Reggianito Argentino cheese: influence of Lactobacillus helveticus strains isolated from natural whey cultures on cheese making and ripening processes

International Dairy Journal 12 (2002) 923–931

Reggianito Argentino cheese: influence of Lactobacillus helveticus strains isolated from natural whey cultures on cheese making and ripening processes Mario Ce! sar Candioti, Erica Hynes, Andrea Quiberoni1, Susana Beatriz Palma, Nora Sabbag, Carlos Antonio Zalazar*,1 Programa de Lactolog!ıa Industrial, Facultad de Ingenier!ıa Qu!ımica, Universidad Nacional del Litoral, Santiago del Estero 2829, S3000AOM Santa Fe, Argentina Received 10 January 2002; accepted 16 July 2002

Abstract Reggianito Argentino cheeses were manufactured with three defined single strains Lactobacillus helveticus cultured in sterile whey, and one ‘‘natural’’ whey starter. Gross composition of cheeses did not significantly differ, and viable starter cell counts were similar for all cheeses. Soluble nitrogen at pH 4.6 was also alike for cheeses made with natural or selected starters, but soluble nitrogen in trichloroacetic acid 12% and phosphotungstic acid 2.5% showed significant differences. Electrophoretograms showed that g casein bands increased during ripening, while b casein band decreased; as1 casein was also cleaved to as1 -I. Acid degree values of fat for control and experimental 0-day old cheeses were slightly different, but were similar at 90 and 180 days of ripening. All cheeses were good quality Reggianito, but control and experimental samples differed in aroma and texture. Natural whey starter replacement by selected single starter of Lactobacillus did not alter cheese making and primary proteolysis or lipolysis, but it modified secondary proteolysis and sensory characteristics. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Lactobacillus helveticus; Natural whey starter; Proteolysis; Lipolysis; Sensory characteristics

1. Introduction Lactic acid bacteria (LAB) are added to milk used for cheese-making mainly to metabolise lactose to lactic acid, a process that improves clotting and curd strengthening and helps provide an environment that safeguards the final product. The starter culture also contributes to cheese aroma and flavor development during ripening, by means of carbohydrate metabolism, proteolysis and—in minor degree—lipolysis (Fox, Law, McSweeney, & Wallace, 1993; Choisi, Desmazeaud, Gripon, Lamberet, & Lenoir, 1997). There are several forms of inoculating cheese-making milk with starter culture; these methods have been *Corresponding author. Tel.: +54-342-4530-302; fax: +54-3424571-162. E-mail address: azalazar@fiqus.unl.edu.ar (C.A. Zalazar). 1 Researchers of Consejo Nacional de Investigaciones Cient!ıficas y ! Tecnologicas.

modified with the time and vary from one cheese type to another. Lyophilised or frozen, defined or mixed strain/species direct-to-vat lactic cultures are now widely used for cheese manufacture, and their use continues to increase in some countries. Nevertheless some cheeses, generally protected by legislation # e!e or D!enomination (D!enomination d’Origine Control d’Origine Prot!eg!ee), are still produced with ‘‘natural’’ cultures. ‘‘Natural’’ cultures are so-called because they are obtained from a previous batch of fermented product (milk or whey) to inoculate a new batch. The predominant genera consist of streptococci and thermophilic lactobacilli for milk and whey cultures, respectively. The milk or whey are incubated overnight and used in the next day cheese-making as the starter culture (Bozzetti, 1993). ‘‘Natural’’ whey starters are widely used in Italy for Grana Padano and Parmigiano Reggiano (Mucchetti, Addeo, & Neviani, 1998; Bottazzi et al., 1992), in France for Comte! and Beaufort (Mahaut, Jeantet, & Brule! ,

0958-6946/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 2 ) 0 0 1 1 5 - 2

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2000), and in Argentine, for traditional Reggianito Argentino cheese (Zalazar, Meinardi, & Hynes, 1999), among others. Reggianito Argentino cheese was developed by Italian immigrants in late XIX and early XX centuries, inspired by Italian hard cheeses, but then it was modified and adapted all along the last century to Argentinean raw materials and environmental conditions, to give a distinctive product. One of the characteristics of Reggianito Argentino that distinguish it from other hard cheeses like Parmesan is the microflora of Argentinean ‘‘natural’’ whey cultures. Argentinean ‘‘natural’’ whey cultures are composed of B66% of Lactobacillus helveticus strains and B33% of Lactobacillus delbruecki subsp. lactis strains (Reinheimer, Quiberoni, Tailliez, Binetti, & Sua! rez, 1996), while the Italian cultures consisted of the same species in the inverse proportion (Bottazzi, 1998). Reinheimer et al. (1996) also reported that Streptococcus thermophilus was o 1% in Argentinean ‘‘natural’’ whey cultures. This is similar to occurrence of S. thermophilus in Italian cultures, and it is not surprising given the low pH (3.15–3.50) of the environment. Natural whey starters have advantages and disadvantages. Among the advantages are the contribution to final product typical flavour and aroma, attributed to the complex microflora, and the resistance to phage attack due to the multi-strain culture (Giraffa, Mucchetti, Addeo, & Neviani, 1997; Bottazzi et al., 1992). On the other hand, poor quality standardisation and eventual contamination mainly with yeasts has been identified as disadvantages in Argentinean ‘‘natural’’ whey cultures (Reinheimer et al., 1996). The objective of many researchers has been to find cultures that gather the advantages of ‘‘natural’’ whey cultures and the microbial purity provided by selected starters strains (Bottazzi, Parisi, & Cocconcelli, 1999; Paleari, Soncini, Beretta, & Aldrighi, 1996; Bosi et al., 1991). The addition of selected bacteria to ‘‘natural’’ whey starter before incubation has been proposed as a strategy to standardise and improve starter quality, but the contamination problem persisted. On the other hand, direct-to-vat starters seem not to be appropriate for hard cheesemaking processes, because these technologies require an initial lactic acid concentration in the milk before coagulation, which is provided by the liquid ‘‘natural’’ whey starter. On the contrary, ‘‘natural’’ milk starter cultures were easily replaced by defined starter cultures in enzymatic curds like Cremoso Argentino cheese, in which acidification takes place in mould after coagulation. The objective of the present work was to compare the behaviour of single strains of Lactobacillus helveticus with a ‘‘natural’’ whey starter culture for Reggianito Argentino cheese making, and their influence on the ripening process.

2. Material and methods 2.1. Strains Single strains used in the present study were isolated and studied in previous works. Isolates, obtained from ‘‘natural’’ whey cultures used in dairy industry, were screened for technological parameters such as acidifying and proteolytic activities, resistance to NaCl and phage attack (Reinheimer et al., 1996; Reinheimer, Sua! rez, Bailo, & Zalazar, 1995; Quiberoni, Candioti, Meinardi, Palma, & Reinheimer, 1997). The most suitable strains from a technological point of view, identified as Lh SF133, Lh SF138 and Lh SF209, were chosen for the present study. These strains were also characterised by molecular techniques (Quiberoni, Tailliez, Que! nee, Sua! rez, & Reinheimer, 1998). The single strains were cultured in sterilised (1101C– 10 min), reconstituted (10% w/v), low-heat skim milk powder at 451C/24 h. 2.2. Starter cultures Whey samples were taken from Reggianito Argentino cheese-making in a nearby (30 km) dairy plant after the cooking step, and immediately carried to our laboratory under refrigerated conditions (bottles were placed into a container with ice). Once in the laboratory, whey pH was adjusted to 6.3 with NaOH 40% w/v under controlled microbiological conditions, and divided into two fractions of 1.8 L. One of the fractions was incubated for 24 h at 451C in order to obtain control ‘‘natural’’ whey starter. The remaining fraction was heated for 5 min at 851C, in order to destroy all vegetative cells. After cooling to 451C, it was inoculated (2% v/v) with Lh SF133, Lh SF138 or Lh SF209 milk cultures and incubated as the control. 2.3. Cheese making Reggianito Argentino cheeses were manufactured at our pilot plant according to the standard process (Gallino, 1994). 170 L of raw bulk milk, pH 6.6070.05 acidity 18711D were supplied by a near dairy plant every cheese-making day, and divided into two vats. One cheese of about 7 kg was obtained from each vat. The milk was standardised to 2.50% fat matter, and batch-pasteurised at 651C for 20 min. After cooling to 331C, CaCl2 was added at a final concentration of 0.02% (w/v). A volume of ‘‘natural’’ whey starter enough to increase cheese-making milk initial acidity by 41D (400 mg lactic acid L
1) was inoculated. The procedure of measuring the acidity of the cheese-making milk instead of the starter culture volume is traditional for hard cheeses like Reggianito, and it provides a rough uniformity in the number of viable cells per mL of milk.

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The inoculum volume usually represents B4% v/v of the milk if the whey culture is in the usual range of pH or acidity. After 10 min mechanical stirring (100 rpm.), milkclotting enzyme was added (0.29 mL L
1 of milk of adult bovine coagulant 230 IMCU mL
1, Naturen, Chr. Hansen, Quilmes, Argentine). After 18–20 min the curd was cut to the adequate grain size (approximately half a rice grain) and the mixture of the curd particles and whey was gently stirred and heated at 0.51C min
1, till it reached 441C, in order to reduce humidity in curd grains. The mixture was then more rapidly heated to 511C (11C min
1), then stirring was stopped. The curd was separated, put into moulds and the remaining whey discarded. The curd was pressed during 24 h after which it was brined in 20% (w/v), pH 5.4 brine at 121C for 6 days. During this time the cheese was inverted every other day. Ripening was carried out at 121C and 80% relative humidity for 6 months. Control cheeses were made with ‘‘natural’’ whey starter. Three types of experimental cheeses were obtained, using Lh SF133, 138 or 209 single strain cultures. There were two experimental units (cheese vats) every cheese-making day. Three replicate cheeses for each type were made, which gave a total number of 12 cheeses, divided into 6 cheese-making days. The distribution of control and experimental cheeses in cheese-making days was randomised.

925

2.6. Proteolysis assessment Proteolysis was assessed on 0-, 90- and 180-day-old cheeses, by determination of soluble nitrogen at pH 4.6, in tricloroacetic acid (TCA) 12% and in phosphotungstic acid (PTA) 2.5. Electrophoresis was also performed. Soluble nitrogen. Cheese samples were treated to obtain crude citrate extract and soluble fraction at pH 4.6, in TCA 12% and PTA 2.5% according to Hynes et al. (2001). The crude cheese extract was obtained by adding 20 mL of sodium citrate 0.5 m to 10 g of cheese and grinding to homogeneity using a pestle. Deionised water was added to B90 mL and the pH was adjusted to 4.6. After centrifugation (3000g/15 min), the soluble fraction volume was adjusted to 100 mL. TCA 12% and PTA 2.5% soluble fractions were obtained from 4.6 soluble fraction according to Gripon, Desmazeaud, Le Bars, and Berge" re (1975). The nitrogen content was determined in duplicate by the macro-Kjeldahl method according to the IDF method (1993). Electrophoresis. The insoluble residue at pH 4.6 was analysed by Urea-PAGE in a Mini-Protean II cube (BioRad Laboratories, California, USA) by the Andrews (1983) method, with a concentration of acrylamide of 7.5% (Hynes, Delacroix-Buchet, Meinardi, & Zalazar, 1999). Proteins were stained by Coomasie Blue G-250. Samples of cheese casein were prepared by precipitation at pH 4.6 and purified. 2.7. Lipolysis assessment

2.4. Proximate composition of the cheeses Dry matter and protein content were analysed according to International Dairy Federation (IDF) standards (IDF, 1962, 1993), and fat and pH by American Public Health Association (APHA) methods (Bradley et al., 1993) on 6-month old cheeses. 2.5. Microbiology analysis The population of total thermophilic lactic flora present in the cheese products after 0 (fresh curd), 1 (curd after press), 90 and 180 days of ripening was determined by plating sample dilutions on skim milk agar (SMA) and counting plate colonies after 48 h of incubation at 371C according to APHA standards (Frank, Christen, & Bullerman, 1993). Coliforms were enumerated on Bile Red Violet Agar (BRVA) and the plates were incubated for 24 h at 301C (Christen, Davidson, McAllister, & Roth, 1993). Fresh whey was also analysed by a modified Weinzirl method (Annibaldi, 1969) to determine sporulated anaerobic organisms. Mould and yeasts were determined according to APHA (Frank et al., 1993) on the four starter cultures.

Lipolysis was assessed on 0-, 90- and 180-day-old cheeses, by titration of total free fatty acids according to Bernal, Perotti, Zalazar, Cardell, and Zalazar (1998). Lipid extraction was carried out on a sample of grated cheese acidified to pHo2.0. The extraction was performed with hexane at high temperature (hexane boiling point, B691C), in a continuous extractor. The ether extract was titrated with NaOH 0.1 n, in a two-phase system (isopropanol–water). Acid degree value of fat (ADVF) was expressed as mmol of oleic acid per 100 g fat. 2.8. Sensory analysis The sensory characteristics of all the control and experimental 180-day old cheeses were assessed by a panel of eight members selected and trained for the sensory analysis of hard cheeses. Typical Reggianito Argentino cheese from the local market was used as reference for panel selection and training steps. Samples identified by random numbers were presented in individual trays to the panelists. Sensory analysis was performed in a conditioned room. A quantitative descriptive method was carried out. Descriptors were developed by the panel in round-table

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discussion led by a coordinator, until consensus. Each descriptor was quantified by pointing in a 10 cm unstructured scale, anchored at each extreme. The scales were set as follows: genuine aroma (1=weak, 9=very intense), colour (1=weak, 9=very intense), visual texture: eyes and mechanical cracks (1=many, 9=none), grain cut (1=not grainy, 9=very grainy), fracturability (1=none, 9=very much), oral texture (1=rough, sandy, pasty, 9=not rough, not sandy, not pasty), genuine flavour i.e. savoury, slightly spicy and salty (1=extremely weak, 9=normally intense), salty taste (1=weak, 9=intense), residual flavour (1=absent, 9=very much). The analysis was performed in duplicate sessions. The second session began 20 min after the end of the first. 2.9. Statistical analysis Data were processed by one way ANOVA with Statistix 7 (Analytical Software, Tallahasse, USA). When differences were found, means were compared by the least significant difference test (LSD) using the same tool.

protein and pH were appropriate for Reggianito variety according to the Argentinean codex (ANMAT, 1999). Fig. 1 shows changes in LAB counts during ripening. Starter number was about 107 colony forming units (CFU) g
1 in curd before moulding for cheeses made with ‘‘natural’’ culture and single Lactobacillus helveticus strains SF133 and SF209, while the SF138 strain had an initial cell count of about 108 CFU g
1. On the other hand, starter cell numbers reached a maximum of B108 CFU g
1 at 24 h for all the cheeses, and then decreased by one or two log orders during ripening. Strain SF209 showed the lowest final count, about 106 CFU g
1. This result may suggest that the SF209 strain decreased somewhat faster than the other two Lactobacillus helveticus strains (SF133 and SF138), but differences were very slight. Coliform bacteria counts were o102 CFU g
1 for control and experimental 180-day old cheeses. Sporulated anaerobic organisms were not detectable by the Weinzirl method in all fresh whey samples. After incubation, counts of yeasts and moulds on natural

12

Control Lh SF133 Lh SF 138 Lh SF209

10

3.1. Cheese composition and microbiology Starter cultures reached slightly different pH and acidity after incubation (24 h), i.e. 3.1570.05 and 1407101D (11D=100 mg lactic acid L
1) respectively for natural whey starter, and 3.2570.05 and 1307101D (11D=100 mg lactic acid L
1), respectively, for selected single strain cultures. Nevertheless, inoculation, coagulation time and gel strengthening in all cheese-making processes were similar, because according to the acidity balance, a slightly higher volume of single strain cultures was added to the milk. As a consequence, curd firmness and whey removal were the same for all cheeses, and their gross composition did not differ (P > 0:05) among starters, as can be seen in Table 1. Dry matter, fat,

log UFC g −1

3. Results and discussion

8 6 4 2 0 0

1

90

180

Ripening time (days) Fig. 1. Evolution of Lactobacillius helveticus population during ripening of Reggianito cheeses. Control: lactobacilli cell counts on cheese prepared with ‘‘natural’’ whey starter. Lh133, Lh138 and Lh209: cell counts on cheeses prepared with single strain cultures of Lactobacillus helveticus SF133, SF138 or SF209, respectively. Values are the means for three replicate cheeses.

Table 1 Proximate composition of 180-day-old cheeses Cheesea

Dry matter Fat matter Total protein pH a

C

Lh SF133

Lh SF138

Lh SF209

62.9770.58 23.5871.09 28.9471.03 5.1770.03

64.5572.53 26.8272.60 28.7371.54 5.1770.03

64.4472.29 25.0072.77 29.6170.26 5.1570.05

63.2373.63 23.2971.26 29.6971.75 5.1770.08

None of the values in the rows showed statistically significant differences. C: control cheeses made with ‘‘natural’’ whey starter culture; Lh SF133, Lh SF138 and Lh SF209 cheeses made with single strain cultures of Lactobacillus helveticus SF133, SF138 or SF209, respectively. Data are means7standard deviations for three replicate cheeses.

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whey cultures were B104 CFU mL
1. In contrast, yeasts and moulds did not occur in selected single strain cultures, because all vegetative cells were destroyed before Lactobacillus helveticus inoculation, as was verified (data not shown). 3.2. Proteolysis assessment Soluble nitrogen at pH 4.6 for 0-, 90- and 180-day-old cheeses is represented in Fig. 2. The content of soluble nitrogen (SN) at pH 4.6 did not differ (P > 0:05) for control and experimental cheeses during ripening. That

25.00

C Lh SF133 Lh SF138

20.00

% pH4.6-SN/TN

Lh SF209 15.00

10.00

5.00

0.00

0

90 Ripening time (days)

180

Fig. 2. Soluble nitrogen (SN) at pH 4.6 as percentage of total nitrogen (TN) (% pH 4.6-SN/TN), for control and experimental Reggianito cheeses at 0, 90 and 180 days of ripening. Control (C): Cheeses prepared with ‘‘natural’’ whey starter. Lh SF133. Lh SF138 and Lh SF209: cheeses prepared with single strain cultures of Lactobacillus helveticus SF133, SF138 or SF209, respectively. Means and standard deviation for three replicate cheeses.

927

is not surprising because SN at pH 4.6 represents mainly primary proteolysis, especially, due to plasmin and possibly residual or reactivated chymosin in cooked cheeses (Delacroix-Buchet & Fournier, 1992). Although Lactobacillus helveticus ssp. have shown one of the strongest proteolytic activities among the LAB, and their cell wall proteinases are able to attack caseins as and b in vitro (Torriani, Vescovo, & Scolari, 1994; M.ayr.a-M.aniken & Bigret, 1998), no evidence of such a breakdown was detected in the cheeses. Fig. 3 shows the evolution of TCA 12% SN during ripening of experimental and control cheeses. There was no significant difference among 0- and 90- day-old cheeses, but at 180 days of ripening SF209 cheeses differed (Po0:01). TCA 12% SN contains mediumsized to small peptides, amino acids and smaller N compounds, such as amines, urea and ammonium . 1999), and thus suggested a higher peptidolytic (Ardo, activity for strain SF209. PTA 2.5% SN evolution during ripening is shown in Fig. 4. This fraction contains very small peptides, amino acids and smaller nitrogen compounds except dibasic . 1999). As SN at pH amino acids and ammonia (Ardo, 4.6 and in TCA 12%, PTA 2.5% SN did not differ for 0- and 90-day-old cheeses. However, it showed significant differences for 180-day-old cheeses (Po0:01). Means for the starter cultures were grouped as follows: SF138, SF133+control and SF209, in increasing order of PTA 2.5% SN. These differences, as well as TCA 12% data, seems to indicate a stronger peptidasic activity of strain SF209. This is coincident with the fact that strain SF209 showed the lowest final counts, suggesting a higher cell lysis and release of peptidases into the cheese mass. Nevertheless, we did not investigate lysis, so that further research on this subject is needed.

25.00

C Lh SF133 Lh SF138

20.00

% TCA-SN / TN

Lh SF209 15.00

10.00

5.00

0.00

0

90 Ripening time (days)

180

Fig. 3. Soluble nitrogen (SN) in trichloroacetic acid (TCA) 12% as percentage of total nitrogen (%TCA-SN/TN), for control and experimental Reggianito cheeses at 0, 90 and 180 days of ripening. Control (C): Cheeses prepared with ‘‘natural’’ whey starter. Lh SF133. Lh SF138 and Lh SF209: cheeses prepared with single strain cultures of Lactobacillus helveticus SF133, SF138 or SF209, respectively. Means and standard deviation for three replicate cheeses.

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928 25.00

C Lh SF133 Lh SF138 Lh SF209

% PTA-SN/ TN

20.00

15.00

10.00

5.00

0.00

0

90 Ripening time (days)

180

Fig. 4. Soluble nitrogen (SN) in phosphotungstic acid (PTA) 2.5% as percentage of total nitrogen (TN) (% PTA-SN/TN), for control and experimental Reggianito cheeses at 0, 90 and 180 days of ripening. Control (C): Cheeses prepared with ‘‘natural’’ whey starter, Lh SF133, Lh SF138 and Lh SF209: cheeses prepared with single strain cultures of Lactobacillus helveticus SF133, SF138 or SF209, respectively. Mean and standard deviation for three replicate cheeses.

γ γ γ

3 2 1

β α s2 α s1 α s1-l

1

2

3

4

5

6

7

8

9

Fig. 5. Urea polyacrylamide gel electrophoresis of 0-, 90- and 180-dayold Reggianito cheeses. 1–3: cheeses at 0 days of ripening made with ‘‘natural’’ starter (control), or with single strain cultures of Lactobacillus helveticus SF133 or SF138, respectively. 4–6: cheeses at 90 days of ripening made with ‘‘natural’’ starter (control), or with single strain cultures of Lactobacillus helveticus SF133 or SF138, respectively. 7–9: cheeses at 180 days of ripening made with ‘‘natural’’ starter (control), or with single strain cultures of Lactobacillus helveticus SF133 or SF138, respectively.

Typical electrophoretic patterns for control and experimental 0-, 90- and 180-day-old cheeses are shown in Fig. 5. Control and experimental cheeses manufactured with single strain starters SF133 and SF138 are presented as examples. Electrophoretic profiles for SF209 experimental cheeses were essentially the same (data not shown). Plasmin activity on b casein, which leads to g caseins, was clearly observed. Bands for g caseins increased from 0- to 90-day-old cheeses, while b caseins band decreased accordingly. The g caseins were not more intense for 180- than for 90-day-old cheeses, even though b casein band decreased, suggesting further

degradations on g caseins. The as2 casein disappeared in 90-day-old cheeses, as was expected because this minor casein is, along with b casein, one of the main substrates of plasmin (Grufferty & Fox, 1988). The as1 casein was partially degraded to as1 -I peptide in 90 and 180-day-old cheeses. This breakdown has been attributed to residual chymosin or to cathepsin D in hard and Swiss type cheeses (Delacroix-Buchet & Fournier, 1992; Fox et al., 1993), but further research on the subject is probably needed. Electrophoretic patterns for control and experimental cheeses were very similar at 0, 90 and 180 days of ripening. 3.3. Lipolysis assessment Data for ADVF for control and experimental cheeses at 0, 90 and 180 days of ripening are presented in Table 2. Means did not differ (Po0:05) after 90 days of ripening. Nevertheless, in 0-day-old cheeses, fat acidity was significantly different. Cheeses manufactured with SF133 and SF209 showed a slightly higher initial fat acidity than control and SF138. This may suggest a different lipolysis in cheese-making milk, even when cheeses with the same strain were manufactured with different milk batches for minimising the impact of uncontrolled variables. Some authors consider that estearase/lipase activity of Lactobacillus helveticus is limited (Torriani et al., 1994) but others have reported that BAL were able to release quite high levels of free fatty acids in over extended ripening periods (McSweeney & Sousa, 2000). Nevertheless, the effect of the Lactobacillus starter does not seem to explain the difference in 0-day-old cheeses, because it appeared too early in ripening when eventual bacterial esterases

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Table 2 Acid degree value of fat (ADVF) expressed as mmoles of acid per 100 g of fat of 0, 90- and 180-day-old cheeses Ripening time (days)

Cheese type C

Lh SF133 a

0 90 180

Lh SF138 b

15827206 35357457a 37917433a

Lh SF209 a

19757355 28277613a 31817124a

22627209b 30077259a 38837248a

16067142 26687550a 38647709a

a,b Means on the same row with different superscript differ (Po0:05). C: control cheeses made with ‘‘natural’’ whey starter culture; Lh SF133, Lh SF138 and Lh SF209 cheeses made with single strain cultures of Lactobacillus helveticus SF133, SF138 or SF209, respectively. Data means7standard deviations for three replicate cheeses.

Table 3 Sensory characteristics of 180-day-old cheeses Attributes

Mean

Significance level

C Genuine aroma Colour Visual texture Grain cut Fracturability Oral texture Genuine flavour Salty taste Residual flavour

Lh SF133 a

5.070.4 5.470.2a 7.970.8a 7.370.7a 8.270.6a 7.570.7a 8.170.7a 5.970.4a 2.370.4a

a

5.070.5 5.570.4b 7.970.7a 7.570.5a 8.070.7a 5.770.6b 8.070.9a 6.070.4a 4.270.3b

Lh SF138 b

4.670.3 4.870.4c 7.970.6a 7.071.0a 7.870.8b 7.770.9c 7.770.8a 5.970.6a 3.670.4b

Lh SF209 5.470.5c 5.070.6d 6.970.6b 7.070.6a 8.370.9c 7.370.8d 7.971.1a 6.170.5a 1.070.2c

n n nn

NS n nn

NS NS **

NS: Non Significant, a,b,c,d: Means in a row with different superscript differ. C: control cheeses made with ‘‘natural’’ whey starter culture; Lh SF133, Lh SF138 and Lh SF209 cheeses made with single strain cultures of Lactobacillus helveticus SF133, SF138 or SF209, respectively. Data are means7standard deviation (n ¼ 48; eight panellists, two sessions by sample, three replicate cheeses of each type). n Po0:05: nn Po0:01:

and/or lipases activities are not likely effective. On the other hand, the differences disappeared along ripening. The increase in ADFV during ripening (B50–100%) is difficult to attribute to one particular enzymatic agent. Indigenous milk lipoprotein lipase was not likely active after pasteurisation and cooking steps, and rennet did not contain any lipase or estearase. Starter and nonstarter LAB may be at the origin of this observation. 3.4. Sensory analysis Results of sensory analysis are presented in Table 3. All the cheeses had a moderate genuine aroma and flavour, characteristic colour, appropriate salty taste and visual texture; thus the panel agreed on their satisfactory overall quality, although the panellist did not score ‘‘overall quality’’ as an attribute. Control and experimental cheeses did not differ for grain cut, genuine flavour and salty taste (P > 0:05). Significant differences were found for the attributes aroma, colour, visual and oral texture, fracturability, and residual flavour. Means for aroma and fracturability were compared, and grouped as follows: Lactobacillus helveticus SF138,

SF133+control and SF209, in increasing order of aroma intensity and fracturability. The same groups were defined by PTA SN data (see above) which is coherent with the fact that free amino acids contribute to flavour, mainly as precursors of flavour compounds (Urbach, 1993; McSweeney & Sousa, 2000). Proteolysis has been reported also as a factor influencing fracture properties of cooked cheeses (Visser, 1991), thus fracturability may also reflect the higher secondary proteolysis. In both cases of proteolysis assessment and sensory analysis, SF209 cheeses showed the most intense characteristics. SF209 cheeses showed also a unique value for visual texture, due to the higher number of mechanical cracks in this type of cheese, probably related to their higher facturability. In contrast, residual flavour for SF209 cheeses was the lowest, followed by control cheeses. SF138 and SF133 showed a more intense residual flavour. The panellists did not consider residual flavour as an off-flavour, because it was slightly spicy and salty. The stronger residual flavour in SF133 and SF138 cheeses did not correlate with higher proteolysis or lipolysis, thus it is hard to explain our results. SF133 cheeses also showed the lowest rate for

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oral texture, but we cannot explain this difference with the studied variables.

4. Conclusions The substitution of ‘‘natural’’ whey culture by single strain starters prepared by incubation of Lactobacillus helveticus SF133, SF138 or SF209 in heat-treated whey, did not cause differences in the manufacture of Reggianito cheese with respect to acid production in the vat and the microbial survival in the final cheese. Also, primary proteolysis and lipolysis during ripening of control and experimental cheeses were broadly similar. However, secondary proteolysis was different for control and experimental cheeses, and some changes on sensory characteristics were observed, though overall quality was high for all cheeses. Our results open an interesting prospective for replacement of ‘‘natural’’ whey cultures by selected strains cultured in whey, without significant modifications in technology or broad characteristics of Reggianito Argentino cheese; specially in large-scale cheesemaking where selected starters are easier to handle. The slight changes in aroma and texture observed between control and experimental cheeses, however, justify the use of ‘‘natural’’ whey starter cultures in small- and farm-scale Reggianito Argentino cheese-makings.

Acknowledgements The authors thank MsCs Cristina Perotti for ADVF analysis and Tec. Silvia Costa for sensory analysis assistance; and are grateful to sensory panellists for their valuable collaboration.

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