SOME THOUGHTS ON CHEESE STARTERS

QENERAL MEETING A General Meeting of the Society was held on 10th May, 1972, in the Edward Lewis Lecture Hall, Middlesex Hospital Medical School, London W 1.

The President, Dr. J. G. Davis, was in the Chair.

MORNING SESSION

SOME THOUGHTS ON CHEESE STARTERS* B Y B R U N O R E I T E R

National Institute for Research in Dairying, University of Reading

This address is not intended to be a comprehensive review of cheese starters but covers some of the research on starter carried out at the National Institute for Research in Dairying mainly since the last review appeared (Reiter & M~ller-Madsen, 1963).

SELECTION OF STARTERS ACCORDING TO THEIR CHEESEMAKING QUALITIES 1. Rate of acid production There has been steady progress in starter technology - protection against bacteriophage contamination, freeze drying, deep cooling, use of starter concentrate, etc. but present-day cheesemaking practice is based on starters with very high activity; the selection of an adequate starter stock remains, therefore, a problem. The rate of acid development which was once acceptable is quite unacceptable now; while a 6 h ‘Cheddar’ (rennet- ting to milling) using 2 per cent of starter and 14-2 h ripening time, may have been the norm once, the aim now appears to be a 4-4+ h make time for Cheddar without ripening and a 3 h make time for Cheshire with some ripening time. It is, therefore, clear that today the high rate of acid production demanded restricts greatly the number of starters available, irrespective of whether they are single strain or multiple strain starters. 2. Flavour production The recognition that starters can produce cheese with off-flavours such as bitter, fruity, yeasty, malty, burnt (see review of Fryer, 1969) further reduces the availability, at least of the single strain starters. Taking these factors into account Law- rence & Pearce (1972) proposed a rotation of starters which are phage unrelated and avoid production of off-flavour. Also, it has now been conclusively shown using aseptically made cheeses (Mabbitt, Chapman & Sharpe, 1959) that starter *An extended version of the address as given at the General Meeting.

itself produces cheese flavour (Reiter et al., 1967; McGugan et aZ., 1968; Reiter & Sharpe, 1971).

The intensity of the flavour in ‘starter only’ cheeses is low and develops late but can be clearly distinguished from cheese made without bacteria using 8-gluconic acid lactone for acid production (Mabbitt, Chapman & Berridge, 1955). Table 1 (Reiter et al., 1967) shows the results of two inde- pendent cheese panels (at the NIRD and Ottawa) who were asked to rank experimental cheeses according to their flavour intensity. The agreement between the two panels is remarkable and their ranking came very near the calculated optimum : (see bottom line of Table 1) the 8-gluconic acid

TABLE 1 Ranking of 4 cheeses according to the intensity of cheese

Ravour, and disregarding any off-Ravours (The figures represent rank totals. Method of scoring is that

used by Elliott & Beckett (1959).)

Cheese made with:

6-gluconic Starter Starter Starter acid lactone SIML, S/924 S1924fRF 7

r 7

Average ranking

Ottawa 16.8 13.4 9 43 8 SO NIRD 16.4 12.0 10.8 8 *s Calculated* 18 14 10 6

*Average ranking: when there is perfect agreement between

S=single-strain starter. (From Reiter et al., 1967.)

lactone cheeses were devoid of any cheese flavour while the ‘starter only’ cheeses showed character- istic Cheddar flavour which was, in the case of S. Zactis MLB, overlaid by an off-flavour (fruity, yeasty) as first shown by Perry & McGillivray (1964). The cheeses made with starter and the so- called ‘reference flora’ - a laboratory maintained mixed bacterial flora isolated from fresh cheese curd at commercial creameries (Reiter, Fryer & Sharpe, 1965) showed the most intensive flavour. We can state, therefore, that starter produces

tasters and consistency within tasters.

Journal of the Society of Dairy Technology, Vol. 26, No. I , January, 1973 3

TABLE 2 Bacterial flora of cheese milk and its cheese curd in a cheese

factorv Group of Organisms Raw Heat Cheese

milk treated curd milk

(155°F/15”) Heat resistant

corynebacteria + + + Sporeformen + + + Coagulase +ve

staphylococci + 0 0 Coagulase -ve

staphylococci + 0 + Pseudomonads + 0 0 Coli aerogenes + 0 + Lactobacilli + 0 + Leuconostoc + 0 0 Pediococci 0 0 + (From Reiter & Sharpe, 1971.)

cheese flavour but the intensity and early development depends on the contaminating flora.

However, the complex bacterial flora of raw milk is nearly completely destroyed by currently practised heat treatment of the cheese milk but subsequently a similar flora is restored through contamination from the air and cheesemaking equipment (Table 2). This agrees with the findings of Franklin & Sharpe (1963) and Ohren & Tuckey (1969) who stressed that in the absence of sufficient numbers of bacteria other than starter organisms a full flavoured cheese is not produced. (We may add perhaps, in a reasonable time.) The improved hygienic quality of the cheese milk, high heat treatment and eventually the advent of truly continuous cheesemaking in a closed system will throw an increasing burden on the starter for the maturation of cheese. We are, therefore, investigating whether the cheese milk can be matured by a reference flora before cheesemaking and are considering whether starters should contain a ready made cheese flora. The concept of starters containing bacteria other than lactic acid streptococci cannot be too strange in the dairy industry because Sharpe et al. (1958) found that a high proportion of starters maintained in the routine laboratories of creameries were contaminated with lactobacilli.

GROWTH PROMOTION AND INHIBITION OF STARTER

1. Proteolysis and amino acid content of milk The lactic acid production of a starter depends also on the milk itself. Auclair & Hirsch (1953) were first to point out that a balance exists between growth promoting and inhibitory factors in milk. It is generally recognized that the ability of a starter to multiply depends at least partly on its

proteolytic activity. When purified sodium casein- ate was employed as the only source of nitrogen it was found that, in general, strains of S. lactis grew adequately whilst the strains of S. crernoris required amino acids (Reiter & Oram, 1960) at the level present in aseptically drawn milk (Deutsch & Samuelsson, 1959). The individual amino acid requirements of lactic acid streptococci were deter- mined in a synthetic medium based on the relative amino acid composition in milk (Reiter & Oram, 1962, Table 3).

TABLE 3 Amino acid requirements of group N streptococci

S. lactis S . crenioris S. diacetilactis

Proline Alanine Tyrosine Lysine Aklille Threonine Tryptophan

- E

(E) Arginine - Glutamic acid, Valine, Methionine. Leucine, Isoleucine and Histidine E. for all strains. - : No requirement. E: Essential. (E): essential for some strains. S: Stimulatory for some strains. (Data extracted from Reiter & Oram, 1962.)

Lindqvist (1968) attempted to correlate the ‘seasonal variations in free and protein-bound amino acids in milk’ with the ‘occurrence of certain difficulties, encountered at particular times of the year, in the manufacture of cheese and butter associated partly with the slow growth of starter cultures in the milk’. In spite of the use of sophisticated methods and an exhaustive com- puter programme no correlations were found. A simpler approach like using the milk of mono- zygotic twins and feeding them different diets (Reiter, Sorokin, Pickering & Hall, 1969) coupled to rigidly controlled cheese experiments, or at least activity tests of starter, could perhaps yield more positive results.

Recently Westhoff, Cowman & Speck (1971) and Westhoff & Cowman (1971) reported the interesting findings that the intracellular proteinase of ‘slow’ mutant differed significantly from the proteinase of the past parent strain. The mutant was found to require for its growth leucine, lysine, valine, phenylalanine, glutamic acid and proline but the latter two amino acids were not released from insulin by the intracellular proteinase. These authors suggested that the poor ability of the mutant to acquire its essential amino acids - which are limited in milk - could be related to its early cessation of growth in milk.

4 Journal of the Society of Dairy Technology, Vol. 26, No . I , January, 1973

2. ‘Lactenins’ We have touched only briefly on the growth promoting qualities of the milk but strangely enough we seem to know more of the growth inhibiting factors in milk. In the late twenties Jones and his co-workers (summarized in Jones & Simms, 1929/30) published their research on milk inhibitors named by them ‘lactenins’, in a series of out- standing papers. Since then it has become evident that milk contains several lactenins which differ in their antibacterial specificities. Moreover, these inhibitors are not confined to milk but occur also in other biological fluids such as saliva, uterine fluid and in white blood cells. It is time, therefore, to abolish the term lactenins, and rather detail them as the peroxidase system, antibodies, lacto- ferrin (Oram & Reiter, 1968a) and lysozyme (for a review see Reiter & Oram, 1967).

(a) Lactoperoxidasel thiocyanatel peroxide In the dairy industry, Wright & Tramer (1957,

1958) began the work of defining the milk inhibitors. They associated the inhibition of a particular strain of S. cremoris with lactoperoxidase and of another strain with agglutinins. Jag0 & Morrison (1962) demonstrated that the peroxidase system required peroxide while Reiter, Pickering & Oram (1964) showed that thiocyanate in the milk was oxidized by peroxidase/peroxide to an inter-

o * 2 t I I,

0 l - u A - 2 9 cu b u s 5 0 ? ? ‘ W d . . . .

0 0 0 0 0 0 0

Concn. o f NaSCN, pg/ml Fig. 1. E i k t of LP and thiocyanate on the growth of Sfrep. cremorls 972 in a lynulaic medium. Appro?. l(r colony-forming chain# were added to 10 ml of thqmedium of Reiter &.Oram (1962), from which c teine and ascorbic wid ware omitted, contaming 10 umts of LP/d and &CN tu indicated in 150 mi conical flasks. The E:;? was measured after 24 h

at 30”. (RSitcr, Pickcring & Oram, 1964.)

mediary oxidation product which caused the inhibition (Fig. 1). The nature of this oxidation product of SCN- is still in doubt because it is ex- tremely short-lived and therefore difficult to identify (Oram & Reiter, 1966a, b; Hogg & Jago, 1970a, b). The inhibitory system requires, therefore, (lacto)peroxidase, thiocyanate and peroxide, the latter being produced by the metabolism of the streptococci under aerobic conditions; alternatively, peroxide can be added to the system for experimental purposes directly or indirectly (glucose plus glucoseoxidase). Most starters are, however, resistant to the peroxidase system although they can give rise to sensitive mutants (Auclair & Vassal, 1963). We found that the lactic acid production of suspension of a sensitive strain was abruptly inter- rupted in a synthetic medium after the addition of all 3 factors, while (a) a resistant strain remained unaffected (Fig. 2a on p. 6) (b) the addition of the cell free extract of a resistant strain to the inhibited strain after 30 min (Fig. 3) reversed the inhibition and lactic acid production gradually proceeded at the same rate as before. The enzyme responsible for the reversal was purified and shown to be present only in peroxide resistant and not in sensitive strains as seen in Fig. 2b on p. 6.

I t has always seemed incongruous to expect a culture to produce acid at a fast rate in the cheese milk in which the milk inhibitors are not inactivated while the cultures are propagated in heated milk in which all inhibitors have been destroyed. A long time ago, I wrote, -&re€ore (Reiter & Moller-Madsen, 1963) ‘This phenomenon under- lines the wjsdom of cheesemakers who insist on - cultivating _ - ~ starters in milk pasteurized at €ow temperatures similar to those used for cheesemilk’.

Sensitive mutants would, of course, be eliminated if the starter was propagated in raw milk or milk heated below the inactivation temperature of peroxidase (less than 70°C for 20 min). It had been shown (Jago & Swinbourne, 1959) that starters continuously propagated in steamed (or auto- claved) milk can become susceptible to the inhibitors of raw milk. It could make sense, therefore, to propagate starters in raw aseptically drawn milk from udders free from bacteria and phage and which could be distributed in freeze dried form for mother cultures. Alternatively, known inhibitors like peroxidase, antibodies, etc., could be added to heat sterilized milk. The effect of the peroxidase/thiocyanate system on cheesemaking was shown in the following experiment: Thio- cyanate was removed from milk with ion-exchange resins and Fig. 4 (p. 7) shows that in that case the peroxidase sensitive strain S. cremoris 972 (Wright & Tramer, 1957) was not inhibited, and lactic acid was produced at a normal rate during cheesemaking. The addition of thiocyanate prevented any appreciable acid development similar to the behaviour of phage infected starter culture. It is

Journal of the Society of Dairy Technology, VoI. 26, No. I , January, 1973 S

C

fa)

O e 8 r 0.6

0.4

0.2

0 0 10 20 30

Time, min 0, Slrep. rremoris 972 (sensitive); A , Strep. cremoris 803 (resistant);

h r

‘2 4 I- 0

(b)

0 2 4 Time, m in

0, Strep. rrenioris E.8 (sensitive).

Fi .2a and 2b. Rslationship between the effect of LP and thiocyanate on streptococci and their possession of the NADH,-oxidizing enzyme. (a) Effect of Le70 units) A, KSCN, (lO#molcs) B and olucose oxidase (!00,#8) C on the 8lycoIysis of suspensions of streptococci in 0.1 M-glucose-lOmM-potassium

phosphate buffer, pH 6.8. (b) NADHI-oxidizing enzyme activities ofextracts of streptoccocci. (Oram & Reiter, 1966.)

‘r

interesting to note that later we discovered an early French paper (BoulangC, 1959) which contained data on the seasonal variation of the thiocyanate in the milk of a herd over two years, compiled for quite other reasons. There exists also an extensive literature on thiocyanate in milk, and its origin from various plants (glucosides) and its alleged goitrogenous effect in children (e.g. Virtanen, 1962).

“ 0 30 60 90 120

Time, min

Fig. 3. Inhibition of the glycolyais of Slrep. cremoris 972 by LP and KSCN and its reversal b an extract of Slrep. cremorls 803. KSCN (5 #moles) A and LP (70 unitrfB were added to a suspension of strain 972 in 0.1 M- glucose-l0mM-potassium hosphatc buffer, pH 6.8, and extract of strain

803&~Oml)Cwasaddedlater. 0, Glycolysia: 0, concn. ofresidual KSCN, corrected for volume changes.

(6) Agglutinins and their origin ,

Of the other inhibitors, we need only concern ourselves with the bacterial agglutinins which affect many lactic acid bacteria. The cocci are swept up with the rising fat globules in whole milk or agglutinate to the bottom of a culture vessel in skim milk (Wright & Tramer, 1958; Emmons, Elliott & Beckett, 1963, 1966). The latter authors realized the significance of this agglutination phenomenon for the production of cottage cheese from skim milk as it can cause slow acid development, ‘sludge’, texture and body faults or complete failure. It is simple to test for agglutinins against a starter by inoculating a tube containing either raw or heat treated litmus skim milk. Affected starters reduce and clot the litmus milk rapidly at the bottom of the tube while unaffected starters reduce and clot the milk evenly throughout the tube.

The mode of action and the reason for the up- sweep of agglutinated streptococci (and other

6 Journal of the Society of Dairy Technology, Vol. 26, No. I , January, 1973

.W/" t

0 1 I I I I I I 1 0 60 120 180 240 300 360 420

Time, min

Fig. 4. Lactic acid development in Cheddar cheese made with peroxidasel SCN- sensitive starter in the presence of SCN- and after removal of SCN-

from milk by ion exchange treatment. 0-0 SCN- removed from milk 0- - -0 untreated milk 0- - - 0 Control (lactic acid production with peroxidase resistant starter

Sfrep. cremoris 803).

bacteria) in whole milk are not well understood. It can be easily proven that fat globules are agglutinated by cold agglutinins (auto antibodies which act only at low temperatures). Dowben, Brunner & Philpott (1967) demonstrated that bovine fat globule membranes cross react with the membranes of jed blood sells (RBC). We found that when packed RBC were added to whole milk and left standing at 48OC or at 37OC overnight and then the RBC removed by centrifugation, the milk kept at 4 8 ° C failed to cream while the milk kept at 37OC creamed as before the treatment. The low temperature treatment of the milk had removed the 'cold agglutinins'. The creaming ability can be restored by resuspending the RBC and incubating for 30 min at 37°C because the RBC adsorbed cold agglutinins are desorbed at this elevated temperature (Reiter, 1967a).

It has been proposed (Stadhouders & Hup, 1970) that the streptococci are agglutinated by antibodies in the milk and become attached to agglutinated fat globules (by cold agglutinins) by yet a third type of antibody. So far this hypothesis is based only on adsorption experiments and heat inactivation of antibodies and needs further confirmation. However, it is possible to put forward another .equally attractive hypothesis. It is well known that bacteria and viruses can adhere to RBC. This reaction requires both antibodies and complement. Considering the antigenic cross reaction between RBC and fat globules (or rather their membranes) it is possible that bacteria adhere to the fat globules in a similar fashion because we know now

that milk also contains complement (Reiter & Oram, 1967). The distinction between complement and antibodies is easy to make because complement is inactivated at a far lower time-temperature combination - 56°C for 30 min - than antibodies and so far Stadhouders and Hup heat-treated the milk at 80°C for 10 min which would inactivate both complement and antibodies.

Auclair and his collaborators (Portmann & Auclair, 1959, Portmann, Plommet & Auclair, 1960 and McPhillips, 1958) showed that agglutinins against lactic acid streptococci occur at low titres in milk throughout the lactation period but at very high titres in the colostrum. Also they established first that the agglutinins are antibodies which can also be elicited in blood serum by vaccination of

Now, of course, we know much more about blood serum antibodies and how they are transferred from the blood to milk but we do not know enough yet about the origin of the antibodies in the blood. I suggested previously (Reiter & Mdler- Madsen, 1963; Reiter & Oram, 1967) that 'rumen bacteria may well be the source of antigens which result in the production of antibodies . . . '. Anti- body production by rumen bacteria has now been well established (Sharpe, Latham & Reiter, 1969; Sharpe & Reiter, in press). However, until recently it was thought that the antibodies against lactic streptococci may arise because of shared antigens with Streptococcus bovis (Sharpe, 1952) which is now known to belong to the dominant rumen flora under certain conditions (e.g. high cereal diet). This may be wrong or only partially true. Sharpe (1952) did once isolate lactic acid streptococci from the rumen. Since then repeated reports have appeared that Group N streptococci were present in the stomach of mice. Dr. Sharpe repeated recently the isolation of Strep. Zuctis from the rumen of a cow (unpublished). This in itself is interesting but there have also been isolated reports of bacteriophage in the rumen. Dr. A. Ritchie, National Animal Disease Laboratory, Ames, Iowa, USA (personal communication) has now reported that he could distinguish 140 different morphological types of bacteriophages. Isolates of streptococci and phages from the rumen may now throw light on the ecology of cheese starters.

cows.

BACTERIOPHAGE While the rate of acid production of the starters and the inhibitory factors in the milk greatly restrict the selection of starter, the phage suscepti- bility of the starters remain the overriding factor in the choice of starter. When Whitehead & Cox (1935) discovered lactic acid streptococcal phages protection against phage contamination was re- garded only as a temporary measure . . . 'from a

Journal of the Society of Dairy Technology, Vol. 26, No. I , January, I973 7

practical point of view it is necessary to find some method of eliminating the phage or of using an organism immune to its action. The isolation of phage-immune variants seems to offer the greater promise of success. . . . ' Whitehead's first line of approach - protection against phage infection - is now well proven and widely practised; methods of physical and chemical protection have been developed in the past but, at least in my opinion, relatively little effort has been made in the second direction (see reviews of Whitehead, 1953; Babel, 1962; Reiter & Moller-Madsen, 1963; Robertson, 1966; Crawford, in press).

1. Phage resistant mutants Brock (1962) reported that streptomycin resistant bacteria (E. coli, Strep. faecium, Strep. faecalis, Strep. liquefaciens and Strep. zymogenes) acquire also resistance to their homologous phage. Later Friend & Slade (1967) showed that chloramphenicol mutants of Group A streptococci developed simultaneously a non-specific resistance to phage. Preliminary attempts to prove this for group N streptococci (Reiter, 1967a) were only partially successful, but warrant further investigation. Later, the phage resistance of chloramphenicol resistant starter was confirmed by Erskine (1970a). The possibility of selecting phage resistant mutants from phage infected cultures in the presence of spermine (Erskine, 1970b) has so far not been confirmed.

2. Phage adsorption and phage lysin After 1963, we became more interested in the phage adsorption process with the ultimate aim of isolating and identifying streptococcal phage receptors. Before then it was too readily assumed that most of the knowledge accumulated from coli phage research is applicable to other phages. This was, of course, before the structure and chemistry of the cell wall of Gram positive and Gram nega- tive organisms became known in any detail.

( i ) Purification of phage Zysin and its activity Our first objective in the project on phage

adsorption was to purify phage lysin (which was first described for lactic streptococci by Czulak & Naylor (1956) in a brilliant series of papers on the nascent phage phenomenon and lysogenicity), and to lyse cell walls of starter streptococci in the hope of isolating the phage receptors. The associ- ation between phage activity and cell walls was established when we showed that the serological grouping of a number of strains of S. cremoris based on agglutination with immune whey (produced by infusion of strains of S. crernoris into the dry udder) was in close agreement with that based on the phage relationships of the same organisms. (Reiter, Di Biase & Newbould. 1964, Table 4).

Our results with the purified phage lysin showed that the enzyme is specific to groups N and D streptococci, none of the other serological groups of streptococci being lysed; it lyses viable and heat killed cocci or their cell walls (in contrast to egg white lysozyme) (Reiter & Oram, 1963; Oram & Reiter, 1965. Fig. 5). Its relative activity is under the genetic control of the infecting phage. The phage lysin purified from phage lysates or from the tail of the phage appeared to be biochemically and biologically identical. However, against ex- pectations, the phage lysin failed to liberate the specific phage receptors from the cell walls as the supernatant of lysed cell walls showed no phage neutralizing activity (Oram & Reiter, 1965). Nevertheless this work opened up further possibilities.

0 1 1 1 1 1 1 1 1 1 1 1

0 20 40 60 80 100 ' 130 Time, min

Fig. 5. L sis of viable cocci and cell walls of Strep. cremorls 972 b m l 3 lyain andllysozme. 10 mg (9 weight) of viablo cocci or cell wali was suapended in 5.0 ml 0.1 M-p opphate buffer, pH 6.7. containing either

m l 3 phage-lysin (12 U N ~ S or lyaozyme (2.5 nag). 0--0 Viable cocci + phage-&rin* A-A viable Mcci + lysozyms: .& cell walls f phage-iysin; A L A , cell walls t

lysozyme.

(ii) Phage lysin and resistant mutants? In the past we had attempted to produce specific

phage antisera which could neutralize phage either in the milk used for starter making or even in the cheese milk (Reiter, 1962). (See also Erskine, 1964). This project was abandoned because the titres of the antisera were too low to be practical and there appeared to be too many serological

8 Journal of the Society of Dairy Technology, Vol. 26, No. I , January, I973

L aJ nw 0 0 1 2 3 4

Time, h

Fig. 7. Reduction in turbidity of cell-wall suspensions of M. lysodeiktlcus strains LS, LRI and.LPR. (Brumfitt 1960.)

X strain LS. , .A strain LRl* &aia LPR; LS-Imomne &nsmve: LR-hsozymo resistant mutant;

LPR-phage resistatit mutant.

acid. The mechanism of phage resistance of the lysozyme resistant strain differs from the phage resistance of a mutant selected by lysing a culture with phage (eliminating the phage susceptible cells) because this phage resistant mutant does not adsorb its phage as it happens with many other phage races.

It appears, therefore, that the mucopeptides are involved in the binding of the phage particle and in triggering the release of desoxyribonucldc acid (DNA). These considerations led us to speculate that we might be able to produce phage resistant mutants. If it proves possible to train lactic streptococci to become resistant to phage lysin (they are already resistant to egg white lysozyme) in the same way as M . lysodeicticus can be made resistant to egg white lysozyme and phage lysin - it can be expected that such mutants will be totally resistant to phage.

(iii) Specific and nonspecific adsorption of phage to cell walls and membranes

So far we had failed to liberate any phage receptors from the cell wall by phage lysin but unexpectedly discovered that contrary to what was commonly accepted in the phage literature, some stre tococcal phages did not adsorb to the cell wal P but to the cell membrane (Oram & Reiter. 1968b).

Six phages were adsorbed to their live hosts or cell walls (5 strains of S. cremoris and 1 strain of

S. lactis) and their ability of adsorption compared with their ability to multiply. The host range of phage multiplication was, as expected, restricted; no phage multiplied in more than two of the streptococcal strains (Table 5) but most phages were also adsorbed to the viable cocci and/or their cell walls of several strains without being capable of multiplying. Adsorption to cell walls was shown to be generally irreversible as with viable cocci, with the exception of two phages, ml, and d,, which adsorbed only reversibly to cell walls. Phage hp was adsorbed not only to the cell walls of S. lactis M1, irreversibly - without multiplication-but also to viable cocci of S. cremoris TR and D, - also without multiplication. Other examples are self-explanatory from Table 5.

loo I

25

0

Time, min Fig. 8. Interaction of Micrococcus lysodeikticus strains LS, LR1 and LPR,

with phage N4. (Bfumfltt,, 1960.) "'-x strain LS;

LS-lysozyme sensitke. A LR-lysozyme resistant mutant, . . . strain LRl, w-0-m strain FPR;

LPRLphage resistant mutant.

(iv) lsolation and identification of phage recep-

When the cells of S. lactis ML, were fractionated into cell wall, cytoplasmic fraction and plasma membrane it was found that only the membrane was capable of adsorbing and inactivating the phage ml, (Fig. 9). The adsorption to the cell membrane was quite specific as phage resistant mutants failed to adsorb phage mla to their membrane (Fig. 10). The phage receptor substance for ml, phage was isolated from the plasma membrane by

tor from cell wall membrane

10 Journal of the Society of Dairy Technology, Vol. 26, No. I , Junuary, 1973

TABLE 5 Phage relationship according to multiplication and adsorption

Strains Phage m

Str cmmoris 699.

KH

TR

799

D9

HP

Str laclis ML,

0 Adsorption > 50% by viable cells Adsorption <50% by viable cells

Adsorption by purified cell walls

which contained five principal and some minor polypeptide components. As digestion with trypsin reduced the receptor activity and the removal of lipid destroyed it, it appears that the vital part of the phage receptor is the lipoprotein fraction (Oram, 1971). -

It is most interesting that at the same time as we studied the phage receptors Dr. A. Hurst (formerly Hirsch) now at the Food & Drug Directorate, Ottawa, Canada, took some electron micrographs of cell walls of a strain of S. la& (nisin producer) and discovered a number of holes on the surface with an approximate diameter of 20 nm which appeared to be filled by conical protrusions from the plasma membrane (see Hurst & Stubbs, 1969; see Fig. l l a and diagrammatic representation of the outer layer of S. la&, Fig. l l b on p. 12). As the tail of phage ml, has a cross-sectional diameter of approximately 9 nm the holes would be large enough to permit the phage to pass through the cell wall and to attach to the receptor in the plasma membrane. The phage ml, and do are so far the only phages known to attach to cell membranes. Hitherto only animal viruses but not phages (bacterial viruses) have been known to attach to a membrane. These findings are academi- cally quite exciting but it is also possible to en- visage a more practical application of these results.

One of the limitations to the continuous use of the same starter (including refills) is the phage

extraction with sodium deoxycholate at 37°C and Partially Purified through a COlumn of SePharose 4B. The receptor activity was Specific for Phage ml, and associated with a lipoprotein fraction

g 106 +J

E .C

E 0

c .r

105 n 7

m V

UI 0) CL

.r

104 0 0.2 0.4 0.6 0.8 1.0 1.2

Amount o f m a t e r i a l , mg

Fig. 9. Inactivation ofphuge m l 3 by cell fractions of S. lacfir ML 3. Phage waa incubated with the plasma membrane. (Oram & Reiter 1968b.)

0, cell wall; 0, or cytoplasmic fraction; 0 in 0.5 ml. of GL broth at 30" for 30 min.

.? 107 E

cc: n

Time, min Fig. 10. Absence of phage receptor activit in plasma membranes of phage-

resistant mutants of S: /actis ML i. (Oram & Reiter, 1968b.) 0, Membrane of phage-senntive stram ML 3, lOOpg; 0, membrane of phage-resistant mutant ML 3/5, 5OOpg; 0, membrane of phage-resistant

mutant, ML 3/15,50Opg.


1 - Inside Ml3 lbWle .I. 100 nm-

Fig. 11. (a) Freezeetched outer membrane surface (or inner wall surface) of a stationary-phasc cell of 8. lacfls.

(b) Diagrammatic representation of tho outer layers of 8. laefls. Wurst & Stubbs, 1969.)

build-up in the creamery. Although the abolition of the ripening time contributed greatly to the protection of the starters, the phage build-up in the cheese milk can reach levels which often causes slow cheese in spite of the use of phage free bulk starter. The irreversible phenomenon of the non- specific phage adsorption could be exploited in a practical way. We could eliminate from the cheese milk a phage specific to the starter we wish to use for cheesemaking by prior non-specific adsorption of the phage to another starter. Thus, the normal acid production of the ‘working’ starter would be safeguarded, e.g. Table 5 shows that the starter HP is lysed by phage hp only; if we want to safe-

guard this starter against its specific phage contaminating the cheese milk it would be necessary to add a small inoculum of any of the starters which are capable of adsorbing hp phage non-specifically, like starter Do or ML,. To safeguard the starter Do against de and tr phage, the starter 799 adsorbs both phages non-specifically. Considering the relative low level of phage titres encountered in cheese milk (even after refilling) a low inoculum of a ‘scavenger’ starter would suffice and of course the activity of this starter is expendable as the cheese would be made with the main starter inoculum, e.g. HP or De (Table 6). Table 5 makes further examples of this approach explicit.

TABLE 6 Absorption of phage by ‘scavenger’ starters

Phme Starter D9 199*

tr

A A

HP” hLp ML8* A D9 A Tr* (A)

“Working starter. +Scavenger starter. L: Specific adsorption leading to lysis. A: Non-specific adsorption, no lysis.

( v ) Transformation and transduction A less immediate practical outcome from these

research results, but with some considerable interest, is the fact that some phages have direct access to the cell membrane. In our 1963 review (Reiter & M~ller-Madsen) we cited two cases of transformation by deoxyribonucleic acid (DNA). No further confirmative work has appeared since. Streptococci are known to be noncompetent for transformation with the exception of Group H streptococci. It is, however, probable that lactic streptococci with ‘exposed’ membranes are accessible to DNA and hence transformation; thus desirable characteristics could be transferred to competent organisms.

Also, as will be seen later, we have now isolated and purified temperate or virulent phages from a lysogenic strain. It is well known that temperate phages become arrested in their cycle of multiplication at an early stage, become part of the host and are transmitted to following generations of the host. When these so-called prophages become fully vegetative in a small number of cocci or by treatment with U V , etc., fragments of the bacterial chromosomes can be disrupted and can become incidentally incorporated in the newly-formed phage particle. After reinfecting another strain such fragments are transferred and can transmit to the host new characteristics, this phenomenon is


called transduction and the isolation of temperate phages now makes this kind of work possible.

(vi) Isolation and properties of polysaccharide

The failure to identify the phage receptors in the cell wall necessitated an attempt to analyse the cell wall and identify the antigens and explore their possible relationship to a phage receptor activity of the cell wall. Using the conventional methods of hydrolysis for cell walls the cell wall polysaccharides were found to contain: rhamnose, glucose, galactose, and glucosamine, part of which is present as glucosamine 6-phosphate but no glycerol' or ribitol. Both serologically active and inactive polysaccharides were isolated but none of them showed any phage inactivating activity (Table 7).

components of cell wall

TABLE I Polysaccharide componenta of group N cell walls

Components Extracts of Serological cell walls activity

+ Galactose 5 % TCA at 90" -

(GA-6-phosphate) 0 : 1 N-HaS04 at 60" -

- Rhamnose 0.01 N-HCI Glucose formamide at 150"

Glucosamine

(Unpublished, Oram & Reiter.)

It seemed, therefore, that we have made little progress towards the identification of the cell wall phage receptors. Since then, however, Yokokura (1971) extracted from the cell wall of a Lacfo- bacillus casei a trichloracetic acid (TCA) soluble fraction which inhibited phage adsorption and was even capable of desorbing adsorbed phage; L-rhamnose by itself showed phage inhibiting activity at 0.0142 molarity. Monosaccharides with similar structure and configuration to L- rhamnose, such L-mannose and L-fucose also inhibited phage adsorption. The inhibition of phage adsorption by a single carbohydrate is rather sur- prising and perhaps encouraging for future work on the cell wall receptors of lactic streptococci. In other Gram positives like staphylococci the cell wall teichoic acid appears to be part of the phage receptor, but both lactic acid streptococci and L. casei (in contrast to all the other lactobacilli) do not possess cell wall teichoic acid but their teichoic acids are situated near the cell membrane (intracellular) (Elliott, 1963). While this analogy makes further work on these lines regarding phage adsorption and the isolation of phage receptors promising, the apparent reversibility of phage adsorption .(desorption) in the case of L. casei indicates that the phage adsorption of streptococcal phages may yet be somewhat dilTerent.

3. The isolation of a lysogenic strain from a multiple strain starter culture

The recent work by Keogh & Shimmin (1969) on defective phage particles liberated after U V irradiation and induction of a strain of S. cremoris (C,-56) revived our interest in lysogenicity (Reiter, 1949; Czulak & Naylor, 1956; Crawford & Gallo- way, 1962). The report of the latter authors is particularly interesting. They described an incident in which after supplying a phage-free multiple strain starter to a factory the starter remained in continuous use for several years without using any phage protection. Throughout that time no slow cheese was experienced in the factory. However, ultimately the culture became contaminated with yeasts and the authors received a request to reissue the same starter to the creamery. When this pre- sumably phage-free culture (propagated in a laboratory of the West of Scotland Agriculture College) was made into bulk starter at the factory it failed completely after one day of cheesemaking. Subsequently, phage was isolated from the old cultures. The same authors surveyed commercial starters propagated in various creameries and detected phage in many of them using some of the well-known single strain starters as indicator strains. They concluded that commercial starters either carried phage or contained lysogenic strains. We obtained a commercial mixed strain culture through the courtesy of Dr. Crawford from a factory which had used the starter without any slow- ness in the creamery for over 10 years. We found (Reiter & Kirikova, in press) that this culture (perhaps because it was not propagated under aseptic conditions) contained lysogenic strains which, after UV irradiation and induction, liberated phage with the same specificity. We also observed the same lytic phenomenon due to the defective phage (unpublished) as first demonstrated by Keogh & Shimmin (1969). As far back as 1956 Czulak & Naylor discussed lysogenicity in their paper and wrote that 'in nature such interaction between phage races and lactic acid bacteria must be con- stantly taking place'. It is indeed possible that the balance of several strains is maintained by this means in multiple strain starter or produced during propagation without protection against phage. Phage sensitive mutants which arise regularly in a culture of lysogenic organisms would be eliminated by the small amount of fully vegetative phage particles released from a few cells in any lysogenic culture.

The thinking on starters has been dominated for over 35 years by the research on lactic phages and single strain starters as pioneered by Whitehead and his colleagues. Perhaps we should reconsider whether the use of multiple strain starters or a mixture of strains composed of lysogenic but unrelated strains of streptococci may not be superior

Journal of the Society of Dairy Technology, Vol. 26, No. I , Janaury, 1973

D

13

to the use of single strain starter with all their setbacks.

4. Obituary or prologue to cheese starter research The last heading may appear somewhat frivolous but there are many signs that dairy research is not expanding but contracting. This is not restricted to the UK; for instance, in the USA practically every former dairy department has been converted into food science departments with dairying a poor second.

There is no doubt that we have made techno- logical progress in starter preparation, particularly with regard to phage protection but could it be that we have failed to consider alternatives? Is it not significant that Crawford and Galloway used some of the best-known single strain starters as ‘indicator strains’ for the detection of phage in commercial cultures? Does this not mean that we try to make cheese with organisms which are an artefact, as they would in the course of nature be wiped out by phage if we did not perpetuate them? A lysogenic culture releases very few infective phage particles - only one coccus in 1-100 million cocci would normally release about 100 phage particles. This would hardly be a threat to cheesemaking, provided of course that we do not use ‘indicator strains’ at the same time or in rotation. On second thoughts why do we put up with rota- tions? When I was responsible for the starter supply in three creameries, the system of rotation was just emerging. However, I soon discovered that certain starters could only be used for a day or two because they built up a whey titre of up to lO-l1/rnl while other starters could be used for longer periods (a whole cheesemaking season) because the phage titres in the whey never rose to more than lO-@/ml. Naturally the high titre phage could be detected in the air on exposure of seeded plates, while the low titre phage was not detected. A fraction of 1 ml of whey containing phage particles/ml could thus infect 100 gal of sensitive starter while with the low titre whey some sabo. teur would have had to empty a bucket of whey into the starter can to produce the same effect. Furthermore, what other respectable fermentation industry chops and changes, or continuously borrows or buys their ‘ferments’? As far as I know strains of yeasts are guarded closely in the brew- eries and used daily. Should we not rather aim for the daily use of the same starter and perhaps a ‘bespoke’ starter for a particular flavour.

Many papers appear now on large scale production of starter either by the batch or continuous process. For this to be successful, however, we ought to know far more than we do about the nutrition and metabolism of the lactic streptococci. By this I do not mean just adding ingredients to milk (yeast extract, peptone, etc.). Many bio-

chemists are presently looking for job oppor- tunities but how many are employed in the whole of the country on starter or, for that matter, cheese research -very few I think.

If we believe that our cheese industry has to change from a salvage operation of surplus milk to a profitable and competitive undertaking, we need to do more research and not less as apparently is the tendency today.

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Documents

SOME THOUGHTS ON CHEESE STARTERS