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Effect of high pressure treatment applied on starter culture or on semi-ripened cheese
in the quality and ripening of cheese in brine
Marianna Giannoglou a,⁎, Zoi Karra a, Eleni Platakou a, George Katsaros a, Golfo Moatsou b, Petros Taoukis a
Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of Athens, Iroon Polytechniou 5, 15780 Athens, Greece
Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

a r t i c l e i n f o a b s t r a c t

Article history: Cheese ripening acceleration is of continuous interest for the industry. High-pressure (HP) treatment of starter
Received 14 April 2016 cultures used in cheese-manufacturing offers the potential to accelerate ripening by increasing the activity of
Received in revised form 11 July 2016 their intracellular peptidases that contribute in the development of desired cheese organoleptic characteristics.
Accepted 25 July 2016
The objective of the present research was the investigation of the effect of HP treatment (200 MPa-20 °C - 20 min)
Available online xxxx
directly on white brined cheese or on the starter culture used for its manufacture (Str. thermophilus:L.
lactis:L. bugaricus 2:1:1). For this purpose, the microbial, textural, physicochemical and organoleptic characteris-
Starter culture tics and proteolysis were assessed during the 2nd stage of ripening in cold stores. Control cheese without any
High pressure treatment was also studied.
Cheese ripening Cheeses made with HP-treated starters had increased secondary proteolysis. Organoleptic scoring of these
cheeses was higher during the whole storage period compared to control and HP-treated cheese. Their superior-
ity was evident even at the early stages of ripening in cold stores, since no bitterness was detected. On the
contrary, although HP treated cheeses showed the highest increase in aminopeptidases activities, this was not
correlated with the studied ripening indices or the organoleptic characteristics.
According to the results, HP-treated starter culture can accelerate proteolysis and potentially the ripening of
Industrial relevance: The data obtained from this work suggest that application of HP treatment under
optimized conditions on cheeses in brine starter cultures or on whole cheeses can be effectively used
for the production of products with reduced ripening time. This is of great importance for the cheese
industries, since the storage period for ripening is long (higher than two months), while applying HP
treatment as suggested in this study, this time may be reduced to less than one month, producing cheeses
of superior quality.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction they are kept at 16–18 °C to be acidified and drained properly (pH ≤ 4.6),
for 7 to 15 days. This period is the 1st stage of ripening. Then, cheeses
White brined cheeses (WBCs) are cheeses ripened and preserved in packaged with brine are transferred to cold stores for the continuation
brine made from curds that are not subjected to any heat treatment, like of ripening, up to at least two months. This cold storage period is the
Feta and similar cheese varieties. Their flavour is slightly acidic and salty second stage of ripening (Moatsou & Govaris, 2011).
that sometimes becomes rancid and piquant. Their colour is pure white, Cheese maturation is a complex process involving many biochemical
when they are made from ewe's and goat's milk. The cheese mass has no changes such as glycolysis, lipolysis and mainly proteolysis (Kalit,
rind, no gas holes or other openings, except for small mechanical Havranek, Kaps, Perko, & Cubric, 2005; Mc Sweeney & Sousa, 2000).
openings and its texture is smooth and rather soft but always sliceable. Proteolysis in cheeses takes place in two stages. In the primary proteol-
Feta and other white brined cheeses ripen in two distinct periods. Firstly ysis, residual rennet enzymes along with endogenous milk proteases,
hydrolyse casein resulting in the production of rather large peptides
or medium-sized peptides. In the secondary proteolysis, proteins and
⁎ Corresponding author at: Laboratory of Food Chemistry and Technology, School of large peptides are gradually hydrolysed to smaller peptides and amino
Chemical Engineering, National Technical University of Athens, 5, Iroon Polytechniou,
acids due to the action of intracellular and extracellular enzymes of
Zografou, 15780 Athens, Greece.
E-mail addresses: (M. Giannoglou), starter cultures or other cheese microorganisms (Addeo et al., 1992;
(G. Moatsou), (P. Taoukis). Gagnaire, Mollé, Herrouin, & Léonil, 2001; Ur-Rehman, Fox, & Mc
1466-8564/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article as: Giannoglou, M., et al., Effect of high pressure treatment applied on starter culture or on semi-ripened cheese in the
quality and ripening of cheese in br..., Innovative Food Science and Emerging Technologies (2016),

2 M. in containers with a capacity of 500 mL each (two cheese blocks in brine contrast to a 6 month ripening period for conventional Cheddar cheese. 2003. Roden. PepX. High pressure processing to those reported by Yokoyama et al.. bulgaricus HP treatment increased the activities of aminopeptidase (AP) and X. & Steele. approximately 108 CFU/g. They reported increased activities for all the was used for HPB. Katsaros. lactic acid. Miyakawa. Mierau et al.. and Beresford (2000) treated Cheddar cheese at Cheese samples were analysed in triplicate after 1.01. was packed in laminated pouches and treated with HP.1016/j. M. (2009). Katsaros et al. Anisa. reaching the late exponential phase. bulgaricus Courtney (2003) studied the activity of aminopeptidases of L. The treatment of the starter cultures and HPC HP-treated at 200 MPa.5 and 3% v/v. are presented below. PepA. 2009. The applied HP conditions were se- similarly to Feta cheese or on the starter culture used for its manufac. in reconstituted skimmed milk sites (Rovere. lactis. that the levels of proteolysis in HP-treated cheeses were higher com- pared to control cheeses.doi.. and Moatsou (2010). Only HP treated starter mixture was used for the manufacture of Mathematical models were developed to describe the effect of HP on HPB samples. inactivation or no effect of proteolytic and glycolytic enzymes A mixture of untreated and HP-treated starter culture (40:60 ratio) depending on the HP conditions and enzyme. The effect of HP on ripening indi. at 25 °C for 5 min and various HP conditions. et al. University of Athens. polyglycol ISO viscosity class VG 15 (Resato International BV.2016. Roden. The pressure transmitting fluid was cheese ripening in cold stores. were vacuum packed separately in sterile laminated pouches. Lactobacillus bulgaricus was grown anaer- High Pressure (HP) treatment induces conformational changes of obically at 45 °C and Streptococcus thermophilus and Lactococcus lactis proteins. 60 various stage of ripening at 50 MPa for 3 days at 25 °C and concluded and 90 days of their placement in cold stores at 4 °C. thermophilus and L. lected according to the findings of Katsaros et al. In the case of HPC samples. vation. According to several research findings.07. the cultured milk precipitate resuspended in appropriate cul- investigation. (2009) and our own ture. 14. cheeses. Katsaros. Giannoglou et al.024 . leads to the maximum activity of the characteristics and proteolysis were assessed during the 2nd stage of starter culture aminopeptidases.. Wick. 21. and Yoshikawa After the 1st stage of ripening (approximately 1 week) all the (1992) studied the acceleration of cheese ripening using HP and report. and (2. ing acceleration. In order to investigate the potential of these treatments to acceler. Anjitsu. They reported acti. Innovative Food Science and Emerging Technologies (2016).. 45. the produced lactic acid per liter of milk was the Str. which can lead terial strains Lactobacillus delbrueckii subsp. Dudley. The starter cultures used in cheesemaking were a mixture of the bac- Giannoglou. Sendra. 2. Please cite this article as: Giannoglou. / Innovative Food Science and Emerging Technologies xxx (2016) xxx–xxx Sweeney. as described by Katsaros The objective of the present research was the investigation of the et al. The bacterial strains and the HPC cheese were treated effect of HP treatment directly on white brined cheese manufactured under 200 MPa. An amount of 2. 30. Chiba. Moschopoulou. and Streptococcus thermophilus/Lactobacillus delbrueckii subsp. was carried out in a laboratory-scale HP system with a maxi- cheese and reported that the use of HP-treated starter increased mum operating pressure of 1000 MPa (Food Pressure Unit FPU 1. which was higher in comparison to control samples. Holland). same (i. physicochemical and organoleptic under these HP conditions. studied HP-treated aminopeptidases which were maximized after The mixture ratio in the cases of HPA and HPB experimental cheese treatment at 200 MPa for 20 min at 20 °C.. mixture compared to untreated culture).e.3.e. Sendra. In the international literature there is a number of pub. cheese was coded as HPC. Cheeses coded as Control samples were manufactured using 2% v/v prolyl dipeptidyl aminopeptidase (PepX). 2000). stage of ripening and the samples were also vacuum packed in sterile Maniou et al. Giannoglou. Two Ishibashi. at 20 °C for 20 min. Mor-Mur. Kelly. to economic and technological advantages for the producers by Streptococcus thermophilus ACA-DC 0022 and Lactococcus lactis ACA- reducing the cost of storage of cheeses (O'Reilly et al. (400 MPa at 30 °C for 10 min) on the activity of Lb. 2000). and Guamis (2001) studied Predetermined volumes of each bacterial strain for HPA and HPB the effect of HP at 500 MPa for 5. Materials and methods ties during cheese ripening comes from the starter cultures. more starter culture was used in the case of HP treated Some indicative studies concerning the effect of HP on cheese ripen. (2013) investigated the effect of a DVI starter mixture laminated pouches. PepN. half of the Control cheese quantity used in Cheddar cheese manufacturing.. An amount of 3% v/v of the pre-mentioned starter culture aminopeptidases' activity. textural. Malone. Yokoyama. 1996). O'Reilly.2.1. according to Feta cheese-making and ripening conditions as described by As regards the effect of HP on aminopeptidases. 2. (2009). Starter culture preparation of these peptidases may accelerate the ripening step of cheese pro- duction (Christensen. 7. http://dx. Although.8% fat content).2. & Guamis.ifset. Feta cheese was cut in pieces after the 1st treated samples. and Shimamura (1994) studied the effect of pressure replicate experiments were carried out within two consecutive weeks. bulgaricus ACA-DC 0105.. (1992). Resato International BV. In all cases. an increase of the activity 2. Cheese manufacture lications on the effect of HP on aminopeptidases of starter cultures and on the effect of HP on the ripening stage of different types of Four cheeses were manufactured using ovine milk (5. Katsaros. Studies have been carried out on the application of ture medium (MRS broth for lactobacilli and M-17 broth for cocci) HP in the starter cultures used in cheese manufacturing or directly showed an absorbance of A600nm = 4. after treatment at various HP produced. in order to investigate the effect of HP on cheese proteolysis. & Taoukis. the microbial. two cheesevat replicates were studied for each experimental cheese. Murphy. Mató cheese and reported that the total nitrogen content of the HP. Greece). At ces of cheeses in relation to potential ripening acceleration is under this stage. Taoukis. helveticus peptidases. i. part of proteinase and peptidase activi. car. These changes can lead to enzyme activation (10% TS). 1999. was used for the manufacture of HPA cheeses. Shellhammer. lactis. delbrueckii. 1995). Effect of high pressure treatment applied on starter culture or on semi-ripened cheese in the quality and ripening of cheese in br. was defined based on unpublished data Taoukis (2016) observed similar effect of HP and temperature on the on the effect of HP treatment on the ability of starter cultures to produce activity of the abovementioned aminopeptidases of the starter cultures. the increase was not in accordance 2. Pedersen. 15 and 30 min at 10 °C or 25 °C on samples. unpublished data that show that the subjection of the starter culture ate ripening. respectively). 2000). secondary proteolysis in cheese. i.. The bacteria cultures were used in cheesemakings after or inactivation (Saldo et al. in each container) and were stored at 4 °C for the 2nd stage of ripening. who applied similar treatments. DC 0049 (kindly provided from Collection ACA-DC of the Agricultural Saldo. 1:1. After the pre-maturation stage.e. PepC and PepY from Lb. Capellas. Therefore.5% v/v ried out a kinetic study on the effect of HP on the aminopeptidases of the pre-mentioned starter culture was used. which are important for the of untreated starter culture containing 50% Lactococcus lactis and 50% acceleration of cheese ripening. affecting directly the enzyme modulation sites or active were grown aerobically at 35 °C. to the cheese itself. This levels. produced cheeses were packed with brine (7% w/w NaCl) in plastic ed that Cheddar cheese could ripen in 3 days by treatment at 50 MPa. 20 °C and 15 min on ripening indices of Feta cheese.

07. The cheeses after pressure build up (20 MPa/s). et al. Merck. To determine the mineral content (ash).4. For data analysis. Merck. A craft knife adapter with a standard load (including starter microorganisms.05463. 3.e.8. Kotoula. Merck. Reduction panel consisted of at least eight members of laboratory personnel of the protein content of the cheeses was observed and could be mainly Please cite this article as: Giannoglou.8. Total fat content was measured using the Smedes method (Smedes. UK). water solu- 2. NSLAB and TVC) was re- 50 mm wide craft blade and blade thickness 0.05) differences between HPA and HPB cheeses Cheeses were organoleptically evaluated at all sampling points by a were observed with regard to physicochemical parameters. 2. as nmoles of p-NA per min per gram of cheese. non-starter bacteria and the total viable count viabilities. Milan. 1a–d.05) thereafter. Innovative Food Science and Emerging Technologies (2016). are using Mohr method (IDF. Data analysis oven at 100 °C for 24 h. and Alichanidis (2001).6 mm was used for deter. Pressure and temperature in the chamber were constantly monitored and recorded in 1 s intervals during the process. This were also evaluated regarding the ripening perception. Decagon devices.1. M-17 agar acid (PTA-SN) was performed as described by Nega and Moatsou (1. The desired value of pressure was set and ness and general opinion by means of a 1 to 9 hedonic scale. put into pre-weighed glass dishes and kept in 2. Analysis of physicochemical characteristics and 2 mm/s for pre.. 2. Moschopoulou. Organoleptic evaluation two replicate experiments ± stdev). Plakas. A slight decrease of viable cells within the first 20 days of cold storage was evident while the Cheese texture was analyzed using a TA-XT2i Plus texture analyzer counts did not change significantly (p N 0. 7 cm in width and 2. Soluble nitrogen fractions For the determination of the soluble nitrogen fractions.2. all cheeses apart from HPC showed the same trends 2. Italy). according to the method described by The color of the samples was measured using a colorimeter Minolta Desmazeaud and Juge (1976).15029. Assessment of proteolysis surization. 14. Effect of high pressure treatment applied on starter culture or on semi-ripened cheese in the quality and ripening of cheese in br. Nega and Moatsou (2012).6. inactivation.1 M in distilled water (1:1 ratio) using an AMEL pH-meter (338. The initial adiabatic with a mature cheese. Osaka. for 48 h. USA). i. For HPC (Stable Micro Systems Ltd. flavour. The nitrogen soluble at 5% phosphotungstic aerobic mesophilic bacteria incubated at 30 °C for 72 h.3. Results and discussion The total protein content of the four experimental cheeses was measured as described by Moatsou. the pressure vessel was isolated.7. adhesiveness. the Texture Expert samples that were subjected to HP 60 and 90 days of defined time. Chuo-Ku. 2. PepA and PepX means of a combined glass electrode in a dispersion of grounded cheese aminopeptidase activities were estimated in cheese extracts in 0. duced. 45. incubated at 30 °C for 4 days. / Innovative Food Science and Emerging Technologies xxx (2016) xxx–xxx 3 Holland).0.5. and were analyzed statistically by one-way ANOVA using least signifi- Water activity of the samples was measured using a 4TEV water cant difference (LSD) at p b 0. for colour. http://dx..5 cm in height.1016/j.and post-test were used throughout the study. elasticity and cohesion were determined. the cheeses were removed from the oven. the 2nd stage of ripening. Germany) for thermophilic cocci incubated at 42 °C (2012). Peptides profiles and non-starter lactic acid bacteria (NSLAB) in Rogosa agar (1. Microbiological analysis Anifantakis. pH 5.. mainly due to the effect of HP that apparently induced microbial mining the textural characteristics of a cheese block of 10 cm in length. Texture analysis for all studied microorganisms.5 L vessel was controlled by liquid trained to grade cheese. Microbiological analysis ble extracts (WSE) of the four experimental cheeses were prepared as described by Zoidou. Probe speeds of 1 mm/s during the test 3. For this purpose point defined the time zero of this process. All measurements of samples were carried out at 25 ± 1 °C and the The physicochemical characteristics of the four cheeses were studied hardness.05 to check the differences. texture.. USA) was used.2. released (release time b 3 s) after a predefined time (according to the ex. Giannopoulou. (2015). No significant (p N 0. The weight loss was expressed as All analyses were performed in triplicates for different parameters moisture content. Statistica 7 activity meter (AQUA LAB.ifset. OK. After the 24 h. 10 g of cheese were grounded. Thermophilic lactobacilli.9. thermophilic The salt content of the four experimental cheeses was measured cocci. 21. M. at 12% trichloroacetic acid (TCA-SN) were also performed as described Plate count agar (1. using Leu-p-NA and Gly-Pro-p-NA Instruments. Kandarakis. Tulsa. compared the experimental cheeses to a mature cheese. the physico- chemical characteristics of the four cheeses are presented (mean of 2.10661. (2010).05413. For the determination of the moisture content of the samples.1. appearance. 7. 30. In Table 1. Process temperature in the 1. Japan). Peptide analysis of SN was performed using RP-HPLC as described by Merck. the initial microbial Exceed Software was used. Samples were compressed twice for a penetration depth of 10 mm. pH 7. Aminopeptidases assays The activities of aminopeptidases PepA and PepX were measured The pH value of the four experimental cheeses was measured by during the ripening stage for all studied cheeses. In general. Giannoglou et al.5. using the pour plate method.8. The analysis of the soluble nitrogen (SN) and nitrogen soluble Moschopoulou et al. respectively. 1988). high perception score means closer organoleptic characteristics perimental design) by opening the pressure valve.. Germany) incubated anaerobically at 42 °C for 72 h 2. AMEL sodium phosphate buffer. bitter- circulation in the outer jacket. as substrates. Germany). The pressure of the vessel was the panellists.doi. and Moatsou Microbiological analysis was performed as described by (2015). presented in Fig. Germany) was used for the total by Zoidou et al. 3. 1999).024 . M. Analysis of physicochemical characteristics 2. during the 2nd ripening stage for both trials. 3 g of grounded cheese Microbiological analysis of all the cheese samples was performed were heated in a muffle furnace at 600 °C for 6 h and weighed after a after the pre-maturation stage at 1. Thermophilic lactobacilli were grown in acidified MRS-agar (1. temperature increase during pressure build up was taken into consider- ation in order to achieve the desired operating temperature during pres.2016. Soft.8.. software (Stat. Apep and PepX activities were expressed CR-200 (Minolta Company.. cooled and weighed.

63 ± 0.75 19.54 ± 0.75 ± 0.49 ± 0.63 ± 0.92 ± 0.40 ± 0.79 ± 0.70 ± 2.18 4.21 19.13 0.72 4. HPA . 2015.64 ± 2.75 53.11 0.45 5.59 ± 0.20 13.61 49.12 4.05) differences were observed for the other physicochemical of water holding capacity (Saldo.03 ± 0.012 45 59.81 4.70 20.21 3.47 14.09 49.68 ± 0.66 14.79 ± 0.41 3.53 2.11 ± 0..11 ± 0.09 4.41 3.15 ± 0.14 18.20 4.41 ± 0.45 5.20 4.38 3.34 3.54 ± 0.965 ± 0.4 M.35 ± 0.936 ± 0.06 4.94 ± 0.58 ± 0.018 30 62.45 6.67 5. 2013).950 ± 0.60 ± 0.80 3.39 ± 1.14 4.13 0.15 ± 0.04 0.42 ± 0.12 14.40 22.70 ± 0.003 7 59.41 ± 1. b Fat in dry matter.03 0.14 21.56 ± 1.96 4.79 ± 0.943 ± 0.62 ± 0.32 ± 0.43 19.11 3.ifset.11 ± 0.99 14.82 ± 0.008 21 61. attributed to their diffusion into the brine (Zoidou et al.21 50.11 0. Ferragut.99 ± 0.90 52.014 60 59.40 ± 1.007 14 61.56 21.90 ± 0.08 55.62 ± 0.98 ± 0.71 ± 0. Cheese Rta (d) Moisture Fat FDMb Protein Ash Salt S/Mc pH aw Control 1 63.55 5.24 4.48 18. HPC samples.20 ± 0.41 51.84 ± 0.57 4.57 5.53 ± 0.940 ± 0.56 4.69 ± 0.98 3.10 4.64 13.024 .54 3.40 ± 1.54 4.72 ± 0.52 ± 0.73 6.64 5.50 ± 0. and Trujillo (2008) et al.85 ± 2.73 17.36 ± 0.38 ± 0.79 6.44 3.012 90 59. for cheeses processed with HP at 300 MPa for 10 min.55 ± 0.89 ± 0.08 ± 0.88 ± 0.07 4.85 17.90 ± 0.68 ± 0.90 ± 0.79 4.942 ± 0.63 ± 0.33 52.07 ± 0.30 ± 1.70 19.14 ± 0.950 ± 0.51 ± 0. Viable bacterial counts (log cfu/g) of a) Thermophilic lactobacilli.09 4.942 ± 0.02 ± 0.01 4.07 ± 2.56 6.41 14.70 ± 0.57 ± 0.18 ± 0.04 4.010 90 58.01 0.06 47.57 ± 0.77 4.82 ± 0.74 ± 0.40 ± 1.03 4.54 19.31 19. 2001).18 4.75 ± 0.001 21 61.11 ± 0.953 ± 0.71 4.46 22.55 ± 0.012 45 60.941 ± 0.45 3. Innovative Food Science and Emerging Technologies (2016).19 ± 0.39 4.16 ± 0.943 ± 0.19 ± 0.81 ± 0.25 4.06 52.96 ± 0.13 18.944 ± 0.99 14.56 ± 0.66 ± 0..24 3.12 ± 0.doi.019 7 62.39 ± 2.47 ± 0. et al.31 ± 0.14 ± 0.06 4.018 HPB 1 63.13 3.959 ± 0.86 ± 0.05 17.70 ± 0.89 ± 1.942 ± 0.76 ± 0.13 ± 0..61 6.56 ± 0.93 ± 0.45 6.30 4.05 0.33 50. Effect of high pressure treatment applied on starter culture or on semi-ripened cheese in the quality and ripening of cheese in br.12 0.93 ± 0.45 ± 0.26 ± 0.016 30 62.13 ± 2.24 4.62 ± 0.09 3.07 0.09 ± 0.60 50.009 HPC 1 61.80 ± 0. HPB and HPC cheeses during the second ripening stage (mean values of two replicate-experiments ± stdev).26 ± 0.005 21 61.02 15.00 53.18 17.59 14.65 ± 0.03 ± 0.60 ± 0.59 ± 0.71 ± 2.05 0.10 0.17 0. mineral content and water activity between higher salt content in accordance to its higher moisture content.66 ± 0.942 ± 0.021 90 61.956 ± 0.76 ± 0.22 ± 0.21 6.017 45 62.04 ± 0.940 ± 0.941 ± 0.67 ± 0.17 ± 0.90 ± 0.011 45 59.07 0.69 ± 0.020 60 62.61 4.2016.41 ± 0. b)Thermophilic cocci.22 4.62 ± 0.74 ± 0.46 ± 1.04 4.30 ± 0.08 4.24 ± 0.003 14 63.13 ± 1.20 50.16 3.19 ± 1.11 14.61 ± 0. Please cite this article as: Giannoglou.19 ± 1.46 50.47 14.98 21.56 ± 0.49 ± 0.17 ± 0.020 90 57.87 4.10 ± 1.59 14.66 ± 0.66 ± 0.35 ± 0.89 ± 0.57 55.63 54.99 ± 0.07 ± 0.91 53.93 ± 1.07 0.61 15.013 60 60.73 ± 0. Guamis.67 ± 2.26 ± 0.93 ± 1.18 ± 0.45 ± 0.73 5.12 4.81 3.009 HPA 1 63.37 ± 1.22 ± 0.77 14.21 ± 0.66 5.42 ± 0.87 ± 0.69 ± 0.48 21.01 0.66 50.97 ± 0.60 ± 1..13 0.86 48.17 3.74 ± 0.946 ± 0.05 53.81 3.57 23.011 30 59.52 ± 0. Maniou results are also presented by Juan.43 ± 0.43 5.56 21.79 ± 1.06 0.30 3.55 ± 1.60 4.62 ± 0.49 ± 0.74 ± 0.008 14 60.35 ± 0.39 19.20 4.020 60 58.69 ± 1.07 ± 0.54 ± 0.83 ± 0. maintained their initial moisture content.36 4.76 49. HPB and HPC cheeses during the second stage of ripening (mean of two replicate-experiments ± stdev).19 ± 0.58 ± 0. Similar the samples.07 0.10 ± 1.20 47.49 ± 0.01 14.80 ± 0.941 ± 0.34 ± 0.51 ± 2.97 ± 0.28 4.04 0.56 ± 2.61 ± 0.29 ± 0.15 0.18 14.57 4.42 51.04 14.32 ± 1..957 ± 0.72 ± 0.55 ± 0.05 0.28 6.69 5.56 ± 0.18 3. M.26 ± 0.04 6.07.38 ± 0.50 ± 0.67 21.54 5.28 3.56 52.01 0. c Salt-in-moisture.35 5.75 ± 0.22 ± 0.48 13.60 ± 0.938 ± 0.18 ± 0.70 4. No significant probably due to high pressure processing that may result in an increase (p N 0.56 ± 1.007 14 63.01 0.67 4.942 ± 0.69 ± 0.71 ± 0.03 ± 0.16 13.71 4.10 4.95 ± 0.79 3.60 ± 1.43 19.947 ± 0.13 ± 0. Giannoglou et al.03 0.83 13.88 ± 1.26 ± 1.017 a Ripening time (days of second ripening stage). & Guamis.26 ± 0.07 ± 0.943 ± 0.25 ± 0.15 6.015 7 64.946 ± 0.46 2.83 ± 1.62 14.43 ± 0.64 ± 0.49 3.02 0.90 ± 0.57 ± 0.86 ± 1.97 48.83 ± 2.90 ± 0.935 ± 0.08 ± 0.76 ± 0.71 ± 2.61 ± 0.96 ± 0.45 ± 0.79 ± 0.94 4.83 4.02 6.86 4.07 6.10 0.67 4.63 ± 0.45 54.53 4.014 30 59.56 ± 0.19 4.31 14.64 ± 0.65 ± 0.34 ± 0.01 0.36 3.41 15.002 7 61. http://dx.33 15.21 ± 0.02 14.06 4..06 15.35 ± 1.70 13.70 ± 0.23 ± 0.26 ± 0.57 21.61 ± 0.06 19.06 3.79 3.01 5.06 19.55 ± 0.97 ± 0.49 3.28 ± 0.13 ± 0.43 ± 0.43 ± 0.05 0. Sendra.87 ± 1.26 4.60 ± 0.37 14.73 ± 0.52 ± 0.99 ± 0.69 4.67 3. / Innovative Food Science and Emerging Technologies xxx (2016) xxx–xxx Fig.. Table 1 Physicochemical characteristics of Control.26 3.11 ± 0.55 3.34 ± 0.014 21 61.40 3.14 22.944 ± 0.62 ± 0.91 ± 0.65 ± 0.97 4.31 4.38 53.05 ± 0.79 6.18 3.941 ± 0.48 15. c) Non-starter bacteria and d) Total viable lactic acid bacteria of Control .32 5.18 ± 0. HPA.26 4.35 4.11 55.73 ± 0.79 3.46 51.23 53.50 ± 0.05 0.45 21.64 ± 0.88 ± 0.01 5.66 4.26 6.66 ± 0.63 ± 0.86 ± 1.97 ± 0.04 ± 3.1016/j.12 5.939 ± 0.03 ± 0.38 ± 0.22 ± 0. 1.56 3.05 4. HPC had parameters such as pH.48 15.70 18.951 ± 0.90 ± 1.75 ± 0.29 4.35 14.95 20.18 0.07 4.15 4.07 ± 0.99 48.943 ± 0.

72% and 0. HPC cheeses were character. 3. store ripening period.07.. during the 2nd stage of ripening in cold stores of the four experimental cheeses is shown. the hardness of all samples is presented. showed the low.05) differences were observed among the observed due to migration of soluble nitrogen compounds into the values of elasticity and cohesiveness for the four experimental cheeses.. followed 12% trichloroacetic acid on total nitrogen (TCA-SN/TN) and the soluble by HPB. 5b–d. cheese produced with HP treated culture had better organoleptic scores compared to the control cheese and cheese treated with HP. et al.6% and 0. nitrogen at 5% phosphotungstic acid on total nitrogen (PTA-SN/TN). the nitrogen soluble at hardness and adhesiveness were observed for HPA cheeses. 2015.. 7.05) of the soluble first days of storage.05) during the studied period. 2013). HPA . which is usually observed in not fully ripened cheeses. respectively. higher than the reference sample at 90 days of cold storage by 6.4. In the case of HPC cheeses a parison to the other cheeses. Less consequently the fastest proteolysis was observed in HPB cheeses mechanical incisions were observed for HPB and HPC samples in com. the changes of %WSN/TN. In the case of HPC samples. than in control samples by 4. Peptide analysis faster reduction of the bitter taste. HPA . respectively. HPB and HPC cheeses for the specific time in- HPC cheeses' bitterness was less profound compared to the Control tervals during the 2nd ripening stage were analyzed and the RP-HPLC Fig. Reduction of %TN of the cheeses during storage was No significant (p N 0. 3a). HPA. 2001)..2016. In general. The fastest accumulation of the soluble HP treatment on cheese structure (Saldo et al. b show the scores of ripening perception and the general opinion of the panel for the four cheeses at 30 days of ripening. 21.024 . 2001). 3.5. They had the highest scores in structure. Maniou et al. Innovative Food Science and Emerging Technologies (2016). est hardness. compared to control. The bitterness of the unripened cheeses was another characteristic studied and evaluated organoleptically. However. in cheese.49%.ifset. ized as softer compared to other samples. 2. Organoleptic evaluation of Control ■. respectively. 4a. Similarly to instrumental results. Proteolysis progress All the samples were analyzed during the 2nd stage of ripening. 3b). 30. HPA and HPB cheeses were similar to a mature cheese even from the In contrast to %TN. Fig. as shown by the increased levels of nitrogen soluble in 12% tic scoring of all HP-treated samples (HPA. / Innovative Food Science and Emerging Technologies xxx (2016) xxx–xxx 5 sample. stage is depicted. HP treatment can activate starters' proteolytic enzymes that contribute to the further degradation of particular peptides responsible for the bitter taste (Casal & Gomez.22%. In general. M. compared to HPC and Control cheeses (Fig.3. In Proteolysis progress was evaluated using the ratio of the water Fig. especially in HPA and statement. HPA and HPB cheeses showed 3. compared to all the other samples. was observed in HPA cheeses compared to Control and HPC samples. Higher values in hard. nitrogen fractions during the 90 days of the 2nd stage of ripening and The mechanical incisions of the four cheeses were also studied. %TCA-SN/TN and and HPC samples. Effect of high pressure treatment applied on starter culture or on semi-ripened cheese in the quality and ripening of cheese in br. HPC samples. M. appearance-color and flavor.1016/j. this phenome. Faster proteolysis during the whole period. Hardness of Control . starter culture (HPA and HPB samples) increased secondary proteolysis 60 and 90 days of cold storage using a 1–9 hedonic scale. 1999). ness were observed for HPA and HPB compared to control samples In particular the SN/TN%.6. 5a. 14. Please cite this article as: Giannoglou. HPB and HPC) was higher trichloroacetic acid and 5% phosphotungstic acid.doi. The texture of these cheeses confirmed the above nitrogen fractions was observed in all samples. a significant increase (p b 0. The organolep. HPB and HPC cheeses during the second stage of ripening for their a) hardness and b) bitterness (mean of two replicate- experiments ± stdev). 2. Organoleptic evaluation WSN/TN and TCA-SN/TN of all cheeses were significantly increased (p b 0. HPB and HPC cheeses during the was similar to that of a mature cheese even at the beginning of the cold second stage of ripening (mean of two replicate-experiments ± stdev). PTA-SN% in HPA cheeses were (Fig. Giannoglou et al.. apparently due to the effect of 5. 45. Control and finally HPC samples.. WSE of Control. The highest values of soluble nitrogen over total nitrogen (WSN/TN). The scoring of the ripening perception was higher for HPA and HPB cheeses and slightly higher for HPC samples compared to Control samples. the change of the %TN of the cheeses during the ripening capacity that may affect their final hardness (Saldo et al. 3. brine (Zoidou et al. In Fig. moderate acceleration in cheese proteolysis took place compared to non is also attributed to the HP processing which results in trapped gas the reference samples. %PTA-SN/TN. SN/TN% and PTA-SN% at 90 days were higher liquefaction.. http://dx. HPA and HPB samples showed the highest organoleptic score during the whole studied period. since HPA and HPB cheeses were harder compared to control HPC cheeses. the use of HP-treated All the cheeses were organoleptically evaluated at 1.. No bitterness was detected while the organoleptic ripening perception of the HPA and HPB cheeses Fig.45%. Texture analysis 3. as the HP processing of cheese increased the water holding In Fig.

Similarly to the results phobic and/or large peptides in the soluble extracts. Innovative Food Science and Emerging Technologies (2016). The aminopeptidase activities The comparative RP-HPLC profiles (A220) of the soluble N fraction were higher in HPB cheese at the first day of cold storage compared to profiles of HPA. stage of ripening of HPB samples could be attributed to an increase of Fig. part II to 16–32% acetonitrile and behavior in all cheeses. Giannoglou et al. HPB and Control cheeses (Fig.1016/j. soluble extract that are not retained in the C18 column.. 1a–d). most clearly in the profiles of HPB cheeses made exclusively with HP. Monitoring of the a) Total nitrogen (%TN) b) Water soluble nitrogen to total nitrogen (%WSN/TN) c) Nitrogen soluble in 12% TCA to total nitrogen (%TCA-SN/TN) and d) Nitrogen soluble in 5% PTA to total nitrogen (%PTA-SN/TN) of the Control ■. 6a–c. HPA . Effect of high pressure treatment applied on starter culture or on semi-ripened cheese in the quality and ripening of cheese in br.. 20 °C for 20 min resulted in a significant increase was markedly reduced (Table 2). with ≥48% acetonitrile in the elution mixture.024 .e. particularly at the day 60. tive characteristics are presented in Table 2. increase compared to untreated samples) in the HPA and HPB mentioned effect of HP on starter peptidases. According to their report. ripening stage. Individual caseins and whey proteins control samples but in HPA cheese was more active during the whole are also eluted in part III. (2016).6 M. profiles were divided in four parts.. up to 40 days. In particular. / Innovative Food Science and Emerging Technologies xxx (2016) xxx–xxx Fig. i. HPB and HPC cheeses during second stage of ripening (mean of two replicate-experiments ± stdev). the profiles of The increased aminopeptidases activity was not related to HP- the four cheeses had the same peaks in variable quantities. b shows the PepA and PepX activities The part 0 corresponds to the first 10 min of analysis. elution in Control. 5. HP treatment after 90 days of cold storage the peak area ratio of 55–100/10–55 min. In general.e. HPB and HPC vs control at day 90 of the second ripening control and HPA cheeses but they were constantly reduced thereafter stage are shown in Fig. standard mixtures of individual caseins and whey proteins are eluted PepA and PepX activities were measured during the 2nd stage of after 85 min. Activity of aminopeptidases PepA and PepX according to Nega and Moatsou (2012). of Katsaros et al. et al. while both enzymes presented similar corresponds to 0–16% acetonitrile. http://dx. The increased activity of aminopeptidases at the first day of the 2nd treated starter.. HPA and HPB cheeses during the 2nd ripening stage in with 0% acetonitrile and includes mainly components of the cheese cold store. some Significant differences in aminopeptidases behavior among the free amino acids and non-nitrogen soluble components. (2009) and Giannoglou et al.2016. HPA aminopeptidases activity was similar to part III to 32–48% acetonitrile.7. This effect was depicted samples. HPA. Please cite this article as: Giannoglou. It was induced autolysis. at 200 MPa. M. as the microbial load appeared to be similar for evident that the HP treatment of either cheese or starter reduced hydro. 7a. 4. HPC.e.. Fig.ifset... respectively.05) of starter culture aminopeptidases' activity (3-fold findings of proteolysis analyses and apparently are due to the above. Organoleptic evaluation of the a) ripening perception and the b) general impression of the Control ■. while the particular quantita. i. The interpretation of the profiles was 3. ripening for all the cheeses.. HPB and HPC cheeses during the second stage of ripening (mean of two replicate-experiments ±stdev).doi. i.07. HPA . The part I different samples were respectively. These findings coincide with the (p b 0.

cell membrane permeability.5 0.1 30. M.27 90 8. peared to be much higher even when the microbial load was similar to Table 2 Parts of RP-HPLC profiles of SN fractions of the four experimental cheeses for 0 and 90 days of cold storage expressed as mean percentage of the total area (mean values of two replicate- experiments).9 28.9 1. 6.5 0.. such as peptidases.8 27.9 27.21 90 8.. to the activity up to 45 days of cold store ripening.7 35.09 1. M.4 34.19 0. Comparative RP-HPLC profiles (A220) of the soluble N fractions for 90 d cold storage of a) Control and HPA cheeses. Aminopeptidase activity ap- medium (Trujillo et al.94 1.64 1.ifset.5 33.8 0.7 36.5 29. b) Control and HPB cheeses and c) Control and HPC cheeses.81 1.1 32. Effect of high pressure treatment applied on starter culture or on semi-ripened cheese in the quality and ripening of cheese in br. 1992)..2016. caused by HP treatment (Cheftel.14 HPA 0 11. HPC cheeses appeared to have the most increased aminopeptidase favoring the release of intracellular material.03 90 10.10 HPC 0 10.75 1.30 HPB 0 12.doi..2 43. 2000).1 20. Cheese Ripening Part 0 0–10 min Part I 10–40 min Part II 40–70 min Part III 70–100 min 55–100 min/ 40–70 min/ time (d) 0% ACN 0–16% ACN 16–32% ACN 32–48% ACN 10–55 min 10–40 min Control 0 7.3 23. Giannoglou et al.5 1.3 31.024 .. http://dx.1016/j.8 0. Innovative Food Science and Emerging Technologies (2016).99 1..14 Please cite this article as: et al. / Innovative Food Science and Emerging Technologies xxx (2016) xxx–xxx 7 Fig.6 0.89 90 11.7 26.3 20.7 25.1 0.7 35.7 34.3 27.9 23.07.5 30.85 1.3 24.8 28.

M. scaling up of the findings of our study. http://dx. Le Lait. Moschopoulou. L.. Kaps. (2001). Murphy. Katsaros. Guamis. A. & Guamis. Innovative Food Science & Emerging Technologies. especially tion of flavor compounds in cheeses during ripening: A review. Hard cheese structure after a high hydrostatic overview. R.. Multiple-peptidase mutants of Lactococcus lactis are severely starter cultures (small volumes) is more cost efficient than applying impaired in their ability to grow in milk.. IDF (International Dairy Federation) (1988). J.. This conforms with the potentiality of 293–324. High pressure effects soluble in 12% trichloroacetic acid and 5% phosphotungstic acid and on proteolytic and glycolytic enzymes involved in cheese manufacturing. Anjitsu. optimal ripening time of Tounj cheese. Havranek. J. as shown by the increased levels of nitrogen 219–225.. K. 80. Journal of Dairy Research.. A. 4402–4413. Ishibashi. Journal of Food Science. M. E. H.. (1996). & Govaris. Brussels. & Sousa. 109–117. & Shimamura.. 11(3). L. & Taoukis. J. M. proteolysis in cheese. significantly affect the bacterial counts of Feta-type cheese. / Innovative Food Science and Emerging Technologies xxx (2016) xxx–xxx Fig. 86(4). & Malorni. A.. The third dimension of food technology. T. J. (2001). (2010). E. Oliveira. Pedersen. Hellendoorn. D. The fastest accumula.. 65(4). M. No bitterness was detected from the be. D. Miyakawa. 101. E. Proteolysis and related enzymatic activities in ten Greek cheese varieties.. A. Paschos and Mr.. P.. R. conduction of the research. G. E. Auty. vol. G. J. V. Proteolysis and the cheeses were harder compared to control and HPC samples. J. Small Ruminant Research. A. Kelly. France: John Libbey Eurotext. I. C. E. C. Le Lait. 11–20. Application of high-pressure treatment on ovine brined cheese: Effect on composition and microflo- Grammatikas from the Laboratory of Dairy Research of the Agricultural ra throughout ripening. T.. C. Cappuccio... I. Belgium: International ginning of the cold storage period.. P. the use of effect of high hydrostatic pressure and temperature on the activity of Lactobacillus HP-treated starter culture (HPA and HPB samples) increased secondary delbrueckii subsp. & Léonil. In C. Journal of the peptidic profiles of the water soluble extract. V. Journal of Bacteriology.07. (1994). Innovative Food Science & Emerging Technologies. Addeo. The ripen. A. A. & Beresford. Conclusions Gagnaire. & Beresford. N. Kok. Giannoglou et al..doi. &amp... International Journal of Dairy Technology. since HPA and HPB Kalit. P. E. 58(3). (2005). Dairy Science. E. International Dairy Federation IDF Standard 12B.. J.. (2012).... 4. (Eds.1016/j. T. Peptidases and amino acid catabolism in lactic acid bacteria.. Poolman. the other samples. 81. 165–173. 24. M. 2–8). Effect of high pressure on the viability and enzymatic Saldo. Wick. Kunji. Giannoglou. cheeses treated with HP (HPC). & Courtney.. Technology. 69–77. Moschopoulou.. (2013). Montrouge. 11(4). Dairy Science. Moschopoulou. Please cite this article as: Giannoglou. G. M.. Dairy Science. O'Reilly. Tetra International Dairy Journal. In general. & Steele.. (2008).. 1092–1098. The texture of ment at 300 MPa on ripening of ewe's milk cheese.4. 93. pressure treatment at 50 MPa for 72 h applied to cheese after brining.. C. (pp. G. 2794–2803. directly to cheeses (larger volumes) for same HP vessel filling ratio. et al. C. Technologie alimentari.. Effect of high pressure treatment applied on starter culture or on semi-ripened cheese in the quality and ripening of cheese in br. The effect of high pressure treat- cheese even at the early phase of cold store ripening. Changes in activity of (a) aminopeptidase A (PepA) and (b) X-prolyl dipeptidyl aminopeptidase (PepX) of Control .. Tsala.. J.. B.. S. Ferragut.. Desmazeaud. P. The increased aminopeptidase activity could be Christensen. International Journal of Dairy Technology. (1992). T.024 . O'Reilly. (1992). Ef- We thank Professor Tsakalidou E. High hydrostatic pressure for accelerating ripen- activity of mesophilic lactic acid bacteria isolated from caprine cheese. P. S. P. Herrouin.. E. B.. 619–624.. M. C... B. processing on physico-chemical characteristics of fresh goat's milk cheese (Mató)... Cheftel. Katsaros. when applied to starter cultures. M. & Moatsou. & Juge.. the results show that HP technology could be a potential Mc Sweeney. Sacchi. Chianese. M.. (2000). ing indices studied such as WSN/TN and TCA-SN/TN were significantly 15(6–9).. P. T. G. Journal of ing of goat's milk cheese: Proteolysis and texture. Salzano.2016. Mor-Mur.. P. Determination of salt content (Mohr meth- od). Katsaros. & Guamis. Rovere. G. Dairy Science and Technology. 178.. K. (1976). & Taoukis. leptic evaluation of all cheeses showed that the cheeses produced with Food and Bioprocess Technology. F. H.. 195–209).. E. However. 1139–1146. ception of the HPA and HPB cheeses was similar to that of a mature Juan. 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Effect of high pressure 277–284. Biochemical pathways for the produc- treatment for the acceleration of ripening of cheeses in brine. S. A. Antonie Van Leeuwenhoek. since applying HP technology to . Pak Report. Journal of Application of HP treatment (200 MPa at 20 °C for 20 min) did not Agricultural and Food Chemistry. attributed to both HP processing and autolysis. (2009). P. Effect of to all the other samples. Effects of pressure on enzyme activities of Lactobacillus helveticus LHE-511.. I.. P. Dairy Federation.. Innovative Food Science and Emerging Technologies (2016). A. A. Sendra. Nega. We also thank Mr. (2000). Sendra. M. Effect of high pressure References on proteolysis during ripening of Cheddar cheese. ripening of Cheddar cheese. L. M.. 56. tion of proteolysis products took place in HPB cheeses when compared Maniou. A. Moatsou. Le Lait.doi. E. 129–138. cheesemakings..1007/s11947-016-1781-3 HP treated culture were superior compared to the control cheeses and (FABT-D-16-00165). 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