ORIGINALRESEARCH Influence of an autochthonous starter culture and a

commercial starter on the characteristics of Tenerifepasteurised goats’ milk cheese

LORENA GONZALEZ and VICTORIA ZARATE*Departamento de Microbiologıa y Biologıa Celular, Universidad de La Laguna, 38206 La Laguna, Tenerife, Spain

*Author forcorrespondence. E-mail:vzarate@ull.es

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Two batches of Tenerife cheese were produced from pasteurised goats’ milk, one with a commercial star-ter and another with an autochthonous starter consisting of three selected strains of lactic acid bacteriaisolated from artisanal Tenerife cheese. Influence of starter on the characteristics of the cheeses wasevaluated. Owing to the composition of the starters, lactobacilli and leuconostocs varied significantlybetween both cheeses. However, the type of starter did not significantly affect the physicochemical andproteolytic properties of the cheeses. In general, cheeses made with autochthonous starter receivedhigher scores for sensory attributes, especially for aroma, than those made with commercial starter.

Keywords Lactic acid bacteria, Autochthonous starter culture, Goats’ milk cheese.

INTRODUCT ION

Cheese ripening is a very complex biochemicalprocess involving glycolysis, proteolysis, lipolysisand production of volatile aroma compounds, thesereactions being catalysed by residual rennet, indig-enous milk enzymes and enzymes of microbialorigin (Fox et al. 1993).In cheesemaking, the use of raw milk allows the

manufacture of high-value traditional varieties,where the ripening processes are produced mainlyby wild strains of lactic acid bacteria (LAB)derived from milk or environment. Also, cheesesare manufactured on an industrial scale from pas-teurised milk. Pasteurisation allows the standardi-sation of the cheese processing as well as theimprovement of hygienic and sanitary quality butalso eliminates autochthonous microbial flora ofmilk that is replaced by commercial starters. Ingeneral, raw milk cheeses are considered to havemore intense aroma and flavour than those madefrom pasteurised milk because of the inactivationof indigenous milk enzymes and the poor biodiver-sity of the LAB present in the commercial starters(Leroy and de Vuyst 2004).Because of the growing interest in preserving the

typical sensory properties of traditional cheeses,there is an increasing demand for autochthonousstrains to replace or complement existing commer-cial starters currently used by the dairy industry

(Broome 2007). In fact, many studies have beenpublished in the past few years which describe theuse of autochthonous starter cultures in the manu-facture of cheeses, including Spanish varieties suchas Ibores (Gonzalez et al. 2003), Tetilla (Menendezet al. 2004), Armada (Herreros et al. 2007) andManchego (Gomez-Ruiz et al. 2008).Tenerife cheese is a variety manufactured

throughout the island of Tenerife (Spain) fromgoats’ milk. It can be prepared in farms from rawmilk without the addition of starter cultures accord-ing to the traditional method (artisanal cheese) asreported by Zarate et al. (1997) or in cheese dairiesfrom pasteurised milk with the addition of com-mercial starter (industrial cheese).In previous work, we have designed an autoch-

thonous cheese starter culture that included threeLAB isolated from good quality raw Tenerife goats’milk cheese and that were selected on the basis oftheir technological properties (Perez 2001; Perezet al. 2003). Lactococcus lactis subsp. lactis TF53is the primary starter organism because of its highacidifying and proteolytic activity while Lactobacil-lus plantarum TF191 and Leuconostoc mesentero-ides subsp. mesenteroides TF756 are flavour ⁄aromaproducer organisms because of the diacetyl produc-tion and high peptidolytic activity of the former andbecause of the high esterolytic activity of the later.The aim of this study was to compare the

microbiological, physicochemical, proteolytic and

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sensory characteristics of pasteurised Tenerife cheeses madewith either a commercial starter or the autochthonous starterdescribed above. This is necessary to produce industrial cheeseswith good microbiological quality while preserving the originalTenerife cheese characteristics.

MATER IAL AND METHODS

Starter culturesThe autochthonous starter culture used in this study comprisedLac. lactis subsp. lactis TF53, L. plantarum TF191 andLeu. mesenteroides subsp. mesenteroides TF756. Before theirinclusion as starter cultures, the inability to produce biogenicamines was assessed with the procedure described by Bover-Cid and Holzapfel (1999). Furthermore, these strains did notproduce antibacterial substances against each other followingthe method described by Fleming et al. (1985).Freeze-dried preparations of autochthonous LAB strains

were prepared. An overnight culture of each LAB strain inMRS (de Man, Rogosa and Sharpe) was inoculated in a 2.5-Lfermentor (Minifors Infors HT, Allerød, Denmark) containing1100 mL of sterile MRS. The temperature was adjusted to30 �C, and the stirring speed was set at 100 rpm. No aerationwas performed during the fermentation, and the pH wasadjusted automatically by addition of sterile 1 N HCl and 5 NNaOH to 6.0 (for the Lactococcus and the Leuconostoc strain)and to 5.7 (for the Lactobacillus strain). After 20–22 h, the cul-ture medium was cooled to 4 �C, and cells were harvested at12 000 g for 5 min at 4 �C (Avanti J-E, Beckman-Coulter,CA, USA). Cells were washed three times with 20 mM sodiumphosphate buffer, pH 6.5 and suspended to one-tenth of the ori-ginal volume with the lyophilisation medium composed of10% w ⁄v sterile reconstituted skimmed milk (RSM) containing12.5 g ⁄L glutamic acid monosodium salt hydrate (Sigma, St.Louis, MO, USA). After 1 h at room temperature to allowequilibration between cells and added compounds, cellular sus-pensions were frozen at )80 �C and subsequently desiccatedunder vacuum in a Drywinner lyophilizer (Heto-Holten, Bottm-inger, Switzerland). Dried cells, with about 1010 viablecounts ⁄g, were stored in closed containers at 4 �C in darkness.Choozit TM RA21 (Danisco, Copenhagen, Denmark), com-

posed of Lac. lactis subsp. lactis, Lac. lactis subsp. cremoris andStreptococcus thermophilus, was used as the commercial startersince it is the starter commonly used in Tenerife cheese dairies.

Cheese manufacture and samplingTwo replicate cheesemaking trails were performed on differentdays in the pilot plant of the Escuela Superior de CienciasAgrarias (Universidad de La Laguna, Tenerife, Spain) underthe conditions of Tenerife cheese manufacture. In each trial,two batches were manufactured using the same pasteurisedgoats’ milk: Batch C was made using the lyophilised commer-cial starter added at the manufacturer¢s recommended dose(9.47 g ⁄1000 L of milk) and Batch A was made with the

lyophilised autochthonous Tenerife cheese starter inoculated invat milk at a final concentration of about 106 colony-formingunits (cfu) ⁄mL for Lac. lactis subsp. lactis TF53 and of about104 cfu ⁄mL for L. plantarum TF191 and Leu. mesenteroidessubsp. mesenteroides TF756.For each batch, 20 L of pasteurised goats’ milk containing

CaCl2 (20 g ⁄100 L final concentration) was set to 28–30 �C,inoculated with the starter, stirred for 10 min and held for30 min at 28–30 �C. Commercial rennet (25 mg ⁄L, CHY-MAX; Chr. Hansen Laboratory, Copenhagen, Denmark) wasadded, and after agitation, the milk was left to settle at 32 �Cuntil coagulation (approximately 30 min). Curd was cut intograin size (approximately 5 mm) while the temperature wasincreased to 37 �C with agitation. The curd was placed in cylin-drical moulds of 1 kg and pressed (2 kg ⁄cm2) until a pH of5.3–5.5 was reached. Cheeses were salted dry, applying saltdirectly on their surface, and placed again in the moulds. Thefollowing day, cheeses were removed from the moulds, washedto eliminate the excess of salt and transferred to a ripeningroom where they remained at 10 �C and 80% relative humidity.Milk for each trial before inoculation with the starter cultureand one cheese (approximately 1 kg) from each batch at differ-ent days of maturation were sent to the laboratory under refrig-eration conditions. Microbiological and physicochemicalanalysis of cheeses was performed upon arrival. Protein contentand proteolysis assessment was performed with a portion of thecheese sample stored at )20 �C until analysis was in order.

Microbiological analysisCheese homogenate, decimal dilutions, plating procedures andcounts of total viable micro-organisms, faecal coliforms, lacto-cocci, lactobacilli and leuconostocs were determined as previ-ously described (Zarate et al. 1997), except that lactobacilliwere incubated at 37 �C instead of at 30 �C. All analyses wereperformed in duplicate.

Physicochemical analysisDuplicate cheese samples were subjected to compositionalanalysis. Acidity was determined by the AOAC method920.124 (AOAC 2006a), moisture by the IDF Standard 4A(IDF 1982) and NaCl content according to standard of Ameri-can Public Health Association (Bradley et al. 1993). The pH ofgrated cheese (5 g) that homogenised with 45-mL distilledwater was determined using a Crison GLP21 pH-meter (Crison,Barcelona, Spain). Fat content was determined by the VanGulik method (IDF Standard 222 2008). Protein (total nitrogen·6.38) was determined using 0.3-g samples by the micro-Kjel-dahl method 2001.14 (AOAC 2006b).

Assessment of proteolysisWater-soluble nitrogen (WSN), nitrogen soluble in 12% trichlo-roacetic acid (TCA-SN) and nitrogen soluble in 5% phospho-tungstic acid (PTA-SN) were determined according to Sousaand Malcata (1997). For obtaining the water-soluble extract

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(WSE), 25 g of cheese were homogenised with 50-mL distilledwater (40 �C) using a T8 Ultra-Turrax (IKA-Werke, Staufen,Germany). The homogenate was held at 40 �C for 1 h, centri-fuged at 12 000 g for 10 min at 4 �C and filtered throughWhatman No. 1 filter paper (Whatman International Ltd, Maid-stone, UK). The trichloroacetic acid (TCA) soluble extract wasprepared by adding 5 mL of 48% TCA to 15 mL of WSE. Themixture was allowed to stand for 2 h at room temperature andfiltered through Whatman No. 40 paper. Phosphotungstic acid(PTA) soluble extract was prepared by adding 3.5 mL of3.95 M sulphuric acid and 1.5 mL PTA (33.3% w ⁄v) to 5 mLWSE. The mixture was allowed to stand overnight at 4 �C andsubsequently filtered through Whatman No. 40 paper. Thenitrogen content of these fractions was determined by themicro-Kjeldahl method and was expressed as percentage oftotal nitrogen. All analyses were carried out in duplicate.

Sensory analysisCheeses were graded at 30, 60 and 90 days by a team of 10–15panellists selected among staff and students of the Universityon the basis of their general liking and interest in cheeses. Thepanel was then trained in the sensory characteristics of Tenerifegoats’ milk cheese. Following the IDF recommendations (IDF1995), outer appearance, texture, aroma, taste and generalacceptability were scored by using a 7-point scale (1, very poor;and 7, very good).

Statistical analysisPASW Statistics 18 software package (SPSS Inc., Chicago, IL,USA) was used for the statistical analysis. One-way analysis ofvariance (ANOVA) was performed on the results obtained at dif-ferent stages of ripening, with the batch and cheese age as maineffects. Differences were considered significant at P < 0.05.

Table 1 Microbial counts (log cfu ⁄ g) during ripening of cheeses made with commercial (C) or autochthonous (A) starter

Batch

Counts

Time (days) Total counts Lactococci Lactobacilli Leuconostocs

C 2 9.45 ± 0.10 9.44 ± 0.43 <1 <230 9.60 ± 0.19 9.49 ± 0.36 6.60 ± 0.12 <260 9.35 ± 0.46 9.47 ± 0.19 6.62 ± 0.14 3.70 ± 0.1390 8.57 ± 0.24 8.39 ± 0.23 5.72 ± 0.23 3.08 ± 0.26

A 2 9.53 ± 0.33 9.41 ± 0.35 6.43 ± 0.33 5.40 ± 0.2830 9.69 ± 0.54 9.42 ± 0.12 7.69 ± 0.12 7.04 ± 0.1360 9.36 ± 0.19 9.51 ± 0.55 7.62 ± 0.29 7.20 ± 0.1490 8.86 ± 0.17 9.00 ± 0.41 6.50 ± 0.13 5.43 ± 0.39

ANOVAa

Batch NS NS * ***

Ripening time *** *** NS NS

Data are mean ± SD of duplicate determinations in two cheesemaking trials.aSignificance levels: *P < 0.05; **P < 0.01; ***P < 0.001; NS, non significant.

Table 2 Evolution of physicochemical parameters during ripening of cheeses made with commercial (C) or autochthonous (A) starter

Batch Time (days)

Physicochemical parameters

pH Acidity (%) NaCl (%) Moisture (%) Fat (%) Protein (%)

C 2 5.18 ± 0.08 1.21 ± 0.21 1.40 ± 0.08 54.00 ± 0.96 21.50 ± 0.25 17.50 ± 0.0830 5.17 ± 0.02 1.22 ± 0.09 1.44 ± 0.01 52.30 ± 0.70 25.30 ± 0.41 21.18 ± 0.4260 5.32 ± 0.04 1.32 ± 0.05 2.20 ± 0.07 36.70 ± 0.50 25.50 ± 0.41 25.01 ± 0.0790 5.54 ± 0.04 1.28 ± 0.05 2.25 ± 0.06 36.20 ± 0.57 27.50 ± 0.29 25.90 ± 0.19

A 2 5.27 ± 0.06 1.24 ± 0.02 1.53 ± 0.02 52.00 ± 0.82 22.00 ± 0.85 17.64 ± 0.2830 5.26 ± 0.05 1.26 ± 0.07 1.51 ± 0.04 49.70 ± 0.63 26.30 ± 1.10 22.78 ± 0.1760 5.29 ± 0.15 1.50 ± 0.08 2.06 ± 0.01 36.50 ± 1.60 26.00 ± 0.29 23.86 ± 0.3190 5.33 ± 0.14 1.37 ± 0.09 2.27 ± 0.04 36.30 ± 1.28 28.00 ± 1.15 25.41 ± 0.26

ANOVAa

Batch NS NS NS NS NS NSRipening time ** ** *** *** *** ***

Data are mean ± standard deviation of duplicate determinations in two cheesemaking trials.aSignificance levels: *P < 0.05; **P < 0.01; ***P < 0.001; NS, non significant.

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RESULTS AND DISCUSS ION

Microbiological and physicochemical composition ofcheesesThe evolution of microbial counts for cheeses made with com-mercial (C) or autochthonous (A) starter throughout matura-tion is shown in Table 1. Faecal coliforms were not detectedin any sample analysed because of the correct pasteurisationof milk and the hygienic practises followed during

manufacture and ripening of cheeses. Lactococci were themost abundant micro-organisms throughout ripening. Lacto-cocci counts in cheeses of 2 days were around 9.4 log cfu ⁄gfor both batches, remained almost stable during the first60 days and decreased from 60 to 90 days. Their developmentgenerally matched the total counts on PCA. Total counts andlactococci significantly (P < 0.001) varied throughout matura-tion. As expected, significant variation in lactobacilli(P < 0.05) and leuconostocs (P < 0.001) counts was foundwith batch starter composition. No lactobacilli were detectedin 2-day-old C cheese but did appear in 30-day C cheese withmean counts of 6.60 log cfu ⁄g. From this point onwards, lac-tobacilli counts of C cheese declined up to the end of ripening,going down to mean values of 5.72 log cfu ⁄g. Similarly, leu-conostocs appeared in 60-day-old C cheese with mean countsof 3.70 cfu ⁄g and slightly decreased by the end of ripening.The presence of nonstarter lactobacilli and leuconostocs in Ccheeses was probably due to post-pasteurisation contaminationof the milk and ⁄or survival in a heat-shocked state as has beendescribed (McSweeney et al. 1994; Ortigosa et al. 2006).Mean counts of lactobacilli and leuconostocs in 2-day-old Acheese were 6.43 and 5.40 log cfu ⁄g, respectively, thenincreased to more than 7 log cfu ⁄g until 60 days and thereaf-ter decreased reaching at 90-day values similar to those of2 days. The decrease in LAB counts during ripening has beenobserved and described by various authors (Poveda et al.2003; Herreros et al. 2007; Awad et al. 2010) and may be aconsequence of the decrease in aw and the increase insalt ⁄moisture ratio that occurs during maturation.Table 2 shows the evolution of physicochemical parameters

along the ripening of cheeses. pH, acidity, NaCl, moisture, fatand protein content did not vary significantly with the type ofstarter culture (P > 0.05) although they did with the ageingtime (P < 0.01). Moisture decreased with time, as a result ofwater exchange with the environment, with a consequentincrease in fat and protein content. NaCl content was almost

Table 3 Variation in nitrogen fractions of cheeses made with commercial(C) or autochthonous (A) starter

BatchTime(days)

Nitrogen fractions

WSN ⁄TN (%)TCA-SN ⁄TN (%)

PTA-SN ⁄TN (%)

C 2 12.23 ± 0.16 2.97 ± 0.16 2.05 ± 0.1530 23.67 ± 0.56 6.14 ± 0.05 2.35 ± 0.1560 22.96 ± 0.60 8.13 ± 0.44 3.28 ± 0.0890 24.88 ± 0.08 8.46 ± 0.10 4.00 ± 0.19

A 2 12.12 ± 0.30 2.92 ± 0.21 2.12 ± 0.2430 24.01 ± 0.52 6.04 ± 0.15 2.37 ± 0.1660 23.81 ± 0.52 8.54 ± 0.14 3.46 ± 0.1290 24.05 ± 1.02 8.66 ± 0.20 4.26 ± 0.33

ANOVAa

Batch NS NS NSRipeningtime

*** *** ***

TN, total nitrogen; WSN, water-soluble nitrogen; TCA-SN, 12%

trichloroacetic acid soluble nitrogen; PTA-SN, 5% phosphotungstic acid

soluble nitrogen.

Data are mean ± standard deviation of duplicate determinations in two

cheesemaking trials.aSignificance levels: *P < 0.05; **P < 0.01; ***P < 0.001; NS, non

significant.

Table 4 Sensory characteristics of cheeses made with commercial (C) or autochthonous (A) starter at 30, 60 and 90 days of ripening

BatchTime(days)

Sensory characteristics

Outer appearance Texture Aroma Taste General acceptability Total

C 30 5.30 ± 0.82 5.20 ± 1.23 4.50 ± 1.08 5.50 ± 1.51 5.30 ± 1.16 25.80 ± 4.9060 4.91 ± 1.38 5.18 ± 1.33 4.64 ± 0.92 5.55 ± 0.92 5.55 ± 0.52 26.18 ± 4.0590 5.64 ± 0.81 5.82 ± 0.60 4.86 ± 1.19 5.41 ± 0.92 5.68 ± 0.64 27.41 ± 2.29

A 30 5.60 ± 0.52 5.60 ± 0.84 5.50 ± 1.08 5.80 ± 0.79 5.70 ± 0.48 28.20 ± 2.8660 5.00 ± 0.89 5.27 ± 1.01 4.73 ± 0.90 5.09 ± 1.58 5.45 ± 1.21 25.55 ± 3.9690 5.55 ± 0.82 5.68 ± 0.78 5.73 ± 0.90 6.09 ± 0.94 6.00 ± 0.81 29.04 ± 3.39

ANOVAa

Batch NS NS * NS NS NSRipening time NS NS NS NS NS NS

Data are mean ± standard deviation from 10 to 15 trained panellists on two experiments using a 7-point scale.aSignificance levels: *P < 0.05; **P < 0.01; ***P < 0.001; NS, non significant.

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constant during the first 30 days with mean values around1.4% and 1.5%, respectively, and then gradually increased toaround 2.2% at the end of ripening. The mean pH of bothcheeses was approximately 5.2 during the first 30 days,increasing slightly towards the end of ripening to a value of5.3–5.4. Similarly, acidity increased from the initial meanvalue of about 1.2% to values at 60 days of 1.3% and 1.5%,respectively, for C and A cheese and then declined slightly atthe end of ripening. The maintenance of pH and the increasein acidity observed in the first 60 days of ripening are attrib-uted to lactic acid and hydrogen formation, while the increasein pH and the decrease in acidity found after 60 days may bedue to the combined effect of the utilisation of lactic acid, for-mation of nonacidic decomposition products and the produc-tion of alkaline nitrogenous compounds during proteolysis(Webb et al. 1983).

ProteolysisThe evolution of the nitrogen fractions of the cheeses is shownin Table 3. WSN fraction contains large water-soluble peptidesderived from the action of the residual rennet on casein, pep-tides and free amino acids. TCA-SN contains mainly small pep-tides (2–20 amino acid residues) released by the action of bothrennet and starter enzymes upon large peptides. PTA-SN con-tains low molecular weight peptides and free amino acids andreflects the aminopeptidase activities of the starter bacteria(Sousa and Malcata 1997).Nitrogen fractions did not vary significantly with the type of

starter culture, although they significantly increased over time(P < 0.001). In the 2-day-old cheeses, WSN ⁄TN was around12% for both batches and reached 24.88% in batch C and24.05% in batch A at 90-day-old samples. Similar evolutionhas been described for Turkeys’ Beyaz cheese manufacturedwith pasteurised cows’ milk inoculated with two different sin-gle Lactococcus lactis subsp. lactis starter culture (Hayaloglu2007). TCA-SN ⁄TN and PTA-SN ⁄TN at 60 days were slightlyhigher in the autochthonous cheese compared to cheese withcommercial starter (8.66% and 4.26% vs 8.46% and 4.00%).Similar mean contents of TCA-SN ⁄TN and PTA-SN ⁄TN havebeen found for ewes’ milk Kefalograviera-type cheese manu-factured with multiple-species starter and ripened for 90 days(Michaelidou et al. 2007).It has been reported that TCA- and PTA-nitrogen-soluble

fractions are higher in cheeses manufactured with lactobacillirelative to control batches containing only lactococci (Menen-dez et al. 2000). In our study, similar values were found for Ccheese (containing only Lacctococcus and Streptococcus asstarter) and for A cheese (containing one Lactococcus, one Lac-tobacillus and one Leuconostoc strain a starter). These resultscould be explained if one takes into account that the nonstarterlactobacilli and leuconostocs found in batch C may contributeto proteolysis mainly via their peptidolytic potential, increasingthe amount of small peptides and free amino acids (Lynch et al.1999; Briggiler-Marco et al. 2007).

Sensory evaluationThe results of the sensory characteristics of cheeses are given inTable 4. The high values of standard deviation found reflect theheterogeneity among the group of panellists. In general, thebest evaluated cheeses throughout ripening were those fromautochthonous batch which noted for its creamy texture with anintense aroma and a slightly acidic and bitter taste. Autochtho-nous batch received the higher scores for all attributes on day30; for outer appearance, texture and aroma at 60 days and foraroma, taste and general acceptability at the end of the ripeningperiod. Furthermore, according to the analysis of variance,autochthonous cheeses obtained significantly higher (P < 0.05)scores in aroma than those made with commercial starter. Simi-larly, other authors have reported aroma intensity to be higherin cheeses made with a adjunct culture of lactobacilli (Lynchet al. 1999; Ortigosa et al. 2005).

CONCLUS ION

Pilot plant Tenerife cheeses manufactured with pasteurisedgoats’ milk with the autochthonous starter have similar micro-biological, physicochemical and proteolytic characteristics thancheeses manufactured with commercial starter, with the advan-tage of enhanced sensory features, especially aroma. Therefore,the use of the selected autochthonous starter could be an inter-esting way to produce on an industrial scale, a uniform productof constant quality with improved sensory characteristics.

A C KNOWL EDG EMEN T S

This research was carried out with the financial support of CabildoInsular de Tenerife, Spain. The authors are also grateful to Departa-mento de Bromatologıa and Ingenierıa Quımica from La LagunaUniversity for their collaboration.

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