 in rennet whey 3 : 30 5.10 5.10 5.09
12 Trace
:' See Experimental Procedures. ,~. ~)AIBY SCIENCE 3~OL. 49. N(). 11
TABLE 4 Observations durh~g Cottage cheese curd formation in vats for 2(I strains of S. eremoris and S. laetis
Strain
Thickness of sediment ~
p H at end of readings ( ~ )
(m m)
Time to end of readings
Ratio of bacterial c'stimateg~ ( 2B ) ~
A p H in vat ~ / . T d- M \ ~ B ')
)
Agglutinh~ titers Raw
Past.
Chain length
(hr: mbt)
~. cre,tttori8 t( ML~ HP ML~ I~tt /~ Zs TR IL~ E~ I)Rz US. C~ C~ Cz~ CT Cla
0.79 0.90 0.89 0.80 0.57 0.57 0.50 0.52 0.60 0.48 0.48 0.32
1/ 4.7
0.5t)
1 / 8.4
10 25 °5 25 16 10 ]6 16 13 13 6 6 I0 Trace None Trace None
6.o3 4.74 5.t10 5.51 4.96 5.21~ 5.02 4.81 4.89 4.93 4.91 4.94 4.74 4.64 4.76 4.68 4.78
6 : 45 8 : 25 7 : 15 7 : 35 5 : 00 5 : 00 6 : 57 5 : 23 5 : 2? 4 : 30 4 : 43 6 : 02 4 : 25 4 : 09 4 : 30 4 : 36 3 : 55
227 51 43 38 25 15 ]3 12 12 9.0 6.5 5.3 3.7 1.0 1.0 6,9 0.6
0.00 -0.01 0.0~ 0.(~2
6 !N-one 6
5.~14 4.78 4.78
4:57 4 : 32 4 : 15
5.9 ] .0 1.6
u.3~; 1).1)1 (I.14
S. laetis C~, C,_, C~o
a Observations nlade when T and .X[ were at ca. p K 5.2 unless p}[ at end of readings was higher. T, M, and B. See Experimental Proeedures.
1/11.2 1/13
<1/2 1/4.7 1/3.1
1/ 8.4
1/3.6
1/ 9.7
1/3.6 1/3.6
J/13 1/ 8.4 1 / 9.7 1/ 8.4
~/ 4.7
1/2.7 1/2.7 1/2.7
<1/2
1/13
1/6.3
1/ 8.4
1/3.6 1/3.6
<1/ 2 <~/ 2
<1/2 <1/2
<1/2 <1/2
1/ 3.1 <1/ 2 +control
<1/2 <1/2 +control
Long Medium Long Long Short Short Medium Short Long Medium Short Short Dip]oeocei Diploeoeei Medlmn Medium Medimn Dip]oeoeei Diploeoeei Diploeoeei
CURD FORMATION IN CIIEESE t h a n the s u p e r n a t a n t . Microscopic e x a m i n a t i o n showed n m n e r o u s small clumps of b a c t e r i a in both t y p e s of skimmilk. L i m i t e d n m n b e r s of cheese-making trials also indicated wide v a r i a t i o n s in the seriousness of defects of s h a t t e r e d a n d mealy curd. S t r a i n s K_ a n d ML~ did not form a eoagulum, a n d the defects p r o d u c e d with S t r a i n H P are described above. S t r a i n s DR~ a n d E~ p r o d u c e d s h a t t e r e d a n d mealy curd, but it was not as mealy as t h a t with S t r a i n H P . S t r a i n s C~, C,,, a n d C., p r o d u c e d c a r d w i t h o u t serious defects. The usefulness of the A-C test was m a r k e d l y affected w h e n a g g l u t i n a t i n g eultm'es were used as s t a r t e r s ; in such instances the p I I in the A - C test b e a k e r was lower t h a n in the rest of the vat, t h e r e b y n u l l i f y i n g the test as a direct i n d i c a t o r of the time to eut the curd. I n the recommended p r o c e d u r e (8), the s a m p l e f o r the k - C test is t a k e n j u s t before a d d i n g r e n n e t to the rest of the skimmilk, a f t e r which the latt e r receives more agitation. Thus, the p H of the A-C test sample, reeeiving tess agitation, decreased more r a p i d l y t h a n t h a t in the vat. E])'ect o]" calcium. I n view of the o b s e r v a t i o n s of S a n d i n e et ah (20), CaC1, was added t4) f o u r lots o f p a s t e u r i z e d skimmilk a t the r a t e of 0, 2.5, 5.0, a n d 7.5 m~E/liter. I n the first lot of skimmilk, traces of s e d i m e n t f o r m e d w i t h S t r a i n C,~ in beakers w h e n 5.0 or more m5I CaCI~/ liter were added to skimmilk at three, four, eight, a n d nine days of age, w h e t h e r r e n n e t was added or n o t ; in skimmilk at t h r e e a n d eight days of age, c o n t a i n i n g 7.5 n l ~ CaCl~/liter, 4 - 8 mm of sediment of low p H formed. However, CaCI~
1363
added to the same lot of skinnnilk h a d no effect on the a m o u n t of sediment or ttle r a t e of acid p r o d u c t i o n when S t r a i n H P was used as s t a r t e r . I n the three o t h e r lots of s k i m m i l k o b t a i n e d at different times, no s e d i m e n t f o r m e d with S t r a i n C,_, in skinnuilk c o n t a i n i n g the abo~e levels of CaCI:~ n o r did the caleimn a d d i t i o n s have a n y effect on the a m o u n t of s e d i m e n t f o r m e d b y S t r a i n H P . I n view of the low p H of the sedi m e n t f o r m e d with S t r a i n C~ a n d the lack of effect of the added CaCI., with S t r a i n H P , it would a p p e a r t h a t the a d d e d CaCI. m a y have destabilized the eells of S t r a i n C2, p r o m o t i n g a u t o - a g g l u t i n a t i o n . H o w e v e r , a d d i t i o n s of u p to 50 m~I of c a l c i u m / l i t e r did n o t p r o m o t e a u t o - a g g l u t i n a t i o n in s u s p e n s i o n of cells of s t r a i n s C.., t i P , a n d K in E l l i k e r ' s (4) broth. Heat treatmeut o.f skimmilk. H e a t t r e a t m e n t of s k i m m i l k affected r a t e of acid p r o d u c t i o n , a m o u n t of sediment, d i s t r i b u t i o n of p i t , a n d the a g g l u t i n i n t i t e r of the whey with s t r a i n H P ( T a b l e 5). H e a t i n g at 68 C f o r 30 n,in redueed the a g g l u t i n i n t i t e r to < ½ , b u t a p preciable sediment formed. I l e a t i n g a t 71 C a n d above redueed s e d i m e n t to 1 ram. The r a t e of a d d d e v e l o p m e n t increased m a r k e d l y in skimmilk receiving the h i g h e r h e a t t r e a t m e n t s . S i m i l a r results were o b t a i n e d with S t r a i n M L , except t h a t the s e d i m e n t was reduced to a c o n s t a n t a m o u n t of 2 m m a t 74 C a n d above. L a r g e r a m o u n t s of s e d i m e n t f o r m e d in skimmilk h e a t e d at 68 a n d 71 C t h a n at 74 C, even t h o u g h the a g g l u t i n i n titers were <1fi2. S t r a i n s M L , H P , a n d K f o r m e d 1 3 em o f sediment in autoclaved (10 l b / 1 5 rain) skim-
TABLE 5 Curd fornmtion in beakers with Strain ~ P in skimmilk receiving heat treatments up to 77 C (30 rain) Heat t r e a h n e n t of skimmilk
(30rain) ((') Unheated 59 62 65 68 71 74 77 "~ See Experimental
Distribution of p H ~n skimmilk in beaker
Time from setting to reading
Sediment
(hr:min)
(.re,m)
6 : 07 5 : 18 4:58 4:13 5:02 4:20 5:10 4 : 37 3 : 46 3 : 44 3:27 3 : 42 3:15 3:11 3 : 00 3 : 00
16 12 ]8 11 16 ]2 11 9 7 6 1 1 1 1 Trace 1
T"
M:'
]3~'
5.10 5.12 5.08 5.19 5.09 5.10 5.04 5.04 5.01 5.09 5.08 4.90 5.10 5.19 5.25 5.20
5.10 5.12 5.08 5.19 5.09 5.10 5.04 5.04 5.01 5.08 5.08 4.90 5.10 5.19 5.25 5.20
4.30 4.38 4.33 4.48 4.34 4.42 4.38 4.42 4.66 4.69 5.06 4.88 5.30 5.17 5.24 5.19
Agg]utinin ~iter of whey 1/13 1 / 9.8 1/ 5.5 1/ 3.3 ~1/
2
~1/
2
~1/
2
~1/ 2
Procedures. J. DATaY
SCIENOE
~OL.
49.
NO.
ii
1354
D.B.
EMMONS ET AL
milk. This sediment was lower in p H than the supernatant skimmilk and contained about twice as many bacteria. Pasteurization destroyed 50 75% of the agglutinins as measured by the agglutination test (Tables 4 and 5). Moreover, it is evident that the agglutinins are active in promoting defects in curd formation at vmT low levels. Mixed-strain. cull'urea'. Figure 7 illustrates the effect of mixing Strains t;!: and H P as starter on the rate of a d d development in the vat. When t t P formed only 25% of ttle starter, the rate of acid development was normal, and only small amounts of sediment formed. When suspensions of C~ and H P were mixed in tile agglutination test, the number of cells agglutinating was prol)ortional to the amomlt of Strain H I ) present in the mixture. Twenty commercial cultures were tested for ag'glutination and for sediment formation in raw sMmmilk in beakers. Two cultures showed no agglutination, but one of these produced 0.5 I mm of sediment; microscopic examination of slides fram autoclaved skimmilk showed about 50% of the cells of this culture in loose ehnnps. ;Pour cultures auto-agglutinated and produced sediment; three of these clumped extensively in autoel'wed skimmilk; the other showed little clumpino'. The 14 other cultures lind agg'lutinin tilers up to ] / 1 6 and yielded up to 24 mm of sediment of low p i t as compared to 13 mm by Strail, l I P in the same skimmilk; however, in none of these were all of tile cells ag'g'lutinated, indi~,ating' that only one or a few ~t' the strains in the culture agglutinated. ]r~ g'eneral, agglutination or auto-agglutination .1' bm,terial ceils was aeeompanied by f()rmation of ~ediment. Instauees were noted where eul-
6.0
5.5
pH
5.0
"e
4.5
TItlE - HR.
~I(~. 7. Effect of rate of acid development i~ skimmilk of mixing Strains HP m~d Ce in the following respective proportions: 0: 100(I) ; 2 5 : 7 5 ( H ) ; 50:50(III); and 100:0(IV). •~'.
])klR'l" S(*IEN('E V o r . 49, NO. 11
tures auto-agglutinated in skimmilk hut not in the broth of the agglutination test, or vice versa. Discussion
I t is apparent that tile typical antigenantibody agglutination reaetion is the chief cause of serious defects of shattered, mealy curd, large amounts of sediment, and slow acid development. In support of this conclusion, all single-strain cultures that ao'glutinated in the agglutination test resulted in a sediment containing' relatively high numbers of bacteria on the bottom of the vat. From observations made during this study, it is postulated that the following mechanisms are involved: Agglutinated bacteria in skimmilk constitute isolated areas of high cell concentrations and, hence, high rates of acid production. As a result, casein acidprecipitates on these clumps, h~rming fleeks of curd that contain relatively high concentrations or" baeteria and of easein. These dense flecks . f curd settle to the bottom of the vat, lorraine' the sediment and retarding acid development in the rest of the wit; stone remain suspended. forming areas of high casein cm,.entratiou and a nonlutiform eoagulum; this e o a ~ ' n l u m is very fragile and shatters during' handling. These flecks give a mealy body to the conked and washed curd. Strains of agglutinatina' baeterh differed markedly in the sewq'ity of det'e~ts produced in curd. These differences were not related to their a ta'glutinin tilers in whey. For example, Strain K produced w,ry sew,re defevts but had low ag'g'lntinin liters; others (C,, (:,) showed relatively high liters lint defeets were slight. Generally, agglutim, tion and settling wm'e more s~v,,r with strains growing' in hmo'er ehains; this would be logical, because the ,~'vlntination . f hmg chains would effectively bring together more cells tlum agg'lutinntion . f pairs. Also, Whitehead, J o n e s ; a n d } { o b e r t s o n ( 2 2 ) found that strains g'rowing in hmgvr ~.hains showed a o.reater tendency to settle in autodaw'd skimndlk. Auelair and Vassal (2) found that only about 50% of the cells of one mdture ~,f Strait~ ('.,,, were sensitive to agglutinins; thus, suseeptibility to agglutination by only a portion of cells may be a factor in strai, varial)ility. Retardation of acid development in the body of the skimmilk is brought about by reduction of cell numbers through displacement of agglutinated cells to the bottom of the container. Agglutinated cells in the sediment reduce the p H quickly to 4.4-4.3, the limiting p H for acid production by lactic streptococci. These o'bservations, together with tile observed elimination of retardation of aeid production in whole
C U R D FORIt~XTION I N C H E E S E
nfilk (24) and in skimmilk (21) by rapid rennet coagulation, indicate that the agghtinins, per se, do not inhibit, but rather may retard indirectly, acid t)roduction by lactic streptococci. Acid production and cell division by a g g h t i hated cells in large clumps suspended in the skinnnilk may be retarded by low p H if acid cam~ot diffuse away rapidly. Increased periods of agitation of skimmilk containing starter markedly increased the amount of sediment and decreased the rate of acid development with Strain HP. Apparently. agitation promoted a g h t in a t io n which, in turn, resulted in more or larger clumps of bacteria and casein and in more rapid settling. Agitation had previously been observed to be essential in the agghtination test, for ease in detection of agghtination (6). Sediment formed in skimmilk during' the growth of starter bacteria where the antio'cnantibody type of agglutination apparently was not involved. Traces of sediment formed with Strain H P in skimmilk from which the homologous antibodies had been removed by adsorption and centrifngation. Small amounts of sediment formed when Strain H P and some other strains were grown in skimmilk heated to destroy the agglutinins. Sediment was also observed once with agghtination-resistant straim C._., in pasteurized skimmilk containing 5.0 or 7.5 m.~ of added CaClo/liter. In the last two cases, relatively larger numbers of bacteria were present in the sediment, as evidenced by either a lower p H or by actual count. In heated milk, those strains showing sediment also showed some natural tendency to auto-a.gglutinate, although some strains tending to auto-agghtinate did not show traces of sediment. It is a wellrecognized characteristic of some streptococci to show granular growth in liquid media (23). Also, difficulties are often encountered in preparing stable cell suspensions of the lactic streptococeci (3, 6). Thus, auto-agghtination may sometimes he involved in sediment formation, as may the natural tendency of long chains to settle (22), although these aspects of the problem are not clearly defined. However, all instances of serious defects in curd formation in this laboratory were associated with the antigen-antibody type of aggluti~ation. It is uncertain how widespread in commercial practice are defects in Cottage cheese due to agghtinated cultures. They may have been the cause of the grainy and shattered curd in one plant described by Kosikowski (J3), in a survey of commercial Cottage cheese by the same author (12), and the cause of the sediment described by Lueas (15) and hy Sandine et el.
1365
(20). The spongy character of sediment (15) could be explained by the concentration of gasproducing bacteria in the sediment, as could vat~ of cheese in which only a small portion of the curd floats during' cooking. Lundstedt (16) effminated a problem of slow acid production h spring and fall by raising the pasteurization temperature of skinnnilk from 160 to 168 F for 24 see; he attributed the problem to bacterMdal substances secreted by the cow for the protection of calves. One of us has observed a sediment problem in a commercial operation, probably due to agghtinating bacteria, in which curd could not be produced over a 2-wk period. The observation that many commercial cultures contained one or more agghltihating strains indicates that the problem may be quite widespread but unrecognized; the severity of the defects produced would depend on whether the agghtintlting strain became dominant and whether it produced serious or lnild defects. Remedial measures for this problem are to select cultures for Cottage cheese that do not contain agglutinating strains. The present state of knowhdge indicates that agglutinins for susceptible strains are very widespread and are likely present in most or all milk supplies and that resistant strains are not likely to agglutinate in normal milk (7). Frequent renewal of commercial cultures or the use of frozen cultures would help by reducing the probability of dominance by agglutinating strains and of changes in the agghtination charaeteristics of single strains (2). Heat treatment of skimmilk to destroy the agghtinins offers one method of eliminating the problem, but the use of highly heated skim,nilk has not yet been generally accepted commercially; n<~ other practical method has yet been found to remove the agglutinins. References
(1) Auclah', J. 1960. Personal correspondence. (2) Auelair, J., and Vassal, Y. 1963. Occurrence etf Variants Sensitive to Agglutinins and to Laetoperoxidase in a LaeeutinResistant Strain of Streptococcus laetis. J. ])airy Research, 30:345. (3) Briggs, C. A. E., m~d Newland, L,. G. M. 195"~. The Serologienl Classification of Streptoeocc~s eren~oris. ,T. ])airy Research, 19: 160. (4") Elliker, P. R., Andersol~, A. W., and I-Iannesson, G. 1956. A~ Agar Culture Medimn for Lactic Acid Streptococci and Lactobacilli. J. ])airy Sei., 39:1611. (5) Emmons, D. B., Elliott, J. A., u~td Bcekett, D. C. 1963. Agglutination of Starter Bacteria, Sludge Formation, and Slow Acid J . ])kIl~k" SCIENCE ~YOL. 49. No. 11
1366
(6)
(7)
(8)
(9)
(10)
(111) (12) (1:~)
(14)
(15)
D.B.
EMMONS ET AL
Developnlent in Cottage Cheese Manufactm'e. J. Dairy Set., 46: 600. Emmons, D. B., Elliott, J. A., and Beckett, D. C. 1965. Sensitive Test for LacticStreptococcal Agglutinins. J. Dairy Sci., 48 : 1245. Emmons, D. B., Elliott, J. A., and Beckctt, D. C. 1966. Lactic-Streptococcal Agglutins in Milk and Blood. X V I I t h Intern. Dairy Congr., D: 499. Emmons, D. B., Price, W. V., and Swanson, A . M . 1957. The A-C Test (Acid-Coagulation). A New Method of Determinil~g the Time for Cutting Cottage Cheese Cm'd. Ext. Circ. 541, University of Wisconsin. Gillies, A. J. 1959. Inhibitory Factors in Cheese Milk. XVth Intern. Dairy Congr., 2 : 523. Hunter, G. J. E. 1946. A Simple Agar Medimn for the Growth of Lactic Streptococci: The :Role of Phosphate in the Medimn. J. ])airy Research, 14: 283. ]£eogh, B. P. 1958. Variations i~ Lactic Acid Production in Milk. Australian J. Dairy Technol., I3: 132. ];osikowski, F. V. ]959. Better Cottage Cheese. Milk Dcaier, 49(3):44. l':osikowski, F. V. 1963. Some Distributi(m P:~tterns of Cottage Cheese Particles and Conditions Contributing to Curd Shatteri~g. J. Dairy Sci., 46:391. Levowitz, D., m~d Weber, M. 195(;. An Effective "Singie Solution" Stain. J. Milk Food Technol., ]9:121. Lucas, P. S. 1962. What Causes n Spongy Formntio~t in C,ott:lg'e Cheese? Am. Milk Rev., 24(10):74.
J. DAIF~' SeI)~Ne~ V o r , 49, No. ]1
(16)
(17)
(28)
(19)
(20)
(2])
(22)
(23)
(24)
E. 1958. Starter Failures and Their Prevention. Milk Dealer, 47(10):47. Portmann, A., and Auclair, J. ]959. Relation entre ]es Laetenincs et Ies Agglutinines du Lait de Yache. Annal. Inst. Pasteur, 97:590. Portmann, A., Gate, Y., and Auclair, J. 1962. Influe~ce of Lactoperoxidase and Agglutinlns of Milk on the Activity of Thermophilic Starter Bacteria. XVIth Intern. Dairy Congr., B: 729. Randolph, H. E. 1962. Natural Intfibitors in Milk Affecting Lactic Acid Bacteria. Ph.D. thesis, elite State University. Sa~ldi~le, W. E., Anderson, A. W., Elliker, P. R., and Stein, R. W. 1963. Observations on Citrate Uti]~zatim~ by Mixed-Straln Starter Cultures and Protein Stability of Cheese Milk. J. Dairy Sci., 46: 619. Stadhouders, J. ]963. The Inhibitory Effect of L,ctenh, L3 on Acid Production :bl Milk by Streptococcus eremoris 803. Netherlmlds Milk [)airy J., 17:96. Whiteilead, H. ilk, Jones, P. A., and Robertson, P. S. 1958. The Influence of Carben Dioxide o , the Growth of Lactic Streptococci. J. Dairy l,~ese:lreh, 25:24. Wilsm), C. S., :lm] Miles. A. A. 1955. Top]ey and Wilson's Pri.(dp]es of Bacteriology and Immuniiy. 4Ih ed. Edwurd Arnold (Publ.) Ltd., ]~()n,lon. Wright, R. ('., ,11,1 Tr:lmer, J. 1957. The Influelwe of (!,'can, Risi~g upon the Activity of ~qcterbl. in IL,:II Tr('ated Milk. J. Dairy Research, 24 : 174.
Lundstedt,
Shattered~ mealy curd, sediment on the bottom of the vat, an(t slow acid production were assoeiated with agglutinating strains of Cottage cheese cultures. Agglutinated bacteria formed centers of high acid production and resulted in (lense flecks of addprecipitated casein. These flecks either settled as a sediment or were trapped during coagulation and caused a fragile, mealy cm'd. The sediment was low in p H and high in protein and bacteria. Acid production in the supernatant skimmilk was retarded through displacement of agglutinated bacteria to the bottom of the vat, indicating that agglutinating antibodies in skimmilk are not inhibitory, per se. Prolonged agitation of skimmilk and starter promoted agglutination and, after agitation ceased, resulted in more sediment and reduced rates of aeid t)roduction. With agglutinating cultures, the p H of the A-C test sample, receiving less agitation, reduced more quickly than skhnmilk in the vat, thereby nullifying the test as a direct indicator of the time to eat the curd. tteating skimmilk (71 C/30 rain) or removing agglutinins by adsorption on heat-killed cells eliminated the defects. Pasteurization (72 C/16 see) reduced the agglutinating activity of skimmilk by 50-75%. Agglutinating strains differed markedly in the severity of defects produced; two strains settled so extensively that the ski,nmilk (lid not coagulate; whereas, defeets with some strains were evident only as small amounts of sediment. Auto-agglutination of certain strains also appeared to cause small amounts of sediment. Some commercial cultures contained agglutinating strains and produced sediment. Selection of agglutination-resistant strains for Cottage cheese cultures is reeommm~ded. A defect of sediment on the bottom of the vat has been observed during curd formation in Cottage cheese manufacture. Lucas (15) de-
scribed a. spongy sediment, 1/8 1/2 in. deep, and suggested it was due to the use of overheated nonfat dry milk. San(line et al. (20) reported that sediment formed occasionally when mixed-strain starters containing Streptococcw~ diacetilactis were used. This defect occurred more frequently when more than four mM of caleimn were added per liter of milk and when the milk was aged three to seven days. The defect was not observed when aetive, lowgas-producing, mixed-strain starters were used. They suggested that excess calcium ions~ either added or liberated by citrate fermentation, destabilized the casein which subsequently settled. Initial trials in our laboratory using two single-strain cultures ( I I P and E~) as starter resulted in sediment of very low p H on the bottom of the vat. Assodated with the sediment were undesirable shattered, mealy curd, a eoagulum that shattered excessively during cutting and cooking, and slow acid development. The low p H of the sediment indicated the presence of relatively larger numbers of starter bacteria. I n this conneetion, Wright and Tramer (24) observed that "slow (agglutinationsusceptible) starters, which give a better aetivity in separated and homogenized milk (than in whole milk), tend to form a deposit on the bottom of the test-tube after 6 hr at 30 C; whereas, fast (agglutlnation-resistant) starters do not, when grown in either kind of milk. Examination of this deposit reveals enormous numbers of starter organisms, presumably due to agglutination." The association of deposit formation in raw or pasteurized skimmilk with agglutination-suseeptible strains of starter was confirmed by Auelair (1) and Stadhouders (21). Gillies (9), Keogh (11), Portmann, Gate, and Auelair (18), Randolph (19), and Stadhouders (21) showed that acid production by agglutinating strains is retarded in unheated skimmilk. This paper 1)resents evidence linking the agglutination phenomenon to defeets that occurred during the manufacture of Cottage cheese with certain strains of starter ba<'teria (5). Experimental Procedures
Cultures. Sino'le strains were of New Zealand Received for publication June 30, 1966. and Australian origin. Commereial cultures • Contxibution no. 43 from tim Food Research Institute, Research Branch, (!amtda Del)art.me~lt of were donated by firms in the United States. Cultures were reactivated frequently i~rom Agriculture, Ottawa, Cmmda. 1357
1358
D.B.
EMMONS ET AL
freeze-dried or frozen (liquid n i t r o g e n ) stoeli: a n d p r o p a g a t e d usually less t h a n 2 wk. Cult u r e s were inoculated a t the r a t e of 1 % in autoclaved r e c o n s t i t u t e d n o n f a t d r y milk (10 l b / 15 r a i n ; 1 0 % ) a n d i n c u b a t e d 16 h r a t 22 C. Agglutinin titers. I n the a g g l u t i n a t i o n test (6)~ e i t h e r one-hal:f- or t h r e e - f o u r t h s - d i l u t i o n s of whey were used. N e g a t i v e t i t e r s ( < 1/2) represented no a g g l u t i n a t i o n in a n equal nfixture of cell s u s p e n s i o n a n d u n d i l u t e d whey. Reported values are log averages of duplicate determinations. Bacterial estimates. D u p l i c a t e p o u r e d plates of either H u n t e r ' s (10) or E l l i k e r ' s (4) media -were counted following i~mubation f o r 72 h r a t 22 C. ChaiJ~ lengths. Slides of cultures were prep a r e d from autoclaved (10 l b / 1 5 rain) skimnfilk a f t e r i n o c u l a t i o n w i t h 5ff{~ of culture a n d incuhation f o r 3 h r at 32 C. The Levowitz a n d W e b e r (14) s t a i n was used. E a c h s t r a i n was classified as diploeoeci, s h o r t ( 4 - 8 cells), medium ( 9 - 16) cells) or long ( > 1 6 cells) chains. (.',urd formation in ,vats. Coagulum was :formed a t 32 C f r o m 182 kg of p a s t e u r i z e d (72 C / 1 6 se(') skimmilk in 228- or 455-kg v a t s ; 5°//, s t a r t e r was added. Unless otherwise stated, r e n n e t was added ii h r a f t e r the s t a r t e r , at the r'tte o1'1 m l / 4 5 5 kg of s k i m m i l k ; the skimmilk was stirred mechanically f o r 15 lni~l a f t e r a d d i n g b o t h s t a r t e r a n d rennet. S a m p l e s f o r p l l a n d bacterial estimates were t a k e n a) 1 em u n d e r the sm'faee ( T ) , b) o n e - t h i r d of the distance from the b o t t o m of the v a t ( M ) , a n d c) tq'om a m i x t u r e of the b o t t o m 25 m m ( B ) . The bottom samph~ ( B ) was o b t a i n e d from 250 nil beal
over a p e r i o d of 130 rain, a n d washed with w a t e r at 16 a n d 4 C. Curd formation in beakers. C o a g u l m n was fomned at 32 C w i t h 5ff~ of s t a r t e r added to 550 ml of s k i m m i l k in 600dnl Berzelius b e a k e r s ; the d e p t h of s k i m m i l k was 13 era. The skin> milk was s t i r r e d f o r 15 rain a f t e r addb~g starter, a n d a g a i n ] h r later, with a mechanical s t i r r e r at a c o n s t a n t r a t e estimated to be the same as
that in the vats. Samples for pH were taken a) 1 em u n d e r the sm'face (T), b) 4 em f r o m the b o t t o m ( M ) , a n d c) iron1 a m i x t u r e of the b o t t o m 1 em of the b e a k e r ( B ) . F o r the bott o m sample ( B ) , the top was s i p h o n e d away u n t i l a d e p t h of 1 em remained. Results
Deseriptio.u o/' defects i~ curd for.marion. Defects r e s u l t i n g f r o m the a g g l u t i n a t i o n p h e n o m enon are i l l u s t r a t e d with S t r a i n H P . S t r a i n C~ was used f o r c o m p a r i s o n ; it did not agg l u t i n a t e in n o r m a l milk a n d p r o d u c e d curd free of these defects. S e d i m e n t always a p l , e a r c d on the b o t t o m of t h e v a t with S t r a i n I t P . The a m o u n t of sedim e n t increased as the p l [ el! the rest o£ the v a t decreased, first a p p e a r i n g between p I [ 6.2 to 5.6 and 3'eaehiljg the m a x i m u m j u s t before coagulation a t p l l 5.2. Usually the bottom 11.5 1 em of the sediment was a tan or cream color, whereas m,y a d d i t i . n a l sediment above it was white. The. sediment contained relatively large n u m b e r s of bacteria (Table 1 ) ; microscopic e x a m i n a t i o n sh~wed large masses of bacterial cells. The sediment also contained a b o u t twice as much nitrogen, i n d i c a t i n g t h a t casein was remow~d from. the skimmilk a n d c o n c e n t r a t e d in the sediment. This sediment was dense, tough, a n d difficult to cut a n d collected on the c u t t i n g k,,iw~s ( F i g u r e 1). D u r ing curd :formati,m iu a vat:, p l l values were relatively u n i f o r m ahow, the sediment. The p I l oJ' the s e d i m e n t was always h,w, apl,roach-
I AI L E 1 l)istribuiio~J of nitrogen, pit, and bacteria in pasteurized sldmmilk in a vat during Got.rage cheese curd formation using Strain H P ;ts starter
Point
of s'unpllng
Time of sampling
Nitrogen content of skimmi]k or curd 1
(hr :'miO Skimmi]k" and starter ] em below surface 1/3 distance from bottom Mixture of sediment (25 mm in depth)
2
Mean
pH
(rag N/g)
Standard plate count
(× 10~)
0 4:20 4:20
5.16 4.93 4.89
5.18 4.94 4.93
5.17 4.94 4.91
6.40 5.'25 5.25
4:20
10.24
8.57
9.40
4.49
1.6 1.9 58
" Agglutinin titers against l i p cells were 1,/14.2 and 1,/3.6 for the r'tw and pasteurized skbnmilks, respectively, J. DAII~Y SCIENCE VoL. 49, NO. 11
C U R D F O R ~ [ A T [ O N I N CI-~EESE
1359
tLr~m~zh thtid xi
I,'I(;. Ii ~,,!hmml th:ll c~dk.'l,'d ~)1~knhc~ d u r i ~ vIli~'ill~ ill: ('1}~;1~"' ViH',"~C,'[ll'd (~4~]'Hill IIl'). h ~ t h a t ~V the limiti~La pH bw lactic avi;l d c v c l o p l m ' . t ( F i g u r e 2): Just beli, le c,aguhltimL (h'ck.~ o£ curd were obtained , b y drawii~g- a screen thrm~g'h the skiullrliik (Fie'rive 3). Fi~:uve 4 shows a ~ , ~ of bacterial cells f r o m such :t fleck a f t e r dissolviu~' the c-t~ein in dih~te alMdi. The amouut of ag'it:ation of s k i m m i l k a f t e r a d d i n g s t a r t e r markedly affected the r a t e of acid deve]opw_ent (I~igure 5). A p H of 4.7 was reached lit 5 h r when the skimmiiI< was a g i t a t e d for only 10 rain a f t e r a d d i u g the s t a r t e r a n d rennet. P r o l o n g e d a ~ i t a t i o n delayed acid p r o d u c t i o n a~d resulted in more sedime~tt. Contiuuous a g i t a t h m f o r 3 b r resulted
~
m faihu'c ,I' lhc I,tl i - dl'~q~ beh~.~ .3~3 m the ttlq>el' liquid i.,rti,m ,,f the vat amI in a ,~edi.,e~a ,.o.,pvi~in~ .m.-thivd <~t~tlw vt~t c.nt~.ut~. ~,imilar agitati(m tveatment~ with ~trah~ tL. did ~,)t affect rate ~)~ acid (h'veh)l)ment , ran' did any sediment [))rut. At cutting, the em'd f o r m e d with S t r a i n l i p s~plit irvegutarly ( F i g u r e 6, A) a.~ c o m p a r e d to the clean split o[ curd £m'n~ed with S t r a i n (J~ ( P l g u r e 6, 1%). I t was ~requeut/y possible to see dense white flecks on the surface or' the cut curd p a r t M e s . Also, the sur£ace of the v a t of et~rd showed small raised b u m p s ; a % w millimeters of whey usually covered the surface. The dense white flecks on cut curd a n d
t~ 30 - -
4 HR
~
0 6.0
5.5
5 HR
5.0
8 HR
4.5
,-
4.0
~IO. 2. I)istribu~ion of pII at various times dur]ll~' elll'd forlil}t'Liot£ ill ~1 Vf[~ (StI'Hill H - P ) .
Fro. 4. Photomicrograph of mass of agglutinated cells in fleck that had bee~L placed on ~t slide in a small drop of N/10 NaOEI. J. DAIRY S(JIEXC!E VOL. 49, No
~I
1360
D.B.
EMMONS
5.7
pHso 4.5
TIME
- HR.
]?IG. 5. Effect of agitation of skimnfilk 11~ vat on rate of acid development in upper portion of skimmilk in vat, usiug Strain H P as starter. Treatments were: ( I ) agitated 10 rain, starter and rennet added together; ( ] I ) agitated 15 rain, rennet added 1 hr after starter and agitated 15 nfin; ( I I I ) agitated conthmouslv for 75 rain, adding rennet 60 nlin after starter; ( I V ) agitated continuously for 3 hi', addhig re,met 60 nlin after starter. the b u m p s on the surface were p r o b a b l y caused by the flecks observed on the screen j u s t p r i o r to coagulation. The curd formed with S t r a i n H P s h a t t e r e d excessively during; cutti~g a n d cooking, as evidenced by the Cornell g r i t test (13), a n d was
E T AL
very mealy in body a n d t e x t u r e (Table 2). 2fealiness was not due solely to the s h a t t e r e d c u r d p a r t i c l e s ; large i n t a c t c u r d particles, obtained with the 1A-in. sieve in the wash water, were j u d g e d to be as mealy as the bulked curd. Reduction of a g i t a t i o n to a m i n i m u m reduced the s e t t i n g time to a n acceptable value, b u t did not reduce the s h a t t e r i n g a n d mealiness in the finished curd. I n 20 cheese-making trials with S t r a i n H P , the em'd was always very. shattered a n d very mealy. A d s o r p t i o n o]' antibodies from..~t,:i:t~,,milk on heat-killed cells. I t was p o s t u l a t e d t h a t agg l u t i n a t i o n of s t a r t e r b a c t e r i a b y antibodies o c c u r r i n g n a t u r a l l y in the skimmilk caused the above defects. A n t i b o d i e s in skimmi]k were adsorbed on heat-killed H P cells a n d removed b y e e n t r i f u g a t i o n as follows: Each of two 450-ml q u a n t i t i e s of u n h e a t e d skinmdlk was t r e a t e d three times with saline-washed heatkilh,d (63 C / 3 0 rain) cells from 15(1 nfl of incubated ('2'2 C/16 h r ) , n e u t r a l i z e d ( p H 6.5) t t u n t e r ' s b r o t h . Then H P s t a r t e r was added to 550-ml q u a n t i t i e s of t r e a t e d a n d u n t r e a t e d skimmilk in beakers. The a d s o r p t i o n t r e a t m e n t increased the r a t e of acid dew~lopment a n d reduced the a m o u n t
A B VI~. 6. A. ]~agged split in curd formed with Strain t I P at time of cutting. Bumps may be seen on surface of vat of curd. B. Clean split in curd formed with Strain C._, at time of cutti~g. J . DAIRY SCIENCE XC:OL. 49, NO. 11 A
C U E D FOI%MATION ]IN C H E E S E
1361
TABLE 2 Quality and particle size distribution of curd made with st,'aius resistant (C~) and susceptible ( H P ) to agglutination Curd particle size distribution '~ Time
Rep.
(In.)
t~
cutting
> 1/2
1/2 7/4
( hr : rain)
1/4-1/8
1/8-1/16
Curd quality
(%)
Strain C.~--agitatcd for 15 rain; rennet added 1 hr after starter, and agitated 15 rain I 4 : 35 0.8 73.9 23.6 1.7 Cleau cut ; satisfactory curd I[ 4 : 42 0.5 72.3 24.6 2.5 Clean cut ; satisfactory curd Strain H P - - a g i t a t e d for 10 rain only; and starter and rennet added together I 4 : 38 2.1 57.1 33.7 7.1 Ragged cut ; shattered and very mealy cm'd lI 4 : 40 2.1 52.3 36.5 9.1 Ragged cut ; shattered and very mealy curd Strain H P - - a g i t a t e d for 15 rain; rennet added 1 hr after starter and agitated 15 rain l 7:09 1.2 54.6 35.0 9.2 g~gged c a t ; shattered and very mealy curd II 7 : ll0 0.9 54.6 32.9 11.6 Ragged cut ; shattered and very mealy curd Kosikowski's Corne]l Grit Test (13). I n a n o t h e r trial, the a d d i t i o n of heat-killed cells, w i t h o u t removal, a c c e n t u a t e d the defects of settling a n d slow acid development. Si,l~gle-.~train c u l t u r e s . W i d e differences were observed a m o n g 20 s t r a i n s in the thickness of the sediment, r a t e of acid development, distribution of p H values a n d of b a c t e r i a in the skimmilk, a g g l u t i n i n titers of whey f r o m the skimnfilk before a n d a f t e r p a s t e u r i z a t i o n , a n d l e n g t h of b a c t e r i a l chains ( T a b l e 4). I n general, the observations s u p p o r t the h y p o t h e s i s t h a t agglut i n a t i o n of s t a r t e r b a c t e r i a by antibodies caused the observed defects. F i v e strains, C,, C~, C~, C~, a n d C2, p r o d u c e d no a p p r e c i a b l e s e d i m e n t a n d were n e g a t i v e f o r a g g l u t i n a t i o n in whey f r o m both r a w a n d p a s t e u r i z e d s k i m m i l k ; p H values a n d bacterial d i s t r i b u t i o n were u n i f o r m t h r o u g h o u t the skimmilk in the vat. The 15 o t h e r strains, except C~o which autoa g g l u t i n a t e d , were positive f o r a g g l u t i n a t i o n , b u t differed widely in the seriousness of de-
feets produced. Cells of S t r a i n s K a n d ML~ settled so extensively t h a t acid p r o d u c t i o n ill the v a t p r a c t i c a l l y s t o p p e d at p H 6.0 a n d 5.5, respectively. Some a g g l u t i n a t i n g strains, e.g., C~ a n d C,, yielded only small a m o u n t s of sedi m e n t a n d p r o d u c e d acid quickly. T h e r e was no absolute r e l a t i o n s h i p between the a g g l u t i n i n titers of the whey a n d the seriousness of bacterial settling in s k i m m i l k ; f o r example, S t r a i n K yielded the lowest titers b u t settled the most extensively. C h a i n l e n g t h m a y h a v e some b e a r ing on this, because a g g l u t i n a t i n g s t r a i n s growing in longer chains t e n d e d to show more serious defects t h a n those g r o w i n g as p a i r s or s h o r t chains. S t r a i n C,o showed a u t o - a g g l u t i n a t i o n in the a g g l u t i n a t i o n test. I n the b e a k e r test, this s t r a i n p r o d u c e d a b o u t h a l f as much sediment in skimmilk h e a t e d (80 C / 3 0 rain) to destroy the a g g l u t i n i n s as in p a s t e u r i z e d s k i m m i l k ; both sediments c o n t a i n e d h i g h e r n u m b e r s o f h a c t e r i a
TABLE 3 :Formation of Cottage cheese coagulmn with Strain H P iu unheated skimmilk before and after adsorption with heat-killed cells Distribution of pH in skimmilk in beaker Time of observation
T"
M '~
B ~'
( hr : ,mi~ )
Sediment ('##~m )
Untreated skimmilk with an agglutinin titer of 1/10.5 in rennet whey 4 : 25 5.10 5.10 4.42 Treated skimmilk with an agglutinln titer of ~ 1/2 in rennet whey 3 : 30 5.10 5.10 5.09
12 Trace
:' See Experimental Procedures. ,~. ~)AIBY SCIENCE 3~OL. 49. N(). 11
TABLE 4 Observations durh~g Cottage cheese curd formation in vats for 2(I strains of S. eremoris and S. laetis
Strain
Thickness of sediment ~
p H at end of readings ( ~ )
(m m)
Time to end of readings
Ratio of bacterial c'stimateg~ ( 2B ) ~
A p H in vat ~ / . T d- M \ ~ B ')
)
Agglutinh~ titers Raw
Past.
Chain length
(hr: mbt)
~. cre,tttori8 t( ML~ HP ML~ I~tt /~ Zs TR IL~ E~ I)Rz US. C~ C~ Cz~ CT Cla
0.79 0.90 0.89 0.80 0.57 0.57 0.50 0.52 0.60 0.48 0.48 0.32
1/ 4.7
0.5t)
1 / 8.4
10 25 °5 25 16 10 ]6 16 13 13 6 6 I0 Trace None Trace None
6.o3 4.74 5.t10 5.51 4.96 5.21~ 5.02 4.81 4.89 4.93 4.91 4.94 4.74 4.64 4.76 4.68 4.78
6 : 45 8 : 25 7 : 15 7 : 35 5 : 00 5 : 00 6 : 57 5 : 23 5 : 2? 4 : 30 4 : 43 6 : 02 4 : 25 4 : 09 4 : 30 4 : 36 3 : 55
227 51 43 38 25 15 ]3 12 12 9.0 6.5 5.3 3.7 1.0 1.0 6,9 0.6
0.00 -0.01 0.0~ 0.(~2
6 !N-one 6
5.~14 4.78 4.78
4:57 4 : 32 4 : 15
5.9 ] .0 1.6
u.3~; 1).1)1 (I.14
S. laetis C~, C,_, C~o
a Observations nlade when T and .X[ were at ca. p K 5.2 unless p}[ at end of readings was higher. T, M, and B. See Experimental Proeedures.
1/11.2 1/13
<1/2 1/4.7 1/3.1
1/ 8.4
1/3.6
1/ 9.7
1/3.6 1/3.6
J/13 1/ 8.4 1 / 9.7 1/ 8.4
~/ 4.7
1/2.7 1/2.7 1/2.7
<1/2
1/13
1/6.3
1/ 8.4
1/3.6 1/3.6
<1/ 2 <~/ 2
<1/2 <1/2
<1/2 <1/2
1/ 3.1 <1/ 2 +control
<1/2 <1/2 +control
Long Medium Long Long Short Short Medium Short Long Medium Short Short Dip]oeocei Diploeoeei Medlmn Medium Medimn Dip]oeoeei Diploeoeei Diploeoeei
CURD FORMATION IN CIIEESE t h a n the s u p e r n a t a n t . Microscopic e x a m i n a t i o n showed n m n e r o u s small clumps of b a c t e r i a in both t y p e s of skimmilk. L i m i t e d n m n b e r s of cheese-making trials also indicated wide v a r i a t i o n s in the seriousness of defects of s h a t t e r e d a n d mealy curd. S t r a i n s K_ a n d ML~ did not form a eoagulum, a n d the defects p r o d u c e d with S t r a i n H P are described above. S t r a i n s DR~ a n d E~ p r o d u c e d s h a t t e r e d a n d mealy curd, but it was not as mealy as t h a t with S t r a i n H P . S t r a i n s C~, C,,, a n d C., p r o d u c e d c a r d w i t h o u t serious defects. The usefulness of the A-C test was m a r k e d l y affected w h e n a g g l u t i n a t i n g eultm'es were used as s t a r t e r s ; in such instances the p I I in the A - C test b e a k e r was lower t h a n in the rest of the vat, t h e r e b y n u l l i f y i n g the test as a direct i n d i c a t o r of the time to eut the curd. I n the recommended p r o c e d u r e (8), the s a m p l e f o r the k - C test is t a k e n j u s t before a d d i n g r e n n e t to the rest of the skimmilk, a f t e r which the latt e r receives more agitation. Thus, the p H of the A-C test sample, reeeiving tess agitation, decreased more r a p i d l y t h a n t h a t in the vat. E])'ect o]" calcium. I n view of the o b s e r v a t i o n s of S a n d i n e et ah (20), CaC1, was added t4) f o u r lots o f p a s t e u r i z e d skimmilk a t the r a t e of 0, 2.5, 5.0, a n d 7.5 m~E/liter. I n the first lot of skimmilk, traces of s e d i m e n t f o r m e d w i t h S t r a i n C,~ in beakers w h e n 5.0 or more m5I CaCI~/ liter were added to skimmilk at three, four, eight, a n d nine days of age, w h e t h e r r e n n e t was added or n o t ; in skimmilk at t h r e e a n d eight days of age, c o n t a i n i n g 7.5 n l ~ CaCl~/liter, 4 - 8 mm of sediment of low p H formed. However, CaCI~
1363
added to the same lot of skinnnilk h a d no effect on the a m o u n t of sediment or ttle r a t e of acid p r o d u c t i o n when S t r a i n H P was used as s t a r t e r . I n the three o t h e r lots of s k i m m i l k o b t a i n e d at different times, no s e d i m e n t f o r m e d with S t r a i n C,_, in skinnuilk c o n t a i n i n g the abo~e levels of CaCI:~ n o r did the caleimn a d d i t i o n s have a n y effect on the a m o u n t of s e d i m e n t f o r m e d b y S t r a i n H P . I n view of the low p H of the sedi m e n t f o r m e d with S t r a i n C~ a n d the lack of effect of the added CaCI., with S t r a i n H P , it would a p p e a r t h a t the a d d e d CaCI. m a y have destabilized the eells of S t r a i n C2, p r o m o t i n g a u t o - a g g l u t i n a t i o n . H o w e v e r , a d d i t i o n s of u p to 50 m~I of c a l c i u m / l i t e r did n o t p r o m o t e a u t o - a g g l u t i n a t i o n in s u s p e n s i o n of cells of s t r a i n s C.., t i P , a n d K in E l l i k e r ' s (4) broth. Heat treatmeut o.f skimmilk. H e a t t r e a t m e n t of s k i m m i l k affected r a t e of acid p r o d u c t i o n , a m o u n t of sediment, d i s t r i b u t i o n of p i t , a n d the a g g l u t i n i n t i t e r of the whey with s t r a i n H P ( T a b l e 5). H e a t i n g at 68 C f o r 30 n,in redueed the a g g l u t i n i n t i t e r to < ½ , b u t a p preciable sediment formed. I l e a t i n g a t 71 C a n d above redueed s e d i m e n t to 1 ram. The r a t e of a d d d e v e l o p m e n t increased m a r k e d l y in skimmilk receiving the h i g h e r h e a t t r e a t m e n t s . S i m i l a r results were o b t a i n e d with S t r a i n M L , except t h a t the s e d i m e n t was reduced to a c o n s t a n t a m o u n t of 2 m m a t 74 C a n d above. L a r g e r a m o u n t s of s e d i m e n t f o r m e d in skimmilk h e a t e d at 68 a n d 71 C t h a n at 74 C, even t h o u g h the a g g l u t i n i n titers were <1fi2. S t r a i n s M L , H P , a n d K f o r m e d 1 3 em o f sediment in autoclaved (10 l b / 1 5 rain) skim-
TABLE 5 Curd fornmtion in beakers with Strain ~ P in skimmilk receiving heat treatments up to 77 C (30 rain) Heat t r e a h n e n t of skimmilk
(30rain) ((') Unheated 59 62 65 68 71 74 77 "~ See Experimental
Distribution of p H ~n skimmilk in beaker
Time from setting to reading
Sediment
(hr:min)
(.re,m)
6 : 07 5 : 18 4:58 4:13 5:02 4:20 5:10 4 : 37 3 : 46 3 : 44 3:27 3 : 42 3:15 3:11 3 : 00 3 : 00
16 12 ]8 11 16 ]2 11 9 7 6 1 1 1 1 Trace 1
T"
M:'
]3~'
5.10 5.12 5.08 5.19 5.09 5.10 5.04 5.04 5.01 5.09 5.08 4.90 5.10 5.19 5.25 5.20
5.10 5.12 5.08 5.19 5.09 5.10 5.04 5.04 5.01 5.08 5.08 4.90 5.10 5.19 5.25 5.20
4.30 4.38 4.33 4.48 4.34 4.42 4.38 4.42 4.66 4.69 5.06 4.88 5.30 5.17 5.24 5.19
Agg]utinin ~iter of whey 1/13 1 / 9.8 1/ 5.5 1/ 3.3 ~1/
2
~1/
2
~1/
2
~1/ 2
Procedures. J. DATaY
SCIENOE
~OL.
49.
NO.
ii
1354
D.B.
EMMONS ET AL
milk. This sediment was lower in p H than the supernatant skimmilk and contained about twice as many bacteria. Pasteurization destroyed 50 75% of the agglutinins as measured by the agglutination test (Tables 4 and 5). Moreover, it is evident that the agglutinins are active in promoting defects in curd formation at vmT low levels. Mixed-strain. cull'urea'. Figure 7 illustrates the effect of mixing Strains t;!: and H P as starter on the rate of a d d development in the vat. When t t P formed only 25% of ttle starter, the rate of acid development was normal, and only small amounts of sediment formed. When suspensions of C~ and H P were mixed in tile agglutination test, the number of cells agglutinating was prol)ortional to the amomlt of Strain H I ) present in the mixture. Twenty commercial cultures were tested for ag'glutination and for sediment formation in raw sMmmilk in beakers. Two cultures showed no agglutination, but one of these produced 0.5 I mm of sediment; microscopic examination of slides fram autoclaved skimmilk showed about 50% of the cells of this culture in loose ehnnps. ;Pour cultures auto-agglutinated and produced sediment; three of these clumped extensively in autoel'wed skimmilk; the other showed little clumpino'. The 14 other cultures lind agg'lutinin tilers up to ] / 1 6 and yielded up to 24 mm of sediment of low p i t as compared to 13 mm by Strail, l I P in the same skimmilk; however, in none of these were all of tile cells ag'g'lutinated, indi~,ating' that only one or a few ~t' the strains in the culture agglutinated. ]r~ g'eneral, agglutination or auto-agglutination .1' bm,terial ceils was aeeompanied by f()rmation of ~ediment. Instauees were noted where eul-
6.0
5.5
pH
5.0
"e
4.5
TItlE - HR.
~I(~. 7. Effect of rate of acid development i~ skimmilk of mixing Strains HP m~d Ce in the following respective proportions: 0: 100(I) ; 2 5 : 7 5 ( H ) ; 50:50(III); and 100:0(IV). •~'.
])klR'l" S(*IEN('E V o r . 49, NO. 11
tures auto-agglutinated in skimmilk hut not in the broth of the agglutination test, or vice versa. Discussion
I t is apparent that tile typical antigenantibody agglutination reaetion is the chief cause of serious defects of shattered, mealy curd, large amounts of sediment, and slow acid development. In support of this conclusion, all single-strain cultures that ao'glutinated in the agglutination test resulted in a sediment containing' relatively high numbers of bacteria on the bottom of the vat. From observations made during this study, it is postulated that the following mechanisms are involved: Agglutinated bacteria in skimmilk constitute isolated areas of high cell concentrations and, hence, high rates of acid production. As a result, casein acidprecipitates on these clumps, h~rming fleeks of curd that contain relatively high concentrations or" baeteria and of easein. These dense flecks . f curd settle to the bottom of the vat, lorraine' the sediment and retarding acid development in the rest of the wit; stone remain suspended. forming areas of high casein cm,.entratiou and a nonlutiform eoagulum; this e o a ~ ' n l u m is very fragile and shatters during' handling. These flecks give a mealy body to the conked and washed curd. Strains of agglutinatina' baeterh differed markedly in the sewq'ity of det'e~ts produced in curd. These differences were not related to their a ta'glutinin tilers in whey. For example, Strain K produced w,ry sew,re defevts but had low ag'g'lntinin liters; others (C,, (:,) showed relatively high liters lint defeets were slight. Generally, agglutim, tion and settling wm'e more s~v,,r with strains growing' in hmo'er ehains; this would be logical, because the ,~'vlntination . f hmg chains would effectively bring together more cells tlum agg'lutinntion . f pairs. Also, Whitehead, J o n e s ; a n d } { o b e r t s o n ( 2 2 ) found that strains g'rowing in hmgvr ~.hains showed a o.reater tendency to settle in autodaw'd skimndlk. Auelair and Vassal (2) found that only about 50% of the cells of one mdture ~,f Strait~ ('.,,, were sensitive to agglutinins; thus, suseeptibility to agglutination by only a portion of cells may be a factor in strai, varial)ility. Retardation of acid development in the body of the skimmilk is brought about by reduction of cell numbers through displacement of agglutinated cells to the bottom of the container. Agglutinated cells in the sediment reduce the p H quickly to 4.4-4.3, the limiting p H for acid production by lactic streptococci. These o'bservations, together with tile observed elimination of retardation of aeid production in whole
C U R D FORIt~XTION I N C H E E S E
nfilk (24) and in skimmilk (21) by rapid rennet coagulation, indicate that the agghtinins, per se, do not inhibit, but rather may retard indirectly, acid t)roduction by lactic streptococci. Acid production and cell division by a g g h t i hated cells in large clumps suspended in the skinnnilk may be retarded by low p H if acid cam~ot diffuse away rapidly. Increased periods of agitation of skimmilk containing starter markedly increased the amount of sediment and decreased the rate of acid development with Strain HP. Apparently. agitation promoted a g h t in a t io n which, in turn, resulted in more or larger clumps of bacteria and casein and in more rapid settling. Agitation had previously been observed to be essential in the agghtination test, for ease in detection of agghtination (6). Sediment formed in skimmilk during' the growth of starter bacteria where the antio'cnantibody type of agglutination apparently was not involved. Traces of sediment formed with Strain H P in skimmilk from which the homologous antibodies had been removed by adsorption and centrifngation. Small amounts of sediment formed when Strain H P and some other strains were grown in skimmilk heated to destroy the agglutinins. Sediment was also observed once with agghtination-resistant straim C._., in pasteurized skimmilk containing 5.0 or 7.5 m.~ of added CaClo/liter. In the last two cases, relatively larger numbers of bacteria were present in the sediment, as evidenced by either a lower p H or by actual count. In heated milk, those strains showing sediment also showed some natural tendency to auto-a.gglutinate, although some strains tending to auto-agghtinate did not show traces of sediment. It is a wellrecognized characteristic of some streptococci to show granular growth in liquid media (23). Also, difficulties are often encountered in preparing stable cell suspensions of the lactic streptococeci (3, 6). Thus, auto-agghtination may sometimes he involved in sediment formation, as may the natural tendency of long chains to settle (22), although these aspects of the problem are not clearly defined. However, all instances of serious defects in curd formation in this laboratory were associated with the antigen-antibody type of aggluti~ation. It is uncertain how widespread in commercial practice are defects in Cottage cheese due to agghtinated cultures. They may have been the cause of the grainy and shattered curd in one plant described by Kosikowski (J3), in a survey of commercial Cottage cheese by the same author (12), and the cause of the sediment described by Lueas (15) and hy Sandine et el.
1365
(20). The spongy character of sediment (15) could be explained by the concentration of gasproducing bacteria in the sediment, as could vat~ of cheese in which only a small portion of the curd floats during' cooking. Lundstedt (16) effminated a problem of slow acid production h spring and fall by raising the pasteurization temperature of skinnnilk from 160 to 168 F for 24 see; he attributed the problem to bacterMdal substances secreted by the cow for the protection of calves. One of us has observed a sediment problem in a commercial operation, probably due to agghtinating bacteria, in which curd could not be produced over a 2-wk period. The observation that many commercial cultures contained one or more agghltihating strains indicates that the problem may be quite widespread but unrecognized; the severity of the defects produced would depend on whether the agghtintlting strain became dominant and whether it produced serious or lnild defects. Remedial measures for this problem are to select cultures for Cottage cheese that do not contain agglutinating strains. The present state of knowhdge indicates that agglutinins for susceptible strains are very widespread and are likely present in most or all milk supplies and that resistant strains are not likely to agglutinate in normal milk (7). Frequent renewal of commercial cultures or the use of frozen cultures would help by reducing the probability of dominance by agglutinating strains and of changes in the agghtination charaeteristics of single strains (2). Heat treatment of skimmilk to destroy the agghtinins offers one method of eliminating the problem, but the use of highly heated skim,nilk has not yet been generally accepted commercially; n<~ other practical method has yet been found to remove the agglutinins. References
(1) Auclah', J. 1960. Personal correspondence. (2) Auelair, J., and Vassal, Y. 1963. Occurrence etf Variants Sensitive to Agglutinins and to Laetoperoxidase in a LaeeutinResistant Strain of Streptococcus laetis. J. ])airy Research, 30:345. (3) Briggs, C. A. E., m~d Newland, L,. G. M. 195"~. The Serologienl Classification of Streptoeocc~s eren~oris. ,T. ])airy Research, 19: 160. (4") Elliker, P. R., Andersol~, A. W., and I-Iannesson, G. 1956. A~ Agar Culture Medimn for Lactic Acid Streptococci and Lactobacilli. J. ])airy Sei., 39:1611. (5) Emmons, D. B., Elliott, J. A., u~td Bcekett, D. C. 1963. Agglutination of Starter Bacteria, Sludge Formation, and Slow Acid J . ])kIl~k" SCIENCE ~YOL. 49. No. 11
1366
(6)
(7)
(8)
(9)
(10)
(111) (12) (1:~)
(14)
(15)
D.B.
EMMONS ET AL
Developnlent in Cottage Cheese Manufactm'e. J. Dairy Set., 46: 600. Emmons, D. B., Elliott, J. A., and Beckett, D. C. 1965. Sensitive Test for LacticStreptococcal Agglutinins. J. Dairy Sci., 48 : 1245. Emmons, D. B., Elliott, J. A., and Beckctt, D. C. 1966. Lactic-Streptococcal Agglutins in Milk and Blood. X V I I t h Intern. Dairy Congr., D: 499. Emmons, D. B., Price, W. V., and Swanson, A . M . 1957. The A-C Test (Acid-Coagulation). A New Method of Determinil~g the Time for Cutting Cottage Cheese Cm'd. Ext. Circ. 541, University of Wisconsin. Gillies, A. J. 1959. Inhibitory Factors in Cheese Milk. XVth Intern. Dairy Congr., 2 : 523. Hunter, G. J. E. 1946. A Simple Agar Medimn for the Growth of Lactic Streptococci: The :Role of Phosphate in the Medimn. J. ])airy Research, 14: 283. ]£eogh, B. P. 1958. Variations i~ Lactic Acid Production in Milk. Australian J. Dairy Technol., I3: 132. ];osikowski, F. V. ]959. Better Cottage Cheese. Milk Dcaier, 49(3):44. l':osikowski, F. V. 1963. Some Distributi(m P:~tterns of Cottage Cheese Particles and Conditions Contributing to Curd Shatteri~g. J. Dairy Sci., 46:391. Levowitz, D., m~d Weber, M. 195(;. An Effective "Singie Solution" Stain. J. Milk Food Technol., ]9:121. Lucas, P. S. 1962. What Causes n Spongy Formntio~t in C,ott:lg'e Cheese? Am. Milk Rev., 24(10):74.
J. DAIF~' SeI)~Ne~ V o r , 49, No. ]1
(16)
(17)
(28)
(19)
(20)
(2])
(22)
(23)
(24)
E. 1958. Starter Failures and Their Prevention. Milk Dealer, 47(10):47. Portmann, A., and Auclair, J. ]959. Relation entre ]es Laetenincs et Ies Agglutinines du Lait de Yache. Annal. Inst. Pasteur, 97:590. Portmann, A., Gate, Y., and Auclair, J. 1962. Influe~ce of Lactoperoxidase and Agglutinlns of Milk on the Activity of Thermophilic Starter Bacteria. XVIth Intern. Dairy Congr., B: 729. Randolph, H. E. 1962. Natural Intfibitors in Milk Affecting Lactic Acid Bacteria. Ph.D. thesis, elite State University. Sa~ldi~le, W. E., Anderson, A. W., Elliker, P. R., and Stein, R. W. 1963. Observations on Citrate Uti]~zatim~ by Mixed-Straln Starter Cultures and Protein Stability of Cheese Milk. J. Dairy Sci., 46: 619. Stadhouders, J. ]963. The Inhibitory Effect of L,ctenh, L3 on Acid Production :bl Milk by Streptococcus eremoris 803. Netherlmlds Milk [)airy J., 17:96. Whiteilead, H. ilk, Jones, P. A., and Robertson, P. S. 1958. The Influence of Carben Dioxide o , the Growth of Lactic Streptococci. J. Dairy l,~ese:lreh, 25:24. Wilsm), C. S., :lm] Miles. A. A. 1955. Top]ey and Wilson's Pri.(dp]es of Bacteriology and Immuniiy. 4Ih ed. Edwurd Arnold (Publ.) Ltd., ]~()n,lon. Wright, R. ('., ,11,1 Tr:lmer, J. 1957. The Influelwe of (!,'can, Risi~g upon the Activity of ~qcterbl. in IL,:II Tr('ated Milk. J. Dairy Research, 24 : 174.
Lundstedt,