Influence of starter cultures on the proteolysis of Teleme

cheese made from different types of milk

Eleni C. Pappa, Ioannis G. Kandarakis, Gregory K. Zerfiridis, Emmanuel M.

Anifantakis, Kyriaki Sotirakoglou

To cite this version:

Eleni C. Pappa, Ioannis G. Kandarakis, Gregory K. Zerfiridis, Emmanuel M. Anifantakis,Kyriaki Sotirakoglou. Influence of starter cultures on the proteolysis of Teleme cheese madefrom different types of milk. 2006, 86 (4), pp.273-290.

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273Lait 86 (2006) 273290 INRA, EDP Sciences, 2006DOI: 10.1051/lait:2006010

Original article

Influence of starter cultures on the proteolysis of Teleme cheese made from different types of milk

Eleni C. PAPPAa*, Ioannis G. KANDARAKISb, Gregory K. ZERFIRIDISc, Emmanuel M. ANIFANTAKISb, Kyriaki SOTIRAKOGLOUd

a National Agricultural Research Foundation, Dairy Research Institute, 452 21, Katsikas, Ioannina, Greece

b Laboratory of Dairy Technology, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, 118 55, Athens, Greece

c Laboratory of Dairy Technology, Department of Food Science and Technology, Faculty of Agriculture,

Aristotle University of Thessaloniki, 541 24, Thessaloniki, Greeced Department of Mathematics, Agricultural University of Athens, Iera Odos 75, 118 55, Athens, Greece

Received 20 June 2005 Accepted 3 April 2006

Abstract Teleme cheese was made from ovine, caprine and bovine milk with thermophilic (Strep-tococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus), mesophilic (Lactococcus lac-tis subsp. lactis, Lactococcus lactis subsp. cremoris) and mixed thermophilic-mesophilic (1:1) cul-tures. Nine lots of cheese each time (five repetitions) were made in order to study the proteolysis ofcheeses during ripening and storage up to 6 months. All cheeses showed similar profiles of proteo-lysis. However, the rate of proteolysis was different. The thermophilic and the mesophilic culturesresulted in cheeses with the highest and the lowest degree of proteolysis, respectively. Proteolysiswas of a descending order in cheeses made from sheeps, goats and cows milk when estimated onthe basis of nitrogenous fractions (total nitrogen, water soluble nitrogen, nitrogen soluble in 5%phosphotungstic acid and nitrogen soluble in 12% trichloroacetic acid), whereas it was highest incows milk cheeses and lowest in goats milk cheeses when estimated on the basis of electrophoresisand RP-HPLC. Multivariate analysis of the data revealed that cheeses could be more correctly clus-tered by the type of milk or culture than by the stage of ripening.

Teleme cheese / white-brined cheese / proteolysis / starter culture

- Teleme) (Teleme cheese),

(Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus)(Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris) (1:1) 3

9 5 6 5%

12% / / /

* Corresponding author (): instgala@otenet.gr

Article published by EDP Sciences and available at http://www.edpsciences.org/lait or http://dx.doi.org/10.1051/lait:2006010

274 E.C. Pappa et al.

Rsum Influence du type de lait et de levain sur la protolyse de fromage Tlm au coursde laffinage. Du fromage Tlm a t fabriqu avec du lait de brebis, du lait de chvre et du laitde vache en utilisant des levains lactiques thermophiles (Streptococcus thermophilus, Lactobacillusdelbrueckii subsp. bulgaricus), msophiles (Lactococcus lactis subsp. lactis, Lactococcus lactissubsp. cremoris) ou mixtes thermophiles-msophiles (1 : 1). Neuf fabrications (cinq rptitions) ontt ralises pour tudier la protolyse des fromages au cours de laffinage pendant 6 mois. Tous lesfromages avaient un profil protolytique similaire, mais le degr de protolyse tait diffrent. Lesfromages fabriqus avec des levains thermophiles taient les plus protolyss et ceux fabriqus avecdes levains msophiles les moins protolyss. La protolyse observe, estime par les fractions azo-tes (azote total, azote soluble en phase aqueuse, azote soluble dans le PTA 5 % et azote solubledans le TCA 12 %), diminuait dans lordre lait de brebis, lait de chvre et lait de vache mais elletait la plus forte pour le fromage prpar avec du lait de vache et la plus faible pour le fromageprpar avec du lait de chvre quand elle tait estime par lectrophorse et HPLC phase inverse.Lapplication de lanalyse multivarie a rvl que la classification des fromages tait plus correcteavec le type de levains lactiques ou de lait utiliss quavec le degr de maturation.

Tlm / fromage en saumure / protolyse / levain / maturation

1. INTRODUCTIONProteolysis is an important biochemical

process in the ripening of most cheese vari-eties. Proteolysis, which contributes to thedevelopment of cheese texture and flavor,is due to the action of enzymes from (a)coagulant, (b) milk (general plasmin), (c)starter bacteria, (d) the adventitious non-starter microflora and (e) secondary starter(in some cheese varieties). The proteolyticsystem of lactic acid bacteria (LAB) con-sists of proteinases, which hydrolyzecaseins, and large peptides and peptidases,which are responsible for the liberation ofsmall peptides and free amino acids (FAA),an essential step in cheese ripening [43].

White-brined cheeses, in which Telemecheese is included, form a special categoryof cheeses. Their main characteristic is thatthey are ripened and stored in brine. Tradi-tionally, Teleme cheese was manufacturedfrom non-pasteurized milk, but nowadays itis manufactured on an industrial scale. Rig-orous control of the production and matu-ration process is necessary. The selection ofthe starter culture is one of the most impor-tant steps in the manufacture of high-qual-ity cheeses. In traditional Teleme cheesemanufacture, yogurt is used as a starter. Amixture of commercial starters, includingmesophilic starters, are used in industrialproduction [2]. Studies concerning theeffect of the starter cultures used on the rip-ening of Teleme cheeses have not been pre-viously reported in a systematic way [20,

45]. In this study we investigated the effectof three commercial concentrated starters,i.e. a thermophilic, a mesophilic and a mix-ture of a thermophilic-mesophilic culture,on the proteolysis of Teleme cheese madefrom sheeps, goats or cows milk duringripening (at 19 C) and storage (5 C ) of upto 180 d.

2. MATERIALS AND METHODS

2.1. Experimental designThe experimental design was that

described in a previous paper [33]. That is,Teleme cheese was manufactured using (a)sheeps milk of the Boutsiko breed fromthe Agricultural Station of Ioannina, (b)goats milk from a local native goat popu-lation and (c) cows milk from a herd ofFriesian cows, near the Dairy ResearchInstitute where the cheese-makings tookplace. Each kind of milk was used in mak-ing Teleme cheese with three starter culturevariables, that is: (i) a freeze-dried yogurtthermophilic culture (T), FYS-11 (Rhodia,France), consisting of the microorganismsStreptococcus thermophilus and Lactoba-cillus delbrueckii subsp. bulgaricus, (ii) afreeze-dried concentrated mixture of ther-mophilic-mesophilic culture (MX), FRC-60 (Chr. Hansen, Denmark), consisting ofthe microorganisms Lactococcus lactissubsp. cremoris, Lactococcus lactis subsp.lactis, Lactobacillus delbrueckii subsp.

Proteolysis of Teleme cheese 275

bulgaricus and Streptococcus ther-mophilus - the ratio of thermophilic to mes-ophilic microorganisms was 1:1, and (iii) afreeze-dried concentrated mesophilic cul-ture (M), R-703 (Hansens Laboratorium,Denmark) consisting of the microorgan-isms Lactococcus lactis subsp. lactis andLactococcus lactis subsp. cremoris. Forone week, on a day after day basis, onebatch of cheese-making of sheeps, goatsor cows milk took place. On the day of thecheese-making, the milk (sheeps, goats orcows) was divided into three equal partsand in each part one of the three cultureswas used. Therefore nine lots of cheesewere obtained in one week. This procedurewas repeated during five subsequent weeks.The reported results are the averages of fivetrials.

2.2. Cheese-making and sampling

Teleme cheese was manufactured asdescribed previously [33]. Briefly, the milkwas standardized to casein to fat ratios of0.75, 0.64 and 0.73 for the sheeps, goatsand cows milk, respectively, to obtain afirst quality cheese that was 56% moistureand 43% fat in dry matter [15]. The stand-ardized milk was pasteurized at 63 C for30 min and then cooled at 37 C. Aftercooling, one of the cultures mentionedalready was added to it, at a quantity of0.5% (v/v). To assist curdling of the milkCaCl2 solution (40%, w/v) was added. Rennetpowder HA-LA (Hansens Laboratorium,Horsholm, Denmark) was added at a quantityof 3.0 g per 100 kg milk, at 33 C. Drainagewas under pressure and salting took placein brine (14% NaCl, w/w) for 16h at 19 C.

After manufacture, cheeses were rip-ened in a warm room (19C), until pH andmoisture values reached the limits of 4.6and 56%, respectively, and afterwardscheeses were transferred to a cold room(5 C) for up to 6 months (period of sam-pling). The relative humidity of the roomswas medium to avoid rusting of the tins,since Teleme cheese is ripened and storedin sealed tins. Teleme cheeses made fromsheeps milk using thermophilic, ther-mophilic-mesophilic or mesophilic culturewere transferred to the cold room at 24, 16

and 12 days, respectively, whereas cheesesmade from goats milk using thermophilic,mixed or mesophilic culture were trans-ferred to the cold room at 22, 13 and 11 days,respectively, and cheeses made from cowsmilk using thermophilic, mixed or mes-ophilic culture were transferred to the coldroom at 23, 17 and 15 days, respectively.

Cheeses were sampled for analysis ondays 1, 60, 120 and 180, as well as on theday they were transferred to 5 C (cold room).Due to Teleme cheese being ripened andstored in brine, the excess of brine was drainedoff by leaving the sample on an absorbingpaper for 12 min during cheese sampling.

2.3. Nitrogen fractionsCheeses were analyzed for total nitrogen

(TN), water-soluble nitrogen (WSN), nitro-gen soluble in 5% phosphotungstic acid(PTA-N) and nitrogen soluble in 12%trichloroacetic acid (TCA-N) as describedby Pappa and Anifantakis [32].

2.4. Urea-polyacrylamide gel electrophoresis (Urea-PAGE)

Pure reference caseins (CN) frombovine, ovine or caprine milk as well ascheese samples and their water-solubleextracts were analyzed in duplicate by urea-PAGE as described by Mallatou et al. [25].From the densitograms the levels of resid-ual s- and -casein in the aged cheeseswere calculated in comparison with thelevel present in reference samples of 1-day-old cheese. The zones of pure whole caseinsamples of the corresponding milk wereused in electrophoresis for the identifica-tion of different bands. The levels of pep-tides in the WSN extract of cheeses werecalculated in comparison with the levelpresent in the reference sample of 1-day-oldWSN extract of cheese.

2.5. Reversed-phase high performance liquid chromatography (RP-HPLC)

Peptide profiles of the water-solublefraction of the cheeses made from thedifferent kinds of milk and culture usedwere determined by RP-HPLC using a

276 E.C. Pappa et al.

Waters HPLC system (WATERS Associ-ates, Milford, MA, USA), as described byMallatou et al. [25]. After each run, the inte-gration area of peptides was determined anddivided into two regions with the criterionbeing the elution time of the peaks. The firstgroup consists of the hydrophilic peptides(HL) with retention times from 0 to 67.5 min(055% eluent B). The second groupconsists of hydrophobic peptides (HB)with retention times from 67.6 to 110 min(55.1100% eluent B).

2.6. Statistical analysisThe statistical analysis of results was

done by a multifactor analysis of varianceusing the software STATGRAPHICS Plusfor Windows v.5.2 (1995, Manugistics, Inc,Rockville, MD, USA). The least significantdifference of the data is reported (P < 0.05).Principal component analysis (PCA) andlinear discriminant analysis (LDA) werealso applied to the data using the same sta-tistical program.

3. RESULTS The composition of cheeses made with

different kinds of milk and cultures hasbeen reported in a previous paper [33]. ThepH of all mature cheeses ranged between4.26 and 4.38.

The changes in nitrogenous fractionsduring ripening of Teleme cheese made fromdifferent kinds of milk and cultures are shownin Table I. The values of TN (g100 g1cheese on a wet basis) of mature cheeses(over 60 days old) made from sheeps,goats or cows milk ranged from 2.222.48, 2.372.55 and 2.162.36, respec-tively, regardless of the kind of culture used(Tab. I). Mature cheeses made with the ther-mophilic culture had lower TN values(2.662.16) than cheeses made from theother two cultures, regardless of the kind ofmilk used (P < 0.05). Cheeses made withthe mixed (2.552.30) or mesophilic (2.532.29) cultures did not show any significantdifference (P > 0.05) in their TN values.From Table I, it can also be seen that thelevel of the %WSN, %PTA-TN and%TCA-TN increased during ripening(P < 0.05). The % WSN/TN content of

cheeses made from different types of milkranged from 3.1019.02%, 3.6913.00%and 4.9917.10% for sheeps, goats andcows milk, respectively, regardless of ageor culture used. The % WSN/TN content ofcheeses made with the thermophilic, mixedand mesophilic culture varied from 8.7519.02%, 4.6012.54% and 3.1010.15%,respectively. The values of PTA-N ofcheeses made from sheeps, goats or cowsmilk ranged from 1.122.69%, 1.102.86%and 1.102.94%, respectively, regardlessof the kind of culture used. The values ofPTA-N of Teleme cheeses made with thethermophilic, the mixed or the mesophilicculture in this study ranged from 1.362.94%, 1.102.42% and 1.102.28%,respectively, regardless of the kind of milkused. The TCA-N content of cheeses madefrom sheeps, goats and cows milk rangedfrom 2.1411.92%, 1.8010.32% and1.8010.31%, respectively, regardless ofthe culture used. The TCA-N content ofcheeses made with the thermophilic, mixedand mesophilic culture varied from 2.8011.92%, 1.999.11% and 1.808.51%,respectively.

The results obtained by urea-PAGEelectrophoresis for Teleme cheeses andtheir WSN, made with the three cultures,were qualitatively similar. The quantitativeresults were different and are presented inTable II. Qualitative results from cheesesand their WSN made from the three typesof milk with the mixed culture are shown inrepresentative electrophoregrams (Figs. 1and 2, respectively). Considering thatcaseins are intact on day 1, then the highests-CNs hydrolysis (estimated as the differ-ence of the lowest percentage of residual s-CNs from 100) ranged from 100 to 54.4%,100 to 73.4% and 100 to 34.4% whilehydrolysis of -CN ranged from 100 to75.9%, 100 to 84.2% and 100 to 70.2% forcheeses made from sheeps, goats or cowsmilk, respectively, regardless of the kind ofculture used. Therefore, Teleme cheeses madefrom cows, goats or sheeps milk had thehighest, lowest and intermediate degree ofhydrolysis, respectively (P < 0.05). Thehydrolysis of s-CNs ranged from 100 to34.4%, 100 to 50.3% and 100 to 61.7% andthe hydrolysis of -CN ranged from 100 to

Proteolysis of Teleme cheese 277Ta

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