Evaluation of cow’s and goat’s fresh cheese preservation under hyperbaric storage at room temperature up to 60 days versus refrigeration regarding nutritional and physicochemical parameters

doi:10.21203/rs.3.rs-1549813/v1

Two highly perishable dairy products (cow’s and goat’s fresh cheese (FC)), were stored under hyperbaric storage at room temperature (HS/RT, 50-100MPa) and compared with RF (4ºC), for 60 days.

HS/RT (≥75MPa) lead to a more stable volatile organic and fatty acid profiles, reducing lipid oxidation rate, particularly for cow’s FC, resembling more to FC prior storage, contrarily to refrigerated cheeses. No variation was observed for total protein, while free amino acids increased over time after 60 days at 75/RT of 13- and 16-fold, and at 100/RT of 14- and 8-fold, respectively in cow’s and goat’s FC, which may have contributed to increased protein digestibility (5.2%) observed for goat’s FC after 60 days at 100/RT.

These results indicate the overall increased preservation performance achieved by HS/RT for cow’s and goat’s FC when compared with RF, slowing down FC matrix degradation, possibly leading to considerable shelf-life extension.

Fresh cheese

hyperbaric storage

volatile organic compounds

fatty acids

free amino acids

protein digestibility

Hyperbaric storage (HS) is new a preservation methodology, based on storage under moderate pressure (between 25-150 MPa), that relies mainly on microbial growth slowdown/inhibition, similar to conventional refrigeration (RF) (Segovia-Bravo, Guignon, Bermejo-Prada, Sanz, & Otero, 2012). While the first studies regarding this new methodology, emphasized the combination of sub-zero or low temperatures with low pressure (Charm, Longmaid, & Carver, 1977, Mitsuda, 1972), when applied at room temperature (RT), HS arises as an environmentally friendlier food preservation methodology compared to RF, with considerable potential to extend foods shelf-life and increase microbial safety (Fidalgo et al., 2014, Santos, Castro, Delgadillo, & Saraiva, 2020). During HS at uncontrolled variable RT, energy employed to maintain the temperature is null, being only applied during the compression/decompression of the storage vessel, what can result in up to 26-fold lower energy used by HS/RT, comparatively to RF (Bermejo-Prada, Colmant, Otero, & Guignon, 2017).

The feasibility of HS at and above RT was studied initially in fruit juices as case study, in more acid ones (strawberry juice) to low acidity juices, more perishable (watermelon and melon juice) (Fidalgo et al., 2014, Queirós et al., 2014, Segovia-Bravo et al., 2012), with the outcomes pinpointing to a possible shelf-life extension, due to microbial inhibition/inactivation during HS, with minimal physicochemical changes reported. Other non-liquid highly perishable food products were also evaluated under HS/RT as a case study, initially for short storage periods with promising results (Duarte et al., 2014, Fernandes et al., 2015). One of these products was whey cheese, which has only a couple weeks of shelf-life at RF, presenting after 8 hours at 100/RT no pronounced changes in colour, pH, and water activity, showing a slightly increased in lipid oxidation values, with a clear microbial growth inactivation in all microbiological groups even at and above RT (25-37 ºC) (Duarte et al., 2014). One a second study, this product presented higher stability under HS (100 MPa) during longer storage periods (10 days) at variable RT, retaining the pH, water activity, and fatty acid profile, while presenting fewer colour losses comparatively to RF, with an additional microbial inactivation effect to undetectable counts (1 log CFU/g) in all the studied microbiological groups (from the 3^rd day of storage onwards) (Duarte et al., 2017). Indeed, in some of the works described just above that were conducted by our research group, pointed to an additional hyperbaric inactivation (HI) effect along storage in various microbiological populations present in different foods, leading in most cases to constant undetectable counts (below the detection limit) until the end of the storage period. This HI effect, reached for several cases over 5 log units for vegetative bacteria, opening the possibility of achieving pasteurization during HS, a possibility that is now being intentionally and systematically studied in our research group.

Fresh cheeses (FC) are highly perishable dairy products, characterized by short shelf-life (a few weeks at RF), mainly due to their high water activity and rich nutritional profile that promote microbial spoilage, leading to increased syneresis, decrease in the pH, lipolysis, proteolysis, oxidation, and off-flavour formation, that crucially limits its shelf-life. In a previous study, FC stored under HS/RT conditions (75-100 MPa) resulted in increased microbial control, leading to an increased microbial shelf-life, while also maintaining most of its basic physicochemical parameters (pH, whey loss, moisture content and colour) at levels comparable to cheeses prior to storage (unpublished results). During that study significant HI was observed, gradually reducing total aerobic mesophiles counts more than 5 log units throughout the 60 days of storage, (initial counts of ~7 log CFU/g), reaching progressively values below the quantification and detection limits (2 and 1 log CFU/g), depending on pressure level and storage time (unpublished results). In the present study, the effect of HS/RT (50-100 MPa) during 60 days was studied on two FC (from pasteurized cow’s and goat’s milk), with a more in-depth approach on nutritional and physicochemical parameters evaluation, regarding in vitro protein digestibility, total protein, free amino acids, lipid oxidation, fatty acids and volatile organic profiles and compared with RF under atmospheric pressure (AP).

2.1 Fresh cheese samples preparation and storage

Two commercially fresh cheeses (FC) one made from pasteurized cow’s milk and the other from pasteurized goat’s milk, were acquired from a local supermarket. These cheeses were produced through enzymatic coagulation, with each kind of cheeses bought from the same company and lot with similar remaining shelf-life. Temperature was kept low (3-8 ºC) during FC transportation being after packaged into low permeability polyamide-polyethylene bags (90 micron, IdeiaPack, Comércio de Embalagens, LDA, Abraveses, Viseu, Portugal), previously sterilized with UV-light, under aseptic conditions inside a laminar flow cabinet (BioSafety Cabinet Telstar Bio II Advance, Terrassa, Spain) and heat-sealed individually.

Fresh cheese samples were stored under different conditions, with the experiments performed under HS (50,75 and 100 MPa) at room temperature (RT) and sampling for analysis at 3, 7, 14, 28, 42 and 60 days, in a high pressure equipment SFP FPG13900 Model (Stansted Fluid Power, Stansted, UK), with a mixture of (40:60) propylene glycol and water used as the pressurizing fluid. As a control, cheeses were also stored under atmospheric pressure (0.1 MPa) at RT (15 - 22 ºC) and refrigeration (4 ºC), during 3 and 7 days, respectively, since samples were microbiologically unacceptable on those periods (unpublished data), and thus those storage experiment were interrupted. Cheeses at AP/RF and AP/RT were kept immerse in the same pressurizing fluid and in the dark, to mimic as much as possible the same environment of HS samples inside the HP vessel, except for pressure.

2.2 Protein profile analysis and digestibility

Protein profile was assessed based on the determination of total nitrogen through the Kjeldahl method, free amino acids (FAA) employing the EZ:Faast Amino Acid Analysis Kit available for GC-FID and also by in vitro protein digestibility. Micro-Kjeldahl procedure was performed with a Kjeltec system 1002 Distilling unit (Tecator, Sweden) and the crude protein content determined by multiplying the total nitrogen content by 6.38 (AOAC Official Method 2001.14, 2002). For FAA determination and quantification, cheese samples were homogenised in the same volume of 0.01 M HCl, centrifuged (17000 × g at 4 ºC for 5 min), and the supernatant was collected and centrifuged again. Then, 100 μL of the second supernatant was used for the analysis of free amino acids using the EZ:Faast Amino Acid Analysis Kit (GC-FID) (Badawy, Morgan, & Turner, 2008) being the results for individual FAA expressed in nmol per g of cheese. Digestibility was performed based on the method developed by Arte et al. (2015) with some modifications. Cheese samples were incubated with 1.5 mg of pepsin in 15 mL of 0.1 M HCl at 37 °C, at 150 rpm for 3 h, then neutralized with 2 M NaOH, 4 mg of pancreatin in 7.5 mL of phosphate buffer (pH 8.0) and 1 mL of toluene were added, followed by incubation for 24 h at 37 °C, at 150 rpm. The enzyme was inactivated with 10 mL of trichloroacetic acid (10%, wt/vol) and centrifuged (5000 × g at RT for 20 min) to separate undigested protein. Nitrogen in the supernatant was determined by micro-Kjeldahl method. Digestibility was performed in cow’s and goat’s FC stored under 100/RT for 60 days, and compared with the respective FC prior to storage, and expressed in % (Eq. 1).

2.3 Fatty Acids Profile

For the fatty acids profile determination, a similar method to the one described by Sobral, Casal, Faria, Cunha, and Ferreira (2020) was performed. Briefly, after cheeses fatty acids extraction and derivatization, determination was carried out by gas chromatography, as fatty acids methyl esters (FAMEs). FAMEs profile was analysed using a GC (Chrompack CP-9001 model, Netherlands) with flame ionization detection (FID). Fatty acids identification and FID calibration was accomplished with a certified reference standard mixture (TraceCert – Supelco 37 component FAME mix, USA) and the results were expressed in relative percentages of their FAMEs.

2.4 Secondary lipid oxidation by-products

Lipid oxidation state was assessed by malondialdehyde (MDA) quantification, using 2-thiobarbituric acid reactive substances (TBARS) method adapted from King (1962). Initially 1 g of fresh cheese was crumbled into smaller pieces and homogenised with 3 mL 7.5% trichloroacetic acid, followed by centrifugation at 4000 × g at 4 ºC for 20 min (Universal 320-R, Hettich Group, Tuttlingen, Germany). The resulting extract was filtered (Whatman nº1) and the same volume of 46 mM 2-thiobarbituric acid was added, vortexed and immersed in boiling water for 40 min, and then cooled down in cold water. Triplicates were measured using a micro-plate spectrophotometer (Multiskan GO Microplate Spectrophotometer, Thermo Scientific, Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 532 nm. Standard solutions of MDA in 7.5% trichloroacetic acid were prepared from 1,1,3,3-tetramethoxypropane and a calibration curve was prepared at a concentration ranging from 0.2 to 10 μg/L. TBARS results were expressed as μg of malondialdehyde per g of cheese.

2.5 Volatile organic compounds

Volatile organic compounds (VOC) profile determination was based on the method described by Yue et al. (2015), through headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS). 2 g of cheese and 50 µL of cyclohexanone (25 µg/mL, internal standard) were added into the vial, then immediately sealed with a polypropylene cap with silicon septum. Compounds were released at 50 ºC during 30 min, then the SPME fiber (DVB/CAR/PDMS, 50/30 µm, Supelco Inc.) was exposed for further 30 min the same temperature. After volatiles adsorption into the fiber, it was inserted in the injection port of the GC equipment, Agilent GC-7890 gas chromatographer equipped with a mass spectrometer Agilent 5977B, and a DB-5 MS Capillary GC column (30 m × 0.25 mm I.D. × 0.25 μm film thickness, Agilent, USA). Thermal desorption was achieved at 260 °C in splitless mode, with helium at a linear velocity 1 mL/min. The oven temperature was set at 35 ºC during 5 min, increasing to 100 ºC at a rate of 4 ºC/min, followed by an increase of 10 ºC/min until 225 ºC and held for 0.25 min (total of 33.5 min). The transfer line was maintained at 280 ºC and the ion source at 230 °C, with ionization energy of 70 eV. Mass spectra were scanned from 20 to 350 m/z in full scan mode. Identification of the volatile compounds was based on computer matching with the reference mass spectra library (NIST 11), retention times, retention index and with individual standards when available. Volatiles’ profile semi-quantitative determination was calculated using cyclohexanone as internal standard equivalents basis, and the results were expressed in μg per 100g of cheese.

2.6 Statistical analysis

All experiments were carried out in triplicate and all analyses were done in triplicate. The different storage conditions were compared using Analysis of Variance (ANOVA), followed by a multiple comparison post hoc test, Tukey’s HSD test, at a 5% level of significance. Additionally, principal component analysis (PCA) was performed in order to identify statistical patterns using the VOC data set obtained for both cheeses.

3.1 Protein profile

Cow’s FC presented a total protein concentration slightly lower compared to goat’s FC (Tables 1 and 2), 15.11 ± 0.49 g/100g and 16.99 ± 0.50 g/100g, respectively, similar to what is reported in the literature(Sant’Ana et al., 2013, Van Hekken, Tunick, Farkye, & Tomasula, 2013). Overall, both cow’s and goat’s FC presented a similar behaviour under the same storage conditions, with no significant changes(р>0.05)observed in all storage conditions, with storage at 75 and 100/RT maintaining the protein content constant throughout the storage(р>0.05), in the two kinds of FC even after 60 days, similarly to what was observed when raw milk was stored under the same HS conditions (75 and 100 MPa) at variable RT for 60 days (unpublished data).

Regarding FAA, cow’s FC was initially richer in glutamic acid, followed by aspartic acid, ornithine, leucine, and glycine with a total FAA of 1.1 ± 0.1 µmol/g (Table 1), while goat’s FC had initially a total FAA of 0.9 ± 0.1 µmol/g (Table 2), mainly constituted by glycine, followed by ornithine, glutamine, glutamic acid, valine, and aspartic acid. Similar compositions in initial FAA were also reported for cheeses made with cow’s and goat’s milk(Atanasova et al., 2021, Teter et al., 2020).

At the 3^rd day of storage, no significant variations were observed regarding individual FAA(р>0.05) of cow’s FC stored at AP/RF, comparatively to the initial ones, while cheeses at AP/RT exhibited(р< 0.05)a 12-fold increase in alanine and a 3-fold decrease in ornithine, while also several amino acids were now undetected such as glycine, isoleucine, threonine, proline and histidine that were initially present, which could have been used in microbial metabolism(Hoskisson, Sharples, & Hobbs, 2003). Despite these small variations in individual FAA, total FAA content remained similar(р>0.05)to the initial ones for both storage conditions.

As for storage under 50/RT, initially at day 3, changes were only detected in ornithine (decrease of 0.5-fold) without significant variations in all the other individual and total FAA(р>0.05). However, on the following storage periods a remarkable increase in the majority of FAA was detected(р< 0.05), with increments of 100-, 48-, 27- and 21-fold, for alanine, histidine, threonine, and valine, respectively, after 28 days of storage. At this sampling period, FAA were majorly composed of alanine, leucine, serine, glutamic acid, valine, and lysine (altogether representing 66% of total FAA), while being also characterized by the presence of serine, phenylalanine, cystine and threonine that were initially absent, resulting in an overall increase of 7-fold in total FAA. This might be due to residual activity of the enzymatic coagulant used for FC production, or plasmin residual activity, initially present in the pasteurized milk, that hydrolyse caseins into intermediate-sized peptides(Enright, Patricia Bland, Needs, & Kelly, 1999). Furthermore, these smaller peptides can be hydrolysed into amino acids by the microbial flora present in the FC, as high microbial loads were observed throughout the storage at 50/RT (around 6.6 and 6.4 log CFU, for total aerobic mesophiles (TAM) and lactic acid bacteria (LAB), respectively, unpublished data), or by extracellular proteinases and peptidases released from that microflora(Abellán et al., 2012). Nevertheless, this proteolytic effect was lower(р< 0.05)for FC stored at 75 and 100/RT, comparatively to storage at 50/RT, with an increase rate of FAA per day of, 93.66, 85.12 and 254.52 nmol/g, respectively (supplementary material Figure S1), resulting in increases in total FAA of 5.9 and 5.7-fold, under 75 and 100/RT respectively, at day 60 of storage. Interestingly the 100-fold increase in alanine observed after 28 days under 50/RT was much higher than the ones observed for storage under 75 and 100/RT after 60 days, of 13 and 14-fold, respectively, which was associated byEugster, Fuchsmann, Schlichtherle-Cerny, Bütikofer, and Irmler (2019)with the microbial activity of added starter cultures in cheese ripening. New FAA such as serine, phenylalanine, cystine and threonine were present in all three HS conditions, with samples stored under 75 and 100/RT showing a higher abundancy in leucine, glutamic acid, valine, and asparagine, reaching a similar total FAA after 60 days of storage of 6.3 ± 0.8 and 6.1 ± 0.5 µmol/g(р>0.05), respectively. Both storage conditions were able to gradually inactivate the microbial load present in FC samples, in a faster rate for 100/RT (with D_p-values for TAM of 17.8 and 13.4 days, for 75 and 100/RT, respectively, unpublished data), which could potentially explain partially at least the results of lower FAA increase.

Regarding goat’s FC, at AP/RF no significant(р>0.05)oscillations were observed in individual or total FAA at the 3^rd day of storage, while a high proteolytic activity (р< 0.05) occurredon cheeses stored at AP/RT, resulting in increments especially in valine, leucine, glutamic acid, proline and serine, responsible for an overall increase of 20-fold in total FAA (р< 0.05), comparatively to the initial cheese, despite the considerable reduction (р< 0.05)in glycine (similar to what was reported for cow’s FC under AP/RT).

Under HS conditions, generally goat’s FC presented signs of proteolysis throughout the storage, however at different rates. Storage at 50/RT resulted in an estimated raise of 641.63 nmol/g FAA per day (supplementary material Figure S2), with significant increases (р< 0.05)observed in almost all FAA, except for glycine, aspartic acid, ornithine, and glutamine that remained in similar concentrations(р>0.05)as the initial ones. A more prominent abundance (р< 0.05)in FAA was observed for alanine, leucine, valine, glutamic acid, and lysine, with leucine, histidine, methionine and valine showing a higher abundance after 28 days of storage, with increments of 380-, 208-, 65- and 61-fold, respectively. Goat’s FC had an initial high microbial load (around 6 log CFU, for LAB, unpublished data), that increased under 50/RT (reaching almost 8 log CFU/g after the 7^th day of storage), which can contribute to increased FAA as LAB are well known to promote proteolysis in cheeses(Abellán et al., 2012), resulting in an increase of 30-fold in total FAA after 28 days. Under 75 and 100/RT, this increase in total FAA was lower (16- and 8-fold increase, respectively), resulting in a proteolysis rate almost 2-fold slower, with increases of 151.57 and 71.73 nmol/g FAA per day (supplementary material Figure S2), respectively, reaching values of 9.5 ± 0.9 and 4.8 ± 0.4 µmol/g for total FAA after 60 days of storage, respectively. Interestingly, TAM and LAB counts were strongly inactivated under those conditions, however the inactivation rate was almost 3-fold faster under 100 MPa (D_p-values for TAM of 9.9 and 3.4 days, and for Lab of 6.3 and 1.9 days, under 75 and 100/RT, respectively, unpublished data), and thus, residual proteolytic activity from microbial proteases seem to be the main factor responsible for the proteolysis observed. Despite the almost half concentration in most FAA between cheeses stored under 75 and 100/RT, both presented a greater abundance(р>0.05)in leucine, valine, aspartic and glutamic acid. Also similarly with storage at 50/RT new amino acids were now present, such as isoleucine, phenylalanine, serine, and tryptophan. Increased proteolysis of FC in prolonged storage under RF is to be expected, as reported bySant’Ana et al. (2013), who observed an increased proteolysis in FC stored at AP/RF after 21 days, attributed mainly to the action of LAB, extracellular proteases, and to a smaller degree to plasmin.

Globally, for both FCs, storage under HS at 75 and 100/RT resulted over time in an increased concentration in FAA, although even after 60 days, values were significantly lower than the ones reported byAbellán et al. (2012)for goat cheese at day 1 of maturation. Still, the possible impact of these increases should be further investigated in the sensory properties of HS cheeses.

Regarding protein digestibility, cow’s (Table 1) and goat’s (Table 2) FC presented values prior to storage of 81.2 ± 2.1 and 75.8 ± 1.1%, respectively. Under 100/RT, after 60 days no significant variations were observed for cow’s FC (81.4 ± 2.7%), while an increase (р< 0.05)to 81.0 ± 1.8% was detected for goat’s FC. As mentioned previously, after 60 days of storage at 100/RT an increase in FAA of 5.7 and 8-fold was observed for cow’s and goat’s FC, respectively, indicating a higher proteolysis in goat’s FC, which could be responsible for the increased protein digestibility.

3.2 Fatty acids profile

Cow’s and goat’s FC fatty acid profile are presented in supplementary materialTable S1 and S2, respectively. Cow’s and goat’s FCs had an overall similar fatty acid content, with slight variations, both with a higher composition in saturated fatty acids (SFA), followed by monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA), overall similar to the composition described byVan Nieuwenhove, Oliszewski, and González (2009). Initially, cow’s FC had a total SFA, MUFA and PUFA of 63.98 ± 0.52%, 31.12 ± 0.38% and 4.42 ± 0.12%, respectively, while goat’s FC had initially total SFA, MUFA and PUFA content of 66.16 ± 0.93%, 27.94 ± 0.83% and 5.09 ± 0.18%, respectively. Regarding SFA, cow’s and goat’s FC were rich in palmitic acid (C16:0, 32.01 ± 0.11% and 27.00 ± 0.35%, respectively), myristic acid (C14:0, 11.49 ± 0.29%, 10.60 ± 0.19%, respectively), stearic acid (C18:0, 10.35 ± 0.19%, 9.37 ± 0.30%, respectively), with the major difference being related with a higher capric acid (C10:0) percentage observed for goat’s FC (9.28 ± 0.82%) comparatively with cow’s FC (2.83 ± 0.20%), similar to what is reported in the literature(Sant’Ana et al., 2013). As for MUFA cow’s and goat’s FC most abundant fatty acids were oleic acid (C18:1c, 22.80 ± 0.36% and 20.98 ± 0.71%, respectively) and elaidic acid (C18:1t, 2.72 ± 0.07% and 2.77 ± 0.12%, respectively), as for PUFA the most representative was linoleic acid (C18:2c, 2.44 ± 0.05% and 3.42 ± 0.13%, respectively).

Throughout the different storage conditions, cow’s FC fatty acid profile presented some variations when compared to the profile prior storage. In general, longer HS periods tended to present increased(р>0.05)values in SFA content, with storage at 75 and 100/RT reaching values of 65.46 ± 1.04% and 64.96 ± 0.65%, respectively. This tendency was more pronounced especially under 75/RT, presenting a tendency for higher amounts of palmitic acid (р< 0.05), stearic acid and myristic acid (р< 0.05). In accordance, for MUFA and PUFA, HS tended to present lower values, with the major differences (р< 0.05) being related to storage at 100/RT after 7 and 14 days, presenting values similar to the initial ones on the following storage periods(р>0.05). Despite the fluctuations (р< 0.05) detected regardingoleic,linoleic, and α-linolenic acids (C18:3c6,c9,c12), overall, the majority of MUFA and PUFA content was not affected during HS(р>0.05).

As for goat’s FC storage, despite some variability in few individual fatty acids during the different storage conditions, under HS no significant changes(р>0.05)were observed after 60 days, comparatively to the initial cheese, although the same tendency was observed similarly to cow’s FC storage, with cheeses at 75 and 100/RT presenting higher values(р>0.05)regarding SFA, accompanied by a decrease(р>0.05)for MUFA and PUFA content. Similar results were also found in HS of raw milk for 60 days, with a more pronounced increase SFA content (р< 0.05)being observed especially for storage at 75/RT, while MUFA and PUFA contents decreased throughout the storage (unpublished data).

Overall, storage under 75 and 100/RT was able to successfully keep a similar fatty acid profile of both cow’s and goat’s cheeses, throughout the duration of the study.

3.3 Secondary Lipid oxidation by-products

Regarding lipid oxidation throughout storage under the different storage conditions, it was clear an overall raise of TBARS values from 1.20 ± 0.11 µg MDA/g to a maximum of 2.78 ± 0.48 µg MDA/g and from 0.67 ± 0.07 to 2.13 ± 0.11 µg MDA/g, for cows’ and goats’ FC, respectively (supplementary materialTable S3). Lipid oxidation was more pronounced (р< 0.05)in cows’ FC stored under 50/RT after 14 days of storage, reaching 2.78 µg MDA/g. Under 75 and 100/RT, lipid oxidation increased slowly up to 2.21 ± 0.16 (1.8-fold) and 1.90 ± 0.11 µg MDA/g (1.6-fold) at the 60^th day of storage, respectively, but with values similar (р> 0.05)to the ones detected at the 3^rd day for each of these two storage conditions. As for goats’ FC, lipid oxidation was overall stable in most of the storage conditions, while at 75/RT a strong increase (р< 0.05)in TBARS values was observed mainly from the 42^nd day of storage, reaching 2.13 ± 0. 32 µg MDA/g (3.2-fold) at the 60^th day of storage. A significant slower lipid oxidation rate was achieved under 100/RT throughout storage, reaching 1.15 ± 0.05 µg MDA/g (1.7-fold) after 60 days of storage, which was comparable the one observed on the 7^th day of storage (0.86 ± 0.11 µg MDA/g). Lipid oxidation can be affected by several factors such as the presence of light, oxygen or enzymes, promoting the formation of several volatile compounds, giving rise to off-flavours, with increasing rate over the storage period(Van Hekken et al., 2013). In fact, increase in lipid oxidation by products in cows’ FC stored under AP/RF were reported byZamora, Juan, and Trujillo (2015), observing increases of 2.5-fold after 13 days, whileErcan, Soysal, and Bozkurt (2019)observed increases around 3.4-fold after 21 days, both higher than the ones reported in the present work for both cows’ and goats’ FC even after 60 days under 100/RT, of 1.6 and 1.7-fold increase, respectively. Significantly higher increases in TBARS values under HS/RT were reported for fish (29-fold) and meat products (4.5-fold), but when a lower temperature (10 ºC) was combined with HS a slower decreasing trend in TBARS evolution was achieved, to 6.6 and 3.9-fold, for fish and meat products, respectively(Fidalgo et al., 2019, Fidalgo, Lemos, Delgadillo, & Saraiva, 2018, Santos et al., 2020). However, results equivalent the ones observed for FCs were obtained in HS of raw milk (unpublished data), reporting a tendency to a more pronounce increase (р> 0.05)in TBARS values under 50-75/RT, while storage at 100/RT delayed lipid oxidation throughout the entire storage (р> 0.05).

3.4 Volatile organic compounds

Initially in cow’s FC a total of 18volatile organic compounds (VOC) were detected (Table 3) and consisted mainly of free fatty acids (FFA), esters, ketones, and aldehydes, without alcohol compounds, an overall similar composition to what is reported for this kind of dairy product(Tunick, Iandola, & Van Hekken, 2013). The composition in FFA consisted of butanoic, hexanoic, octanoic and decanoic acids, with sorbic acid ((2E,4E)-hexa-2,4-dienoic acid) also being detected, added in the form of potassium sorbate as a preservative by the producer, as stated in the product label. Ethyl butanoate and hexanoate were the main esters present, as for ketones, pentan-2-one and heptan-2-one were the most abundant compounds, and nonanal was the main aldehyde, which was only present in the cheese prior to storage.

After 3 days, cheeses under AP/RF presented a similar VOC profile(р>0.05), regarding to the cheese prior storage, with a slight increase(р>0.05)in most FFA, aldehydes, esters, and a decrease in ketones, with alcohol compounds such as pentan-2-ol, cyclohexanol and hexan-1-ol being now detected. Storage under AP/RT after 3 days, resulted in a clear distinguished VOC profile of cheeses, with increased concentrations (р< 0.05) ofFFA, aldehydes, esters, and alcohols. An increase up to 10-fold was observed in almost all FFA and their respective ethyl esters after 3 days, with acetic and nonanoic acid, ethyl octanoate and dodecanoate being now present. As for aldehydes and alcohols the main increases resulted from 2-methylbut-2-enal and hexan-1-ol, respectively, with no significant changes observed regarding ketones(р>0.05). High microbial or/and enzymatic activity can promote lipolysis and the release of FFA, as well as lactose and amino acids degradation, with ethyl esters formed by esterification of the FFA, and alcohols resulting possibly from reduction of aldehydes formed by amino acids degradation(Muñoz, Ortigosa, Torre, & Izco, 2003, Toso, Procida, & Stefanon, 2002).

After 7 days at 50/RT, cheese VOC profile presented overall an evolution similar to storage at AP/RF regarding esters, alcohols, and FFA, that continuously arose over storage (р< 0.05), while ketones and aldehydes decreased on the 28^th day. An overall increase in all FFA was observed especially in hexanoic and octanoic acids, with the now detected acetic and nonanoic acids, contributing to an estimated increase of 52.01 µg/100g of FFA per day (supplementary material, Figure S3). In parallel, esters increased around 21.49 µg/100g per day, mainly due to increases observed in ethyl decanoate, butanoate and hexanoate, and from ethyl octanoate and dodecanoate that were initially undetected. Ketones presented an estimated reduction over time of 0.32 µg/100g per day, possibly due to reduction to alcohols, which increased around 0.67 µg/100g per day (supplementary material Figure S3), mainly due through the formation of heptan-2-ol and butane-2,3-diol. Despite the initial increase in total aldehydes at the 7^th day of storage, since these are transitory oxidation compounds, quick conversion into acids or alcohols can occur(Bezerra et al., 2017), resulting in the significant content reduction after 28 days of storage (р< 0.05). Changes in the VOC profile of cheeses stored at 50/RT can be attributed to the high microbial load under this condition (above 6 and 5 log units for TAM and LAB, respectively, unpublished data), resulting in an overall quality loss of cheeses. Interestingly, storage under 75-100 MPa maintained total aldehydes, esters, ketones and alcohols at constants levels(р>0.05)throughout the storage, with exception for FFA at 100/RT that presented an estimated increase of 6.54 µg/100g per day (supplementary material Figure S3), which was more pronounced on the 42^nd day of storage on forward. And thus, these storage conditions resulted generally in a cheese VOC profile resembling more the initial one prior to storage.

The conducted PCA presented in Figure 1, resulted from multivariate statistical analyses of the VOC detected throughout the storage of cow’s FC. Figure 1 shows the score plots of the different variables, with PC 1 and PC 2 accounting for 52.13% and 22.57% of total variability, respectively. As it can be seen, cheeses from storage at AP/RF, 75 and 100/RT at all storage periods are closer to the cheese prior storage (on the positive PC 1), while cheeses stored under 50/RT are more far apart as the storage period increased, with cheeses under AP/RT being more distant (negative PC 1) from the cheese prior to storage. In the loadings of the two principal components (supplementary materialTable S4), compounds more associated with cow’s FC prior to storage, mainly ketones and aldehydes like pentan-2-one, heptan-2-one, hexanal and nonanal are scored on the positive loadings on PC 1, while the negative PC 1 is related to compounds associated with cheese spoilage, especially higher concentrations of FFA, esters and some alcohols.

In goat’s FC initially a total of 24 compounds were detected (Table 4), most of the VOC belonged to FFA (n=6), followed by esters (n=5), alcohols (n=5), ketones (n=4), and aldehydes (n=1), resembling the ones reported byQuintanilla, Hettinga, Beltrán, Escriche, and Molina (2020).

Storage at AP/RT resulted in a higher VOC content in most major classes, with the exception for ketones and aldehydes that can be easily converted into acids or alcohols. This raise (р< 0.05)was almost up to 10-fold in alcohols, FFA and ethyl esters, resulting in a considerable increase in 3-methylbutan-1-ol, butane-2,3-diol, acetic, butanoic and octanoic acids, and in ethyl butanoate and hexanoate. Under AP/RF this evolution in cheese VOC profile was not so pronounced, despite the significant increases (р< 0.05)observed in acetic and nonanoic acids, ethyl esters remained within the values initially reported(р>0.05), however with a higher alcohol abundance, mainly from 3-methylbutan-1-ol (р< 0.05), while ketones (р< 0.05)and aldehydes concentration were reduced after 3 days.

Under HS conditions, 50/RT promoted significant changes in cheese VOC profile, with an accentuated formation(р< 0.05)of FFA, ethyl esters and alcohols, while ketones and aldehydes were undetected just after 7 days of storage. Prolonged storage at 50/RT resulted in a rise of all FFA, esters and alcohols, contributing to a distinguished VOC profile comparatively to cheeses prior to storage (р< 0.05). Contrarily, storage under 75 and 100/RT promoted a more stable VOC profile over storage, with a reduction in ketones content slower under these storage conditions, while aldehydes increased slightly only after 60 days under 100/RT (р< 0.05). A greater alcohol formation was observed in the first 14^th days (р< 0.05), reaching values similar to the initial ones on the following storage periods, whereas FFA remained constant from the 7^th day on forward, without considerable changes(р>0.05) beingdetected for esters over storage.

Changes in the VOC profile under the different storage conditions allowed the elaboration of a PCA considering the individual VOC, which could explain 71.02% of total variance (Figure 2), with 55.42% and 15.60% corresponding from PC 1 and PC 2, respectively, with ketone and aldehyde compounds scoring on the positive PC 1 (supplementary material Table S5) associated with unspoiled goat’s FC like 3-methylbutanal, butane-2,3-dione, 3-hydroxybutan-2-one, heptan-2-one and nonan-2-one, while FFA, alcohols and esters compounds were more present in spoiled samples, with negative loadings on PC 1, such as heptan-2-ol, octanoic acid and ethyl butanoate (supplementary materialTable S5).

For both cheeses, storage under HS at 75-100/RT, allowed a more stable VOC profile throughout the storage, and resembling more the VOC profile of cheeses prior to storage, even after 60 days at RT, with a better maintenance of FFA, esters and alcohols over storage, when compared to the other storage conditions. It is worthy to note, that even only after 3 days at low temperature (AP/RF), cheeses stored under HS (75-100/RT) after 60 days presented overall a more resembling VOC profile comparatively to cheeses prior to storage.

Overall, HS under 75 and 100 MPa at RT allowed a much better preservation of both cow’s and goat’s FC, during a considerably longer storage period, when compared with RF. The results at these two pressures are very interesting, as throughout storage especially under 100/RT, a slower lipid oxidation rate and a more stable fatty acid profile and total protein was achieved after 60 days. Additionally, it was verified also an increased FAA abundance in HS cheeses, mainly for the longest storage periods, which should be further assessed to fully understand the real impact in the sensorial properties and consumers acceptability of these cheeses. These two storage conditions retained cheeses volatile organic profile similar to cheeses prior to storage, without noticeable formation of undesirable compounds associated with cheese spoilage, even after 60 days at uncontrolled variable RT, which was corroborated by the PCAs conducted for both cheeses.

In conclusion, a much longer shelf-life can be achieved under HS/RT (up to at least 60 days, the maximum period studied in this work), opening new business opportunities, as FC has only few weeks of shelf-life under RF and with additionally enhanced microbial safety due to considerable microbial inactivation (unpublished data). Although further technological development and research regarding HS implementation in the food sector is needed, HS could additionally allow significant energy savings throughout storage, being quasi energetically costless, due to needless constant energy supply to maintain temperature, thus being environmentally friendlier and more sustainable and suitable for example for longer transportation.

Acknowledgements

Thanks are due to the University of Aveiro and FCT/MCT for the financial support for Laboratório Associado LAQV research Units (UIDB/50006/2020) through national founds and, where applicable, co-financed by the FEDER, within the PT2020 Partnership Agreement. We would also like to thank the CBQF scientific collaboration under the FCT project UIDB/50016/2020. Ricardo V. Duarte would like to thank also FCT/MCT for the PhD grant SFRH/BD/121727/2016.

Funding: This work was financially supported by University of Aveiro and FCT/MCT, Laboratório Associado LAQV research Units (UIDB/50006/2020), and through CBQF, under the FCT project UIDB/50016/2020. Additionally, Ricardo V. Duarte was also financially supported by FCT/MCT in a PhD grant (SFRH/BD/121727/2016).

Authors’ Contributions: Ricardo V. Duarte: conceptualization, investigation and writing- original draft preparation. Susana Casal: methodology and resources. Ana M. Gomes: supervision and resources. Ivonne Delgadillo: supervision and resources. Jorge A. Saraiva: supervision, resources, writing-reviewing and Editing.

Data Availability: The data presented in this study are available by request from the corresponding author.

Conflict of Interest: The authors have no conflict of interest to declare.

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Table 1 - Total protein (g/100g), protein digestibility (%), free amino acids (nmol/g) of cow’s fresh cheese prior storage (Initial) and stored under the different conditions (AP/RT, AP/RF and 50, 75 and 100/RT). Different letters (a–g) indicate significant differences (p < 0.05) between the different storage conditions for each parameter.

Condition

Initial

AP/RT

AP/RF

50MPa/RT

75MPa/RT

100MPa/RT

Days

0

3

14

28

3

14

28

60

3

14

28

60

Total Protein (g/100g)

15.11a

13.09a

1.46a

14.08a

14.47a

12.92a

14.57a

13.49a

15.39a

13.78a

15.32a

14.53a

14.85a

14.74a

Digestibility (%)

81.2a

NP

81.4a

FAA (nmol/g)

Alanine

16.7a

207.6bc

24.1a

19.9a

789.7d

1770.5e

24.7a

49.0ab

164.3abc

218.1c

34.0ab

98.2abc

256.3c

227.1c

Glycine

69.6a

ND

83.0a

116.9abc

154.4bcd

247.9e

106.7abc

156.9bcd

188.1de

172.1cd

89.0ab

162.8cd

203.0de

158.5cd

Valine*

23.3a

43.0a

11.1a

35.2a

176.5b

486.6e

25.1a

111.6ab

310.5cd

784.0g

26.5a

219.1bc

382.3de

668.7f

3-Aminoisobutyric acid

18.8

ab

ND

12.6

ab

39.0

abc

69.4

abcde

15.0

ab

56.4

abcd

116.6

def

142.7

f

12.0

a

141.1

ef

88.8

bcde

108.0

de

Leucine*

85.6ab

303.8b

50.6a

66.4a

431.7bc

1333.1e

86.1ab

378.7abc

969.4d

1511.1e

145.8ab

650.7cd

1538.2e

1525.0e

Isoleucine*

4.8a

ND

33.7ab

96.2c

ND

31.7ab

104.0c

278.9e

ND

67.7bc

165.7d

297.2e

Threonine

5.0a

ND

30.2ab

136.6c

ND

46.0abc

63.3abc

ND

nd

121.3bc

105.4abc

Serine

ND

20.2

a

ND

166.4

bcd

605.9

e

ND

39.1

ab

137.9

abc

182.1

bcd

ND

66.5

ab

288.3

d

255.0

cd

Proline

19.8a

69.7abc

ND

41.8abc

76.5abc

ND

104.2bcde

166.3de

185.9e

ND

36.5ab

117.1cde

87.9abcd

Asparagine

13.7ab

36.4abcd

11.7ab

21.6abc

26.4abcd

29.7abcd

10.2a

24.9abcd

48.1cd

57.3d

15.1ab

98.1e

41.5bcd

32.1abcd

Aspartic acid

181.8

abc

56.8

ab

37.4

a

94.6

ab

350.9

abcde

415.7

cde

128.0

ab

363.0

bcde

568.0

e

471.9

cde

113.7

ab

182.8

bcd

473.7

de

488.6

de

Methionine*

10.9a

38.0a

14.1a

ND

42.7a

108.1b

19.6a

43.1a

72.7ab

51.1ab

19.9a

111.6b

114.7b

62.6ab

Hydroxyproline

12.3ab

24.1ab

9.0a

9.6ab

30.3abc

41.3abcd

11.2ab

80.4e

65.8cde

74.1de

14.6ab

39.9abcd

42.5bcd

35.2abcd

Glutamic acid

292.3bcd

224.4ab

209.8abc

58.4a

173.6ab

496.6de

167.5ab

207.3ab

439.8cd

502.4de

255.8abc

481.9de

899.5f

653.8e

Phenylalanine*

ND

19.7a

ND

34.8a

495.7e

ND

60.0ab

219.8bc

269.9cd

ND

92.4ab

269.9cd

390.9de

Glutamine

174.6

abcd

69.4

ab

58.3

a

74.5

ab

294.8

bde

286.6

bcde

117.7

abc

320.0

de

607.2

f

392.2

ef

101.6

ab

175.8

abcd

406.2

e

413.5

ef

Ornithine

167.5b

54.3a

201.2b

84.9a

40.5a

75.6a

54.5a

53.2a

42.8a

72.8a

45.1a

49.5a

48.6a

Lysine*

14.1a

14.2a

15.1a

32.4ab

166.0bc

493.9e

30.7ab

155.8b

298.6cd

348.0de

33.6ab

121.1ab

164.0bc

94.4ab

Histidine*

5.7a

ND

94.8bc

272.2d

ND

57.1ab

144.7c

307.6d

10.1a

110.5bc

265.6d

300.7d

Tyrosine*

2.3a

2.0a

3.3a

4.4ab

6.6ab

4.7ab

2.9a

12.2ab

15.4ab

32.5cd

10.1ab

49.3d

21.3bc

11.7ab

Tryptophan*

ND

33.4ab

4.0a

ND

65.6ab

309.3e

ND

43.4ab

109.2bc

240.2de

ND

63.8ab

170.2cd

270.3e

Cystine

ND

39.7a

168.4c

ND

11.3a

101.1ab

73.3a

ND

48.6a

80.3a

Total FAA (µmol/g)

1.1a

1.2a

0.8a

0.6a

3.2b

7.8c

0.8a

2.3b

4.4c

6.3d

0.9a

2.9b

6.1d

ND and NP - stands for not detected and not performed, respectively. * - essential amino acids

Table 2 - Total protein (g/100g), protein digestibility (%), free amino acids (nmol/g) of goat’s fresh cheese prior storage (Initial) and stored under the different conditions (AP/RT, AP/RF and 50, 75 and 100/RT). Different letters (a–i) indicate significant differences (p < 0.05) between the different storage conditions for each parameter.

Condition

Initial

AP/RT

AP/RF

50MPa/RT

75MPa/RT

100MPa/RT

Days

0

3

14

28

3

14

28

60

3

14

28

60

Total Protein (g/100g)

16.99

abc

15.36

a

17.02

abc

18.84

c

18.33

bc

15.35

a

18.30

bc

16.43

ab

16.83

abc

15.96

a

18.19

bc

16.75

abc

17.13

abc

15.81

a

Digestibility (%)

75.8a

NP

81.0b

FAA (nmol/g)

Alanine

41.4

ab

486.1

g

12.1

a

183.4

cde

841.3

h

1011.1

i

25.1

ab

134.5

abcd

200.4

def

323.0

f

55.9

abc

107.8

abcd

151.0

bcde

265.5

ef

Glycine

202.6

bcde

8.6

a

133.0

b

136.7

bc

225.4

def

276.5

ef

189.2

bcd

285.4

ef

277.0

ef

289.0

f

217.2

cdef

241.2

def

203.8

bcde

215.6

cdef

Valine*

54.4a

1296.1d

17.1a

218.5a

2426.1e

3383.8f

70.0a

733.9bc

1282.0d

2469.5e

98.8a

325.7ab

719.9bc

1168.0cd

3-Aminoisobutyric acid

5.6a

89.2b

ND

16.8a

ND

9.3a

ND

82.0b

195.1c

21.4a

ND

Leucine*

12.6a

1572.3e

14.5a

332.1ab

3175.8f

4799.8g

50.6a

776.9bc

1308.7de

2839.9f

84.1a

455.4ab

1023.1cd

1610.6e

Isoleucine*

ND

149.4bc

ND

28.4a

263.7d

398.0f

ND

86.2abc

118.8bc

355.4ef

ND

58.7ab

137.9bc

276.7de

Threonine

ND

95.4b

ND

10.7a

ND

12.5a

5.6a

ND

5.5a

ND

Serine

ND

1644.7d

ND

566.9c

462.6bc

ND

31.4a

93.4a

219.7ab

ND

16.1a

27.8a

201.1ab

Proline

ND

1488.9c

ND

179.0a

547.9b

724.5b

ND

14.8a

29.7a

85.5a

ND

8.2a

ND

18.4a

Asparagine

13.0a

102.9e

12.6a

32.4abc

68.1d

51.7cd

16.1ab

54.6cd

76.7cd

173.7f

35.1abc

20.2abc

31.8abc

47.2bcd

Aspartic acid

34.6a

77.2a

42.2a

36.9a

51.5a

40.0a

38.9a

100.0a

193.0b

494.6c

93.9a

33.1a

35.0a

62.4a

Methionine*

6.0a

384.7c

ND

28.5ab

341.0c

389.6c

ND

35.6ab

52.0ab

84.5b

ND

24.1b

21.9ab

68.7ab

Glutamic acid

65.7a

1823.3b

ND

171.2a

2329.1bc

2889.7c

ND

86.2a

222.6a

489.7a

42.4a

109.5a

210.9a

456.1a

Phenylalanine*

ND

500.4d

ND

52.6a

831.4e

1108.0f

ND

60.5a

133.6ab

274.1c

ND

26.8a

75.0a

210.4bc

Glutamine

56.3a

176.1bc

50.7a

46.0a

104.8ab

81.4a

48.3a

112.0abc

178.2c

431.6d

85.2a

67.2a

63.1a

81.3a

Ornithine

83.6

bcd

178.3

e

101.3

cd

39.8

ab

58.5

abc

110.0

d

34.9

a

43.3

ab

36.7

ab

72.1

bcd

41.6

ab

50.5

ab

56.0

abc

68.9

bcd

Lysine*

23.1a

575.2c

24.1a

35.1a

999.6d

1208.3d

27.8a

235.3ab

303.6b

250.5ab

48.6a

103.0ab

106.3ab

164.0ab

Histidine*

2.6a

292.7b

ND

8.9a

576.9c

529.8c

ND

75.8a

57.3a

93.1a

ND

89.5a

132.7ab

282.0b

Tyrosine*

1.3a

324.4c

ND

11.0a

157.9b

71.3a

ND

22.9a

4.0a

3.0a

ND

5.3a

2.4a

2.3a

Tryptophan*

ND

367.0cd

ND

81.4ab

467.2d

340.9cd

ND

124.2ab

112.5ab

454.8d

ND

55.8a

77.2a

240.9bc

Cystine

ND

127.3b

ND

27.5a

32.8a

73.4ab

ND

56.2a

Total FAA (µmol/g)

0.6a

11.6de

0.4a

1.6ab

13.9e

17.4f

0.5a

3.1bc

4.8c

9.5d

0.8ab

1.7ab

3.0bc

4.8c

ND and NP - stands for not detected and not performed, respectively. * - essential amino acids

Table 3 - Volatile organic compounds (µg internal standard equivalents /100g) of cow’s FC prior storage (Initial) and stored under the different conditions (AP/RT, AP/RF and 50, 75 and 100/RT). Different letters (a–e) indicate significant differences (p < 0.05) between the different conditions.

Condition

Initial

AP/RT

AP/RF

50MPa/RT

75MPa/RT

100MPa/RT

Days

0

3

7

28

7

14

28

60

7

14

28

60

Free fatty acids

182.10a

2076.29f

318.40ab

602.05d

1654.27e

177.03a

301.29ab

223.26ab

169.55a

252.42ab

247.83ab

402.03bc

572.93cd

acetic acid

nd

284.66d

4.69a

28.67b

137.33c

1.32a

0.69a

0.50a

1.19a

nd

butanoic acid

19.01a

229.34d

32.79a

78.00bc

275.23e

20.73a

43.02a

29.82a

19.37a

39.71a

49.74ab

56.54abc

89.14c

hexanoic acid

28.90a

370.52c

56.60a

153.49b

494.08d

31.04a

86.10ab

56.84a

52.48a

62.86a

92.15ab

105.66ab

169.61b

(2E,4E)-hexa-2,4-dienoic acid

69.19ab

773.42e

115.25bc

135.18c

277.05d

46.35a

46.42a

32.38a

47.12a

48.40a

43.00a

43.57a

59.53a

octanoic acid

47.88ab

315.17e

65.27ab

132.93cd

357.54e

49.36ab

86.07abc

59.90ab

26.93a

60.71ab

82.21abc

120.13bcd

168.53d

nonanoic acid

nd

13.64c

nd

6.56ab

9.57bc

2.70ab

4.11ab

4.87ab

3.17ab

4.14ab

2.64a

2.87ab

3.51ab

decanoic acid

20.53a

96.63d

39.49abc

65.95bcd

102.98d

26.84ab

35.06abc

40.42abc

19.61a

36.65abc

51.14abc

76.06cd

68.75bcd

dodecanoic acid

nd

6.54bcd

4.31ab

7.82cd

10.07d

3.33ab

4.71abc

3.56ab

2.40a

4.09ab

4.28ab

5.05abc

4.82ab

Esters

26.60a

339.46c

63.34a

137.31b

617.04d

23.85a

34.64a

32.07a

22.51a

34.84a

45.17a

39.80a

35.51a

ethyl acetate

4.84ab

58.49d

8.77b

21.45c

19.47c

5.78ab

3.91a

3.45a

3.78a

6.33ab

4.72ab

3.45a

4.63ab

ethyl butanoate

9.97a

121.69c

17.56ab

32.71b

138.47c

7.21a

6.61a

6.18a

3.20a

11.32a

9.20a

8.10a

8.94a

ethyl hexanoate

11.68a

70.85b

16.62a

21.55a

119.34c

8.16a

8.46a

10.95a

5.86a

10.61a

15.08a

12.84a

13.94a

ethyl octanoate

nd

51.88c

14.17ab

37.44ab

128.71d

nd

8.58a

8.45a

9.80a

nd

10.03a

11.18ab

6.64a

ethyl nonanoate

nd

3.13

nd

ethyl decanoate

2.58a

34.65c

7.93a

22.64bc

200.89d

2.70a

5.81a

3.05a

1.15a

6.10a

6.13a

4.41a

3.23a

ethyl dodecanoate

nd

1.90a

0.81a

1.52a

7.04b

nd

Alcohols

nd

12.84c

8.24b

8.95bc

20.05d

1.04a

ND

1.16a

2.30a

nd

0.50a

0.80a

pentan-2-ol

nd

2.63

nd

butane-2,3-diol

nd

5.02a

6.04a

nd

hexan-1-ol

nd

12.84d

1.42ab

3.93c

ND

1.04ab

ND

1.16ab

2.30b

ND

0.50a

0.80ab

cyclohexanol

nd

4.19

nd

heptan-2-ol

nd

14.00

nd

Aldehydes

14.57abc

127.40e

16.26bc

98.85d

19.10c

3.76ab

3.74ab

5.98abc

6.27abc

3.13a

3.39b

5.86abc

3-methylbutanal

nd

12.16b

2.42a

8.95b

nd

0.83a

nd

1.06a

nd

1.28a

2-methylbut-2-enal

nd

90.90c

6.90a

82.63c

19.10b

1.85a

1.70a

2.35a

2.27a

1.19a

1.44a

0.61a

1.13a

3-methylbut-2-enal

3.01a

10.14b

3.13a

7.28ab

nd

2.84a

nd

hexanal

2.07a

nd

2.05a

nd

1.91a

1.21a

1.39a

2.14a

2.24a

1.70a

2.14a

2.44a

heptanal

0.83a

14.20b

2.80a

nd

0.67a

nd

0.84a

1.01a

nonanal

8.97

nd

Ketones

15.51a

16.47ab

7.97a

12.42a

6.23a

10.02a

16.85a

16.04a

12.92a

19.05abc

30.60c

17.08ab

27.96bc

pentan-2-one

4.11a

nd

4.11a

6.45a

6.56a

nd

heptan-2-one

8.46a

9.51a

4.86a

7.07a

nd

7.46a

12.01ab

11.39ab

9.23a

11.57ab

18.40bc

8.00a

21.20c

nonan-2-one

2.94ab

6.96bc

3.11abc

5.35abc

6.23abc

2.56a

4.85abc

4.64abc

3.69abc

3.61abc

5.76abc

2.51ab

6.76c

Others

nd

heptane

25.24c

42.67d

2.43a

13.25b

nd

3.16ab

7.94ab

9.99ab

12.00b

10.31ab

7.34ab

5.61ab

9.34ab

toluene

3.88b

11.86c

2.60ab

2.75ab

nd

2.16ab

1.11a

1.84a

2.23ab

2.60ab

1.75a

1.77a

1.72a

nd – stands for not detected

Table 4 - Volatile organic compounds (µg internal standard equivalents /100g) of goat’s FC prior storage (Initial) and stored under the different conditions (AP/RT, AP/RF and 50, 75 and 100/RT). Different letters (a–f) indicate significant differences (p < 0.05) between the different conditions.

Condition

Initial

AP/RT

AP/RF

50MPa/RT

75MPa/RT

100MPa/RT

Days

0

3

7

28

7

14

28

60

7

14

28

60

Free fatty acids

166.38a

3170.90c

1017.31ab

3282.07c

4239.54c

1007.95ab

1264.32b

1121.78ab

1286.54b

799.95ab

1255.01b

700.41ab

1576.32b

acetic acid

21.64a

422.79d

179.00bc

173.23c

319.21d

74.99abc

71.31abc

46.02abc

48.22ab

56.39abc

56.84abc

40.51ab

37.81a

butanoic acid

14.34a

571.82e

107.32abc

344.72d

535.75e

94.99abc

124.43bc

96.47abc

181.96c

68.04ab

82.43abc

82.85abc

162.53bc

hexanoic acid

27.06a

1013.36c

223.33ab

1029.30c

1294.91c

254.32ab

380.46b

301.62ab

454.01b

193.04ab

267.10ab

260.93ab

470.72b

octanoic acid

61.41a

736.95cd

254.26ab

1062.65de

1198.72e

340.61ab

389.41abc

307.57ab

372.90abc

284.01ab

423.42abc

199.49ab

550.83bc

nonanoic acid

8.81ab

10.24abc

26.39d

21.55cd

17.44bcd

8.39ab

6.69a

8.71ab

5.25a

3.91a

7.88ab

8.62ab

13.65abc

decanoic acid

44.19a

396.63b

168.37ab

710.73c

690.81c

233.13ab

298.83ab

359.02b

122.28ab

164.69ab

336.46b

108.12ab

343.57ab

dodecanoic acid

2.71a

27.18c

7.07a

13.34ab

18.94bc

9.90ab

11.09ab

11.08ab

3.35a

7.77a

12.83ab

6.57a

10.86ab

Esters

55.03a

156.58bc

46.71a

225.24c

563.92d

29.50a

119.02abc

110.92abc

101.84ab

24.83a

107.46abc

64.91ab

117.70abc

ethyl acetate

10.16ab

26.43c

15.21b

9.56ab

10.00ab

8.91ab

7.83ab

5.00a

3.40a

10.14ab

10.96ab

4.88a

4.67a

ethyl butanoate

5.52a

52.20d

12.02ab

27.71bc

75.04e

11.45ab

18.60abc

20.04abc

28.80c

11.50ab

13.30ab

18.82abc

16.29abc

ethyl hexanoate

8.98a

30.22a

7.12a

40.56a

138.36b

8.82a

22.03a

17.46a

20.94a

7.02a

11.93a

12.90a

23.17a

ethyl octanoate

24.94a

41.05a

nd

40.56a

138.36b

nd

18.85a

13.83a

14.80a

nd

14.23a

10.98a

18.52a

ethyl decanoate

3.55a

10.02abc

8.43ab

95.92d

172.44e

nd

51.71c

54.58cd

33.89abc

nd

52.50cd

23.61abc

55.45cd

ethyl dodecanoate

nd

4.43

nd

Alcohols

93.97a

834.88f

364.48d

649.46e

768.53ef

350.06d

329.98cd

175.64ab

166.17a

343.01bcd

362.75d

160.06a

156.15ab

2-methylpropan-1-ol

11.05ab

39.41e

18.99bcd

29.16de

24.53cd

12.78ab

11.48ab

8.77a

8.95a

16.05abc

13.73ab

8.39a

8.63a

3-methylbutan-1-ol

44.09a

482.63c

197.42b

424.03c

555.42c

239.00b

245.46b

103.44ab

105.37ab

212.59b

244.21b

99.50ab

87.33ab

2-methylbutan-1-ol

5.76a

40.86de

14.80ab

30.89cd

54.08e

23.28bc

24.69bc

17.54abc

17.84abc

23.09bc

24.87bc

13.92ab

12.70ab

butane-2,3-diol

29.87a

253.64b

79.79a

81.56a

80.82a

75.00a

48.35a

45.89a

34.01a

104.07a

77.42a

38.26a

56.26a

cyclohexanol

1.73a

15.24b

14.01b

nd

18.69b

nd

heptan-2-ol

nd

4.66ab

3.66a

7.63ab

8.53b

nd

Ketones

98.39b

16.12a

27.54a

nd

37.49a

15.67a

11.77a

19.17a

28.80a

29.96a

9.83a

14.59a

butane-2,3-dione

42.55

nd

3-hydroxybutan-2-one

59.97c

16.12ab

23.71ab

nd

37.49bc

4.90a

3.92a

7.25ab

28.80ab

16.36ab

2.75a

nd

heptan-2-one

7.99a

nd

10.92ab

7.85a

11.20ab

nd

13.60b

7.86a

14.59b

nonan-2-one

5.00a

nd

8.25abc

12.05bc

4.45a

5.47a

4.38a

7.10ab

3.71a

12.96c

Aldehydes

3-methylbutanal

4.75a

nd

3.87a

5.28ab

4.05a

3.30a

7.72ab

10.52b

Others

nd

heptane

20.51abc

38.16bcd

24.55abc

51.21de

5.40a

44.97cde

4.63a

17.93abc

14.70abc

62.12e

10.01ab

6.30ab

42.90bcd

toluene

1.04a

5.85c

nd

5.20bc

5.26bc

2.83abc

1.91ab

3.82abc

1.42ab

3.05abc

4.85bc

3.61abc

2.66abc

nd – stands for not detected

No competing interests reported.

SupplementarymaterialRDetalHSFreshCheese.docx

Evaluation of cow’s and goat’s fresh cheese preservation under hyperbaric storage at room temperature up to 60 days versus refrigeration regarding nutritional and physicochemical parameters

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Fresh cheese samples preparation and storage

2.2 Protein profile analysis and digestibility

2.3 Fatty Acids Profile

2.4 Secondary lipid oxidation by-products

2.5 Volatile organic compounds

2.6 Statistical analysis

3. Results And Discussion

3.1 Protein profile

3.2 Fatty acids profile

3.3 Secondary Lipid oxidation by-products

3.4 Volatile organic compounds

4. Conclusions

Declarations

References

Tables

Competing Interests

Supplementary Files

Status:

Version 1