studies on batchproduction bacterial concentrates from species lactic...

APPLuD MicRomoLoGY, Feb. 1975, p. 133-140Copyright 01975 American Society for Microbiology

Vol. 29, No. 2Printed in U.SA.

Studies on Batch Production of Bacterial Concentrates fromMixed Species Lactic Starters

H.-E. PEYFERSSONChemical Center, Division of Technical Microbiology, University of Lund, S-230 53 Alnarp, Sweden

Received for publication 18 October 1974

Optimum growth conditions for mixed species starter FDs 0172 at constant pHin skim milk, whey, and tryptone medium were investigated. Growth rate andmaximum population were optimal at 30 C. pH values between 5.5 and 7.0 didnot influence the growth rate and maximum population obtainable. Lacticacid-producing activity declined rapidly after reaching the end of the exponentialgrowth phase. The bacterial balance was found to be influenced by the growthparameters: media, pH, temperature, and neutralizer. Skim milk or wheymedium at 25 C, pH 6.5, and neutralized with 20% (vol/vol) NHOH kept thebacterial balance almost constant throughout the cultivation. Grown in tryptonemedium at constant pH, the changes in bacterial balance and other metabolicactivities were striking compared to the other two media tested. The effect oflactate as an inhibitor was found to be complex, changing with the growthconditions. Concentrates made from mixed species starters FDs 0172, FD 0570,CH 0170, CHs 0170, and T 27 were comparable to controls when cultivated at theoptimum conditions found and thereafter centrifuged. Aroma production,proteolytic activity, and CO2 production did not change significantly comparedto controls when cultivated at optimum conditions in skim milk or wheymedium.

Interest in frozen lactic starter concentrateshas grown in the dairy industry during the last 5years. A lot of research work has been done onthe preparation of these concentrates fromstarter bacteria used in the dairies. Most ofthese studies have been carried out on singlestrain cultures, primarily used in the Anglo-Saxan countries for cheddar cheese production(6, 13, 17, 18).The increase in yield of biomass when neu-

tralization was used was confirmed first byKosikowski (12) and later by Bergere and Her-mier (2). This, together with a centrifugationprocedure, has subsequently been the method ofchoice for the production of concentrated start-ers.

Starters used in the northern European coun-tries for their cheese varieties are chiefly of themixed species type. The starter used in Swedenfor most of our fermented dairy products is ofthis type and consists of mixtures of mesophiliclactic acid bacteria. Streptococcus cremoris andStreptococcus lactis are the main lactic acidproducers. Streptococcus diacetilactis and Leu-conostoc citrovorum are responsible for aromaand gas production in the products.There are at least two possible ways to make

concentrates of mixed species starters. One way

would be to make concentrates of the isolatedstrains that compose the mixed strain starter bymethods outlined in the literature (3) and mixthese concentrates afterwards to a mixed typestarter concentrate. Another way would be tomake a concentrate directly from the mixedspecies starter by mass cultivation (constantpH) and centrifugal concentration (20).The first method, although most attractive at

first glance, would have some problems anddisadvantages compared to the second methodoutlined. Sorting out single strains and species,isolated either from commercial mixed speciesstarters or from other sources, which will growin symbiosis with each other without straindominance and changes in important metabolicactivities would be very troublesome and timeconsuming. The greater fermentor capacityneeded for this type of production is anotherdisadvantage of that method.Although cultivation procedures were

thought to be more difficult to optimize whenmaking concentrates from mixed species start-ers, it was nevertheless chosen in this studybecause, if successful, most of the problems anddisadvantages with the other method would beovercome.This paper presents a study of the optimal

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conditions for making a mixed species starterconcentrate directly from a mixed strain starter,with preserved strain balance and metabolicactivities, such as lactic acid production, aromaformation, proteolytic activity, and gas produc-tion.

MATERIALS AND METHODSCultures. Five mixed species starters were used in

this study. FDs 0172, FD 0570, CH 0170, CHs 0170,and T 27 were all commercial mixed species startersobtained from Flora Danica, Odense, Denmark,Christian Hansen Laboratories, Copenhagen, Den-mark, and Viesby Laboratories, Denmark, respec-tively. They were all composed of strains of S.cremorisiS. lactis, S. diacetilactis, and L. citrovorum.To keep the composition constant, the starters wereinoculated in skim milk at 2% (vol/vol) on reachingour laboratory and immediately frozen and stored inliquid nitrogen in stainless-steel tubes to serve as astock culture throughout this investigation. The effectof this preservation procedure on lactic starter FDs0172 can be seen in Table 1. The other starters usedwere equally stable towards freezing. When used formaking concentrate, a tube with inoculated frozenstarter was thawed at 25 C for 10 min and incubatedfor 16 h at 22.5 C. The starters were subcultured twicebefore use.Mass cultivation. The starters [2% (vol/vol)

inoculum] were cultivated in laboratory benchtopfermentors of 1- and 10-liter capacity (Biotec, Swe-den, and Marubishi Ltd., Japan) equipped withpH-stat assemblies (Radiometer, Denmark) to controlthe pH at a desired value by titration with 20%(vol/vol) NH4OH or 5 M NaOH throughout thecultivation. Temperature and stirring speed werecontrolled at desired levels. No aeration was neededfor cultivation of these bacteria. Samples to beanalyzed were taken from the fermentor through asampling port with a sterile pipette.

Media. Three types of media were used for thecultivation at constant pH. (i) Reconstituted skimmilk, 9% reconstituted nonfat milk solids (Semper,Sweden), was sterilized at 110 C for 10 min. (ii) Amedium based on whey with filtrate of papain-treated

TABLE 1. Freezing preservation (liquid N.) of stockcultures of mixed species starter FDs 0172

Relativelactic CO,a

Age acid- Colony- Aroma Diacetyl (om/(months) pro- forming bacteria (g/g) xell(mnh)ducing units/ml (%) xg cell)

activity X1-1

0 100 4.0 x 106 13 2.6 356 100 3.9 x 106 12 2.7 46

12 100 3.8 x 108 14 3.2 3918 100 4.1 x 106 11 2.7 3724 100 4.0 x 10' 14 3.1 41

a CO2 produced during 4 h at 30.0 C.

skim milk, yeast extract (Difco), and MnSO4 wasprepared according to the following procedure: 70 g ofwhey powder (spray dried) and 50 g of skim milkpowder in 1,000 ml of water were treated with 0.5 g ofpapain (12,000 U/g) at 30 C for 20 min, 50 C for 20min, 75 C for 15 min, and 95 C for 15 min. Theenzyme-treated suspension was cooled to 75 C, cen-trifuged (30 min, 5,000 x g), and filtered through amembrane filter (Seitz K 7). After addition of 5 g ofyeast extract and 0.14 g of MnSO4, the medium wassterilized at 121 C for 15 min. This medium was, whenproperly prepared, transparent with a slight opales-cence. (iii) A third medium prepared according toBergere (1) with 40 g of tryptone (Difco), 14 g of yeastextract (Difco), 90 g of lactose, and 0.14 g of MnSO4 in1,000 ml of water was also used. The lactose wassterilized in one-half the water volume at 115 C for 10min, and the other components were sterilized at121 C for 15 min.

Centrifugation. At harvesting time the cell sus-pension from the mass cultivation was cooled to +5 Cand centrifuged at +5 C in a Sorvall SS-3 superspeedcentrifuge at 34,000 x g with equipment for continu-ous centrifugation. The supernatant was discardedand the concentrate was used for analyses.

Analytical methods. (i) Bacterial estimates.Both microscopic and plate count methods were usedto estimate the total number of bacteria. In the milksubstrate, the number of cells, stained with methyl-ene blue, was counted directly in the microscope witha special equipment according to Skar (19). In thetransparent media, direct microscopic count wasmade in a counting chamber (Petroff-Hausser) with aphase contrast microscope. All microscopic countswere true total counts, i.e., the single cells in thestreptococcal chains were counted.

Total viable counts were made with a pour-platetechnique on calcium-citrate agar according to Nick-els and Leesment (14). The results were expressed ascolony-forming units per milliliter. All counts weremade in triplicate. Cell mass in the transparent mediawas as a control estimated by turbidostatic measure-ments in a Beckman model C calorimeter.

(ii) Bacterial balance. The bacterial balance inthe mixed species starter cultures was estimated oncalcium-citrate agar. The different strains were iden-tified according to the scheme of Nickels and Lees-ment (14).

(iii) Lactic acid production. The acid production(mainly lactic acid) was estimated throughout theconstant pH cultivation by reading the amount ofalkali added at intervals during the fermentation. Thereadings were converted into grams of lactic acidproduced.

(iv) Lactic acid-producing activity. The ability ofmixed species starters and mixed species starterconcentrates to produce lactic acid was measured aspH drop versus time with a six-channel registratingpH apparatus (Radiometer, Copenhagen). The mea-surement was made in reconstituted skim milk at30 C with magnetic stirring. The amount of inoculumwas 2% (vol/vol). Lactic acid-producing activity wascalculated from the time to reach pH 5.5 on the pHcurve and the initial number of bacteria in the vessel

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STUDIES ON BATCH PRODUCTION

according to a procedure of Bergere (1). This methodwas shown to be applicable to mixed strain starters, inwhich the lactic acid-producing strains dominated.

(v) Proteinase activity. Proteolysis of starters andstarter concentrates was determined by the method ofHull (11), modified by Citti et al. (5). Cultures orconcentrates were inoculated in reconstituted skimmilk to give approximately 106 cells/ml, 2.5 ml oftoluene per 100 ml of skim milk was added, and theamount of tyrosine and tryptophane liberated at 32 C,determined as tyrosine, was measured at intervals upto 24 h. The result was expressed as milligrams oftyrosine per 100 g of inoculated milk.

(vi) Aroma production. The amount of diacetylformed by the activity of S. diacetilactis and L.citrovorum in the mixed species starters and concen-trates was determined according to the method ofOwades and Jakovac (15), modified for milk culturesby Pack et al. (16). The sample to be tested wasinoculated in skim milk [1% (vol/vol)] and incubatedfor 16 h at 22.5 C. Twenty grams of the 16 h-culturewas used for the diacetyl determination. The resultswere expressed as micrograms of diacetyl per micro-liter in the milk culture after 16 h.

(vii) CO, production. The CO.-producing abilityof concentrated mixed species starters was deter-mined by the Warburg technique. The bacterialconcentrate was diluted in sterile, quarter-strengthRinger solution to a concentration of 101 to 5 x 108cells/ml. Five-tenths milliliter of this bacterial sus-pension was placed in the sidearm of a Warburg cup,which held 2.5 ml of reconstituted skim milk. Afterconnection to a manometer and tempering to 30.0 C,the suspension in the sidearm was mixed with theskim milk. Manometer readings were made every 30min up to 4 h. The CO2 production was calculated asmicroliters of CO, liberated per cell at inoculationtime (21).

RESULTS AND DISCUSSION

Conditions for optimal growth and lacticacid-producing activity. Optimal growth con-ditions for single strain mesophilic lactic acidbacteria have been thoroughly examined previ-ously (2, 3, 6, 17, 18), and the effect of controlledpH on the growth rate and maximum cell yieldis also well documented (2, 17). There was goodreason to believe that growth conditions for amixed species starter composed of mesophilicstrains or species of lactic acid bacteria shouldbe in good agreement with the results obtainedwith single strains. The parameters examined inthis study to determine the optimal growthconditions for cultivation of mixed species start-ers were chosen in the following intervals: 20 to30 C, pH 5.5 to 7.0, and three media, namelyskim milk, tryptone, and whey media. Apartfrom these variables, the effect of differentneutralizers for constant pH was examined.

Preliminary trials to see if there were markeddifferences in maximum cell yields with and

without pH control were in good agreement withprevious work (1, 2). From Table 2 it can beseen that cultivation of the mixed species start-ers at constant pH results in maximum yields 4to 20 times greater than those which are ob-tained with static milk cultures.

Preliminary trials also showed that the re-sponses to different growth conditions for themixed species starters used in this study werequite the same. The mixed species starter FDs0172 was therefore used in the fundamentaloptimization studies, and the results obtainedfor this starter were then tested on the othermixed species starters. The comparison in theoptimization studies was made with the freshcommercial starter (serving as inoculum) withrespect to bacterial balance and important cellactivities.

In Fig. 1-3 growth curves and lactic acid-pro-ducing activity for harvested cells at 20, 25, and30 C are illustrated. The results show that thegrowth rates for mixed species starter FDs 0172increased with increasing temperatures in theinterval examined, and that the maximum cellyield increased with the temperature especiallysignificantly when the starter was cultivated inskim milk. There was no significant differencein growth rate and maximum cell yield withdifferent constant pH in the three media tested.This is illustrated for skim milk in Fig. 4.Comparison of the different media used

shows that the growth rate and maximum cellyield are greatest in the whey and tryptonemedia; the cell yield is about three times greaterin these media than in skim milk. The variationin growth rate of the entire cell mass of themixed species starter at temperatures in theinterval 20 to 30 C, with an optimum at 30 C, isin good agreement with results obtained forpure cultures (3, 6, 18). The insignificant effectof pH on the growth rate and maximum cellcount is in accordance with the results ofPeebles et al. (17) and Pont and Holloway (18),but in contrast to the results of Bergere andHermier (2) and Cogan et al. (6). The smalleffect of pH in this study was probably only acoincidence because the different strains andspecies composing the mixed species starterwere affected in different ways by the constantpH values tested (Fig. 5 and 6).From Fig. 1 and 2, it can further be seen that

the relative lactic acid-producing activity of themixed species starter in skim milk and wheymedia (compared to frozen stock culture) de-clined rapidly after the cells had reached theend of the exponential growth period. Thesefindings show that the harvesting time forconcentrate making is very critical for mixed

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TABLE 2. Effect of growth conditions on maximum attainable population, bacterial balance, and othermetabolic activities at harvest time for various mixed species starters

Maximum Prote-Popu- ~~~~~~~~olytic CO. pro-

latio Time | Aromat Diacetyl activity" ductionb

Starter Medium Method of lationy rah bactroma prd mtf (itescultivation |forming baxtemrum M d |tion |tyrsi/ celtl/units/ml count (h) ml10 X 10-1)X 10'1) mlx 10') sample)

FDs 0172 Skim milk Not neutralized; 5 16 14 2.1 3.7 4022.5 C

Skim milk pH 6.5; 25 C 20 13 20 3.1 4.0 29Whey medium pH 6.5; 25 C 50 13 24 2.0 4.4 68Tryptone medium pH 6.0; 25 C 60 10 50 0.7 2.5 460

FD 0570 Skim milk Not neutralized; 2 15 13 2.5 2.4 5822.5 C

Skim milk pH 6.5; 25 C 25 13 15 2.4 3.6 40Tryptone medium pH 6.0; 25 C 71 11 46 1.0 1.1 380

CH 0170 Skim milk Not neutralized; 4 19 31 2.5 3.0 9622.5 C

Skim milk pH 6.5; 25 C 13 14 27 2.1 2.7 81Whey medium pH 6.5; 25 C 41 13 30 1.9 2.5 102Tryptone medium pH 6.0; 25 C 92 13 56 0.3 0.9 620

CHs 0170 Skim milk Not neutralized; 3 16. 26 3.0 5.1 8422.5 C

Skim milk pH 6.5; 25 C 20 13 28 2.6 4.2 103Tryptone medium pH 6.0; 25 C 40 13 48 0.4 2.2 510

T 27 Skim milk Not neutralized; 8 16 24 3.8 8.7 -

22.5 CSkim milk pH 6.5; 25 C 16 13 27 3.5 7.2Tryptone medium pH 6.0; 25 C 30 13 68 0.9 4.9

a Amount of tyrosine liberated after 6 h at 32 C.° CO, produced during 4 h at 30.0 C.

5 10'Time (hr)

FIG. 1. Growth (open symbols) and relative lacticacid-producing activity by harvested cells (closedsymbols) at (A, A) 20 C, (0, *) 25 C, and (0, 0)30 C. FDs 0172 grown in skim milk at pH 6.5.

species starters, in accordance with the resultsof Bergere (3) and Lloyd and Pont (13), but incontrast to the results of Bergbre and Hermier(2) and Cogan et al. (6). The greater sensitivityof strains Qf S. cremoris, which dominated thisstarter, to inhibitors (lactate, D-leucine) during

prolonged growth has been shown by Bergere(3). This was confirmed by differential counts(14) made in this study during the growthperiod (0 to 20 h) under different conditions.The effect of the cultivation -pH on lactic

acid-producing activity shows (Fig. 4) that the

t IU it . S

Tim (hr)

FIG. 2. Growth (open symbols) and relative lacticacid-producing activity by harvested cells (closedsymbols) at (A, A) 20 C, (0, U) 25 C, and (0, *)30 C. FDs 0172 grown in whey medium at pH 6.5.

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activity at pH 7.0 was constant over a longerperiod of time. This could perhaps be explainedby the differential counts (14), which revealedthat S. cremoris/S. lactis decreased less rapidlyat pH 7.0 than at the other pH values.

In tryptone medium (Fig. 3) the lactic acid-

producing activity exceeded 100% and declinedless markedly after reaching the end of theexponential growth phase. Great changes in themixed species starter occurred during thegrowth at constant pH with respect to bacterialbalance and other cell activities (Table 3; Fig.6). From this it can be concluded that thebacterial balance had changed very much dur-ing the cultivation procedure, and this would bethe explanation for the greatly increased counts

T05o.1

5 6 6.5 -20pH

S.rj

i

20 25 30Tetra (t

Time (hr)

FIG. 3. Growth (open symbols) and relative lacticacid producing activity by harvested cells (closedsymbols) at (A, A) 20 C, (0, *) 25 C, and (0, 0)30 C. FDs 01 72 grown in tryptone medium at pH 6.5.

5 10Timelhr)

15 20

FIG. 5. Specific growth rates of strains of (A) L.citrovorum, (0) S. lactis/S. cremoris, and (0) S.diacetilactis as functions ofpH and temperature. FDs0172 grown in skim milk.

A.

01

?1D-

55 60 65 70

IH

FIG. 4. Growth (open symbols) and relative lacticacid-producing activity by harvested cells (closedsymbols) at (0, U) pH 5.5, (V,V ) pH 6.0, (A, A) pH6.5, and (0, 0) pH 7.0. FDs 0172 grown in skimmilk at 25 C.

FIG. 6. Specific growth rates of strains of (A) L.citrovorum; (0) S. lactis/S. cremoris, and (0) S.diacetilactis as functions ofpH and temperature. FDs0172 grown in whey medium (A) and tryptone me-

dium (B).

TABLE 3. Effect of neutralizer on maximum attainable population, bacterial balance, and relative lacticacid-producing activity

Bacteria/Culturea Neutralizer Time (h) milliliter Aroma Relative

(microscopic bacteria (%) activity (%)count)

CH 0170 NH4OH 12 7 x 109 23 100CH 0170 NaOH 11.5 8 x 109 75 28FDs 0172 NH4OH 10 5.5 x 109 10 100FDs 0172 NaOH 10 5.0 x 109 25 8T 27 NH4OH 13 6 x 109 27 98T 27 j NaOH 12 5 x 109 38 72

FDs 0172, CH 0170, and T 27 were grown in skim milk at pH 6.5 at 25 C.

0.5

= 1,0!20 25 30

0.5 .

m5,5 2D 5 7.0

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and relative activities shown in Fig. 3. Thisdanger of bacterial changes in the cultivation ofmixed species starters in media very differentfrom milk has been postulated by Stadhouderset al. (20).Changes in the bacterial balance during

cultivation. When optimizing the growth con-ditions for a mixed species starter, both thegrowth rate and maximum cell numbers areimportant. The balance between individualstrains and species composing the starter isextremely important for metabolic activities ofthe final cell mass. That balance changes inmixed species starters occur was postulated byStadhouders et al. (20) and has been shown tobe the case by using the phage-typing techniqueby Gilliland (7) on concentrated starters. Nosystematic survey of the changes in bacterialbalance during constant pH cultivation ofmixed species starters in different media hashitherto been undertaken.The method used in this study, differential

counting on calcium-citrate agar (14), couldmerely give a rough picture of what happens tothe strains of S. cremorisiS. lactis, S. diaceti-lactis, and L. citrovorum during cultivation atconstant pH. This had to be complementedwith measurements of important metabolic ac-tivities (Table 2).Growth curves separated on S. kactisiS. cre-

moris, S. diacetilactis, and L. citrovorum re-vealed areas of exponential growth for the threespecies from which specific growth rates werecalculated. These are referred to as "apparent"specific growth rates in this paper because ofthe unknown number of strains of each speciescomposing the starter. In Fig. 5 and 6 changes inbacterial balance due to medium, pH, andtemperature are illustrated.Growth in skim milk at different pH values

and temperatures imposed no significantchanges in bacterial balance except for growthat pH 7. When grown in whey medium thebalance in the mixed species starter did changeduring constant pH cultivation at most of thetemperatures and pH values tested. This waseven more evident for growth in tryptone me-dium. The optimal growth conditions for FDs0172 in whey medium with respect to bacterialbalance were found to be pH 6.5 and 25 C. Intryptone medium no acceptable optimum wasfound. The nearest optimum at which a mini-mum balance change occurred was at pH 6.0and 25 C. This supports the conclusions ofStadhouders et al. (20) and Gilliland (7).Not only the growth rates for strains compos-

ing a mixed species starter, but also the time to

reach maximum population is of importance forthe balance in the final concentrate. Thesetimes were found to be almost equal in thisstudy, i.e., the differences in apparent growthrates illustrated in Fig. 5 and 6 gave a goodpicture of the strain composition of the finalstarter suspensions to be further concentrated.

Effect of neutralizer on growth and activ-ity. Two neutralizers, NaOH and NHOH,which are reported to affect lactic acid bacteriain different ways when used to control pH forconcentrate making (4, 10, 13, 17), were testedin this study to see if such an effect could beobserved on mixed species starters. From Table3 it can be seen that the growth rates andmaximum cell populations did not differ signifi-cantly when either of the two neutralizers wasused. NaOH, however, had a negative effect onbacterial balance and lactic acid-producing ac-tivity. The latter is contradictory to the resultsof Lloyd and Pont (13), who found an increasein activity when using NaOH compared toNH4OH.The decrease in lactic acid-producing bacte-

ria compared to aroma bacteria when NaOHwas used instead ofNHOH could be due to theeffect proposed by Zarlengo and Abrams (24)and Peebles et al. (17), that NH40H (free NH,)could more easily penetrate the cell wall andthereby control the internal pH more accu-rately. When using NaOH the sensitive S.cremoris cells die more rapidly due to too lowinternal pH at times.The size of the cells compared by microscopic

examination by the Skar method (19) did notseem to differ significantly when either of thetwo neutralizers was used.

Factors affecting maximal population. Inthis study, as well as in those reported in theliterature (2, 3, 10, 18), it is not the deficiency oflactose that stops the growth at a certainpopulation but instead the increased concentra-tion of certain inhibitors. Lactate has beenreported to inhibit growth from concentrationsof 2% and higher and definitely stops growth atabout 6% (18) in a pH-controlled cultivation.Gilliland and Speck (9) found an amino acid,D-leucine, which inhibited growth of lacticstreptococci. This amino acid was apparently ametabolite of the streptococci produced undercertain conditions.

In Fig. 7 the maximum lactate concentrationwhen growth ceased at different conditions ofcultivation is illustrated. From this it can beseen that the lactate concentration at max-imum population depended on all parametersinvestigated: temperature, pH, and media. The

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5.0

'730

22

§

55 6D 6.5

pH7.0 20 25 30

Twnpratw.' C

FIG. 7. Lactate concentration at maximum popu-lation as a function ofpH and temperature. FDs 0172grown in (0) skim milk, (0) whey medium, and (A)tryptone medium.

results of Bergere and Hermier (2) and Bergere(3) that a lactate concentration of 6% is inhibi-tory when S. lactis C 10 is cultivated in tryptonemedium at 25 C, pH 6.5, are, however, in goodaccordance with the results presented hereunder the same conditions. The conclusion to bedrawn from the results is that the inhibition oflactic starter growth at certain populationsunder controlled pH is more complex in naturethan previously found. A possibility is thatother inhibitors involved (for example, D-leu-cine) are produced at different rates dependingon the conditions of growth. This, however,must be further investigated before definiteconclusions can be drawn.

Lactic starter concentrates. In view of theresults obtained, mixed species starter bacterialconcentrates were made from the mixed speciesstarters FDs 0172, FD 0570, CHs 0170, CH 0170,and T 27. Cells cultivated at constant pH were

harvested at the late exponential phase ofgrowth and were concentrated by centrifuga-tion. The cell concentration in the concentratevaried between 5 x 1010 and 1011 cells/ml. Theresults of these trials are summarized in Table2.Optimal conditions for controlled pH cultiva-

tion of FDs 0172 with respect to growth rate,maximum population, bacterial balance, andlactic acid-producing activity seemed to bevalid for cultivation of the other starters exam-ined. Analysis of aroma production, diacetyl(15, 16), proteolysis (5, 11), and CO2 production(21, 22) were added to this investigation todetermine whether optimal conditions, accord-ing to the results above, changed other impor-tant metabolic activities necessary for a mixedspecies starter. From Table 2 it can be seen thatcultivation in skim milk and whey medium gavestarter concentrates with almost unchanged

properties compared to the control grown atfalling pH in skim milk. In tryptone mediumthere were marked changes in the parametersexamined, compared to the control. The per-centage of aroma bacteria, particularly S. dia-cetilactis, increased by a factor of 2 to 3, all inagreement with the results in Fig. 6. Theamount of diacetyl produced by the tryptonemedium-cultivated concentrate in skim milkwas very low after 16 h, probably due to thehigher numbers of S. diacetilactis present inthis concentrate or due to the lack of citrate inthis medium, which was shown by Gilliland etal. (8) to impair subsequent diacetyl productionin milk medium. Gas production increased withthe increasing amount of aroma bacteria up to10 times the value of the control. Proteolyticaction of the cell mass was lowered by cultiva-tion in tryptone medium in accordance with theresults of Williamson and Speck (23).The danger of bacterial balance changes and

consequent changes in metabolic activitieswhen mixed species starter concentrates aremade as postulated by Stadhouders et al. (20)and shown for cheddar cheese cultures by Gilli-land (7) has been shown to be valid for mixedspecies starter concentrate production, pre-pared directly from mixed species starters com-posed of acid-producing bacteria and aromaformers. But, as shown in this study, mixedspecies starter concentrates with unchangedproperties compared to inoculum could be pro-duced by choosing the proper conditions forcultivation, such as medium, constant pH,temperature, neutralizer, and harvest time.

ACKNOWLEDGMENTSI wish to thank G. Sjdstrbm, Division of Technical Micro-

biology, for initiating the work on mixed strain starterconcentrates and for stimulating discussions, and also H.Ogura for his skillful analytical assistance.

This work was financially supported by the SwedishDairies' Association.

LITERATURE CITED1. Bergere, J.-L. 1968. Production massive de cellules de

streptocoques lactiques. I. Methodes g6nerales d'6tudeet facteur de la croissance de "Streptococcus lactis"souche C 10. Lait 48:1-11.

2. Bergbre, J.-L., and J. Hermier. 1968. La productionmassive de cellules de streptocoques lactiques. II.Croissance de "Streptococcus lactis" dans un milieu apH constant. Lait 48:13-30.

3. Bergere, J.-L. 1968. Production massive de cellules destreptocoques lactiques. m. Production de differentessouches en culture a pH constant. Lait 48:131-139.

4. Blaine, J. W., W. E. Sandine, and P. R. Elliker. 1970.Preparation of concentrates of lactic streptococci frommilk cultures. J. Dairy Sci. 53:637.

5. Citti, J. E., W. E. Sandine, and P. R. Elliker. 1963. Someobservations on the Hull method for measurement ofproteolysis in milk. J. Dairy Sci. 46:337.

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6. Cogan, T. M., D. J. Buckley, and S. Condon. 1971.Optimum growth parameters of lactic streptococci usedfor the production of concentrated cheese starter cul-tures. J. Appl. Bacteriol. 34:403-409.

7. Gilliland, S. E. 1971. Strain balance of multiple strainlactic streptococcus concentrated cultures. J. DairySci. 54:1129-1133.

8. Gilliland, S. E., E. D. Anna, and M. L. Speck. 1970.Concentrated cultures of Leuconostoc citrovorum.Appl. Microbiol. 19:890-893.

9. Gilliland, S. E., and M. L. Speck. 1968. D-leucine as anauto-inhibitor of lactic streptococci. J. Dairy Sci.51:1573-1578.

10. Gilliland, S. E., and M. L. Speck. 1974. Frozen concen-trated cultures of lactic starter bacteria. A review. J.Milk Food Technol. 37:107-111.

11. Hull, M. E. 1947. Studies on milk proteins. II. Colorimet-ric determination of the partial hydrolysis of theproteins in milk. J. Dairy Sci. 30:881-884.

12. Kosikowski, F. 1966. In Cheese and fermented milk foods,p. 20-21. Ithaca, N. Y.

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140 PETTERSSON


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ERRATAStudies on Batch Production of Bacterial Concentrations

from Mixed Species Lactic Starters

H.-E. PETTERSSONChemical Center, Division of Technical Microbiology, University of Lund, S-230 53Alnarp, Sweden

Volume 29, no. 2, p. 133, 2nd col., line 2: "... mixed strain strater ..." should read "... mixedspecies starter . . ."

P. 134, 1st col., line 2: "... mixed strain starter ..." should read "... mixed speciesstarter . . ."

P. 134, Table 1, last column heading: should read CO 2 (Ml/cell x 108).P. 135, 1st col., line 2: "... mixed strain starters ..." should read "... mixed species

starter . . ."P. 135, 1st col., 2nd para., lines 10-11: "... micrograms of diacetyl per microliter ..." should

read ".... micrograms of diacetyl per milliliter..."P. 139, Acknowledgments, line 2: "... mixed strain starter .. ." should read "... mixed species

starter . . ."

Detection and Growth of Enteropathogenic Escherichia coliin Soft Ripened Cheese

L. D. FANTASIA, L. MESTRANDREA, J. P. SCHRADE, AND J. YAGER

Food and Drug Administration, Brooklyn, New York 11232

Volume 29, no. 2, p. 179, author line: The asterisk indicating author to whom reprint requestsshould be addressed should be inserted beside L. Mestrandrea.

P. 180, 1st col., 4th line from bottom: "... potassium phosphate buffer ..." should read "...Butterfield's phosphate buffer . . "

P. 182, 2nd col., 1st para., 2nd line from bottom: "... cheeses when they were removed ..."should read ". . . cheeses during those times when they were removed . . ."

P. 183, 1st col., line 4: "... F.R.I. Annual Report of ..." should read "... F.R.I. Annual Re-port (1972) of . . "

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studies on batchproduction bacterial concentrates from species lactic...

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