ORIGINAL RESEARCH published: 02 May 2022 doi: 10.3389/fnut.2022.861383 The Effect of Salt Reduction and Partial Substitution of NaCl by KCl on Physicochemical, Microbiological, and Sensorial Characteristics and Consumers’ Acceptability of Semi-Hard and Hard Lactose-Free Cow’s Milk Cheeses Bibiana Juan*, Antonio-José Trujillo and Victoria Ferragut Centre d’Innovació, Recerca i Transferència en Tecnologia dels Aliments (CIRTTA), XaRTA, TECNIO, MALTA-Consolider, Edited by: Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona (Cerdanyola del Maria Cristina Perotti, Vallès), Barcelona, Spain CONICET Instituto de Lactología Industrial (INLAIN), Argentina Increasing consumer demand for healthier foods prompts the development of cheeses Reviewed by: Flavio Tidona, reduced in salt. The aim of his study was to assess the effect of reducing the level of Council for Agricultural and sodium chloride (NaCl) and their partial substitution by potassium chloride (KCl) on the Economics Research (CREA), Italy Veronica Ortiz Alvarenga, biochemical, microbiological, and sensorial characteristics and consumer’s acceptability Federal University of Minas of semi-hard and hard lactose-free cow’s milk cheeses. To improve the possible lower Gerais, Brazil salty taste or the development of bitter taste, the addition to yeast extract, as a flavor *Correspondence: enhancer, was also checked. Different brining times and brine conditions were tested to Bibiana Juan bibiana.juan@uab.cat obtain a reduction of >25% of salt with respect to conventional cheese. Reduced-salt cheeses were elaborated by reducing half the salting time used in conventional cheeses, Specialty section: and a ratio of 60 Na+ :40 K+ was used to reduce Na concentration in substituted This article was submitted to Food Chemistry, cheeses. The results obtained in this study show that the reduction of salt by both a section of the journal methods in semi-hard and hard lactose-free cheeses could be an alternative for the Frontiers in Nutrition production of healthier and sensorial acceptable cheeses, without significantly affecting Received: 24 January 2022 their physicochemical characteristics. The addition of yeast extract, as a flavor enhancer, Accepted: 25 March 2022 Published: 02 May 2022 increased the free amino acids (FAAs) levels but decreased the acceptability of cheeses. Citation: Keywords: sodium reduction, sodium substitution, potassium, yeast extract, biochemical composition, cheese Juan B, Trujillo A-J and Ferragut V microbiota, sensorial quality (2022) The Effect of Salt Reduction and Partial Substitution of NaCl by KCl on Physicochemical, Microbiological, and Sensorial Characteristics and INTRODUCTION Consumers’ Acceptability of Semi-Hard and Hard Lactose-Free Cheese is one of the most diverse varieties of dairy products and has become an important Cow’s Milk Cheeses. contributor to nutritional intake in public health interest because it supplies indispensable Front. Nutr. 9:861383. nutrients for human nutrition, especially proteins, bioactive peptides, lipids, vitamins, and doi: 10.3389/fnut.2022.861383 minerals, essentially calcium, magnesium, and phosphorus (1). However, the high sodium (Na) Frontiers in Nutrition | www.frontiersin.org 1 May 2022 | Volume 9 | Article 861383 Juan et al. Cheeses Reduced in Salt levels in cheese are of major concern for public health worldwide, (30) described a more mature flavor in reduced-fat Cheddar as it has been associated with a high risk of hypertension, cheeses with the addition of yeast extract at the salting step. cardiovascular disease, and other chronic non-communicable Silva et al. (28) revealed that the addition of yeast extract had a diseases (2). The World Health Organization (WHO) has positive influence on the flavor of Prato cheese, minimizing the recommended to food manufacturers to minimize the Na perception of bitterness resulting from the addition of KCl. salt content in their products, particularly in the main food In addition to instrumental results, when modifying food contributors to dietary salt intake, in which recommendation composition, it is essential to evaluate the acceptability of a daily intake is <2 g Na per day [5 g salt per day (3)]. In of consumers. general, cheese and dairy products are listed as the third-largest In Europe, to be able to name a cheese as low or very low contributor of salt intake and are reported to contribute around in salt, cheese may only contain no more than 0.12 g or 0.04 g 10% of the Na in most European countries (4). of Na, respectively, or the equivalent value for salt, per 100 g At present, consumers are more aware of the relationship of product. To be able to name a cheese reduced in salt, it between food and health and are demanding healthier foods. must have a minimum reduction of 25% of salt compared to its Therefore, the dairy industry is continuously looking for counterpart (31). alternative ways to reduce the salt content of cheese while In this study, apart from reducing salt content, cheeses maintaining consumer satisfaction. This task is not easy because were lactose-free, becoming an advantage for most of the adult salt plays an important role on cheese production, affecting population. Approximately 75% of adult humans have low lactase the technological and sensorial aspects of the final product. activity, so the lactose intake can cause gastrointestinal problems, In addition to providing flavor, salt controls microbial growth, such as stomach discomfort, excessive flatulence, and soft stool enzyme activity, and curd syneresis, which in turn affects or diarrhea (32). During cheese making, 90%−98% of lactose is cheese composition (5, 6). In addition, salt can affect proteolysis lost in whey, and the residual lactose is fermented to lactic acid behavior, which may be related to sensory defects, including the (33); therefore, cheeses with 60 days of ripening do not contain development of bitterness and reduction of firmness (7, 8). lactose. However, with shorter or medium ripening time, residual Several strategies for reducing the salt levels in cheeses have lactose could remain in cheeses. Therefore, to ensure lactose- been investigated (7–9). The simple reduction of sodium chloride free cheeses, lactose in cheese milk was previously hydrolyzed, (NaCl) is one of them and can be achieved by reducing brining obtaining lactose-hydrolyzed milk (LHM). At present, various time (10), using brine with different salt concentrations (11), or lactose-hydrolysis technologies are available to produce LHM adding less salt to cheese curd (9). Diverse studies have been (34), being the most used the hydrolysis with soluble enzymes carried out to investigate the lowest salt concentration that can that can be performed either before or after heating (34). In this be applied without affecting cheese quality. In general, salt-free study, β-galactosidase was used during the cool storage of milk cheeses demonstrate undesirable sensory characteristics when before cheese making to hydrolyze most of the lactose present compared to cheeses with intermediate and conventional levels in milk. of salt (7, 8, 12–14). The aim of this study was to determine the effect of Another common technique is the use of salt substitutes Na reduction and partial substitution with KCl on the to reduce the amount of Na. Different mineral salt replacers, physicochemical, microbiological, and sensorial characteristics such as potassium chloride (KCl), calcium chloride (CaCl2 ), and acceptability of cheese made from lactose-hydrolyzed cows’ and magnesium chloride (MgCl2 ), have been studied (15, 16). milk ripened for 30 and 60 days. The use of CaCl2 and MgCl2 resulted in flavor differences compared with the full-Na Cheddar cheese. Only a mixture MATERIALS AND METHODS of NaCl and KCl achieved acceptable results (15, 16). Several studies have shown that it is possible to substitute NaCl with Cheese Making KCl during processing, without affecting the sensory, rheological, Three independent batches of cheese were produced in the and stability of the Nabulsi (17), Minas (18), Halloumi (19, 20), Food Technology pilot plant of the Universitat Autònoma de Cheddar (21, 22), Coalho (23), Prato (24), and Sao Joao (25) Barcelona (Bellaterra, Spain) using 200 L of whole pasteurized cheeses, with a substitution of up to 50%. However, the higher milk (72◦ C, 15 s) from the farm Can Badó SAT (Santa Agnès levels of substitution can affect the flavor because of inherent de Malanyanes, Barcelona). The day before cheese making, 0.5% off-flavors, such as bitter and metallic, making the use of flavor of β-galactosidase (Larbus S.A., Madrid, Spain) was added to enhancers interesting (26). raw milk and held at 4◦ C for 18 h, in order to hydrolyze The effects of different flavor enhancers have been evaluated lactose. The additional level and time required for treating in Cheddar (21, 27) and Prato cheeses (28) produced with cheese milk with enzyme were chosen according to the results a replacement of NaCl by KCl. Results were dependent on of preliminary experiments. the flavor used, modifying the sensory attributes of cheeses Milk was heated at 32◦ C, and 2% (v/v) of a lactic acid starter in a positive or negative way. One flavor enhancer that is culture of Lactococcus lactis ssp. lactis plus L. lactis ssp. cremoris prominent in the international market is the yeast extract (6). (Choozit Superstart M30; Danisco, Barcelona, Spain) was The combination of peptides, amino acids, glutamic acids, and added. Subsequently, 0.01% (v/v) of CaCl2 (35%; Laboratorios nucleotides of the yeast extracts is potentially efficient and enables Arroyo, Santander, Spain) and 0.03% (v/v) of commercial calf labeling with natural additives (29). Shakeel-Ur-Rehman et al. rennet (520 mg active chymosin, strength 1/10,000; Laboratorios Frontiers in Nutrition | www.frontiersin.org 2 May 2022 | Volume 9 | Article 861383 Juan et al. Cheeses Reduced in Salt Arroyo) were added. After 45 min, the curd was manually cut into TABLE 1 | Denomination of cheeses. grains of about 1 cm and heated at 36◦ C for 10 min to facilitate Cheeses Flavor enhancer Brining conditions the separation of whey out of the curd. After heating, all the whey was drained off and curd was transferred to molds (1.2 kg) with Brine Time a cotton cloth. Curd was pressed at 1.2 kPa for 1 h, 1.8 kPa for CN - 20% (NaCl) 4.5 h 1 h, 2.45 kPa for 3 h, and 3.6 kPa for 2 h with a pneumatic press (Garvía, Barcelona, Spain). Cheeses were divided into eight lots CR - 20% (NaCl) 2.15 h for salting with the different procedures described below. After CR0.1 0.1 %a 20% (NaCl) 2.15 h salting, cheeses were ripened in a room at 14◦ C with an 85% CR0.2 0.2 %b 20% (NaCl) 2.15 h relative humidity for 30 and 60 days, in order to obtain semi-hard CS - 20% (60 NaCl:40 KCl) 4.5 h and hard cheeses, respectively. CS0.8 0.8 %2 20% (60 NaCl:40 KCl) 4.5 h CS1.3 1.3 %2 20% (60 NaCl:40 KCl) 4.5 h Salting Treatments and Elaboration of CN, control cheese; CR, cheeses made with reduced-sodium treatment; CS, cheeses Reduced-Salt Cheeses made with substituted-sodium treatment. a Carinarome, b Maxarome. According to Na levels, three different salting treatments of cheese were produced, namely, conventional salting for normal- Na treatment; reduced time of salting by using conventional brine ′ of Na; and salting with brine made with a mixture of potassium (5 -GMP) and inosine monophosphate (IMP) and, as in the (K) and Na. previous case, it was added at the concentrations indicated by the A preliminary study was necessary to determine salting manufacturer and literature review (21, 28). conditions (e.g., salting time and brining composition) to achieve a minimum of the 25% of salt reduction to comply with the denomination of reduced cheeses according to the Cheese Composition and Salt Content European regulations. Determination Conventional salting was performed by immersion in brine Triplicate samples were analyzed for dry matter by a gravimetric (20% w/w NaCl; Sal Costa, S.A., Barcelona, Spain) at 14◦ C for method, drying cheese samples at 102◦ C until constant weight 4.5 h in order to produce control cheeses (CN). To know the (36). pH was measured with a pH meter (Crison Basic 20pH; required time to achieve a minimum of the 25% of salt reduction Crison Instrument, Alella, Spain) on a ratio of cheese:distilled in reduced-Na cheeses (CR), four salting times were tested (3.50, water (1:1) slurry. Fat content was determined by the acid 2.50, 2, and 1.50 h). butyrometric method according to the study by Van Gulik (37). KCl (KaliSel R , Quimidroga S.A., Barcelona, Spain) was used Cheese nitrogen was fractionated according to the method of as a Na replacer to make substituted-Na cheeses (CS). Three Kuchroo and Fox (38) to obtain the water-soluble nitrogen at pH different ratios of NaCl:KCl were tested (i.e., 60:40, 50:50, and 4.6. Total free amino acids (FAA) were determined on the water- 75:25) at 14◦ C for 4.5 h. soluble extracts by the cadmium-ninhydrin method described by All cheeses were elaborated as described in the “Cheese Folkertsma and Fox (39) and were expressed as mg Leu for 100 g making” section. A group of cheeses were salted in brine (20% of cheese. The concentration of lactose was determined using NaCl) for 4.30 h, to be used as CN. RocheTM lactose/D-galactose enzymatic-spectrophotometric kit (R-Biopharm AG, Darmstadt, Germany). Reduced-Salt Cheeses by Reduced-Na Treatment Na and K contents were determined by inductively coupled Three kinds of reduced-salt cheeses (CR) were salted for 2.15 h, plasma optical emission spectrometry (ICP-EOS) method using a time chosen from previous results to obtain the necessary a Perkin-Elmer ICP-EOS equipment (model Optima 4300DV; Na reduction. To improve the possible low salty taste in these Norwalk, CT, USA). Samples were previously digested in a samples, some of them were added with 0.1 and 0.2% of microwave digester (Milestone Ultrawave model, Sorisole (BG), commercial flavor enhancers (CR0.1 and CR0.2, respectively) Italy) equipped with 12 Teflon vessels. A cheese sample was before salting (Table 1). The flavor enhancer was composed of added with 5.0 ml of 70% ultra-pure nitric acid and digestion commercial yeast extract (Carinsa, Barcelona, Spain), and the was carried out at 240◦ C for 20 min. The digested sample quantity added was chosen by the commercial specification and solution was filtered through a membrane filter (pore size literature review (21, 27, 35). 0.45 µm) and made up to 50 g with Milli-Q water. Calibration samples were prepared from single-element standard solutions Reduced-Salt Cheeses by Substituted-Na Treatment (Spectrascan-Teknolab A/S-Norway) that were diluted with 10% Three batches of cheeses were immersed in brine (60% NaCl:40% ultra-pure nitric acid at concentrations ranging between 2 and KCl), and conditions were obtained from previous results. To 120 ppm for Na, and between 1 and 60 ppm for K. The general avoid the possible bitter taste caused by KCl, two batches were experimental conditions used were as follows: injector: alumina previously added with 0.8% (CS0.8) and 1.3% (CS1.3) of flavor 2 mm ID; plasma gas flow: 15 L min−1 ; auxiliary gas flow: 0.2 L enhancer (Maxarome Select Powder AGGL, DSM, Holland) min−1 ; nebulizer gas flow: 0.75 L/min; RF power: 1,300 W; Na before salting (Table 1). This compound is also constituted wavelength: 589.592 nm, and K wavelength: 766.490 nm. Results ′ by yeast extract rich in natural guanosine-5 -monophosphate were expressed as milligram per gram of cheese. Frontiers in Nutrition | www.frontiersin.org 3 May 2022 | Volume 9 | Article 861383 Juan et al. Cheeses Reduced in Salt All analyses were performed in duplicate at 30 and 60 days or positive indicates lower or greater perception). Samples were of ripening. evaluated for color (+: white. –: yellowish), odor intensity, taste (e.g., bitter, salty, acid, and after taste), and texture (e.g., Microbiological Analysis hardness, springiness, adhesiveness, and friability) at 30 and Ten grams of cheese were homogenized in 90 ml of sterile 60 days of ripening. A preference test was also made on a 9- peptone water (Oxoid, Hampshire, UK) for 3 min in a Stomacher point scale from 9 (like extremely) to 1 (dislike extremely). Prior (Stomacher 400 Circulator; Seward Medical, London, UK), to an assessment, the outer rind of each cheese was removed, followed by 1 min in a homogenizer (Pulsifier II R , UK). and cheeses were cut into triangular samples. The order of Serial dilutions were made, and microorganisms were grown in presentation was randomized, and cracker biscuits and mineral different media (Oxoid Ltd., Hampshire, UK) and incubation water at room temperature were served to assessors for palate conditions were as follows: plate count agar (PCA) for aerobic cleansing between samples. mesophilic microorganisms incubated at 30◦ C for 48 h; M17 agar plus 10% lactose for lactococci incubated at 30◦ C for 72 h; Evaluation of the Consumers’ Acceptability Man, Rogosa, and Sharpe agar (MRSA) for lactobacilli incubated and Preference at 30◦ C for 72 h; and Violet Red Bile Glucose Agar (VRBGA) A total of 95 and 88 consumers (for cheeses with 30 and 60 for Enterobacteriaceae incubated at 37◦ C for 24 h. Results were days of ripening, respectively) ranging in age between 18 and 65 expressed as log colony-forming units per gram of cheese (log years participated in the study. Participants were of both genders, cfu g−1 ). namely, 36 men and 59 women for semi-hard cheeses (30 days of ripening), and 35 men and 53 women for hard cheeses (60 days Color Analysis of ripening). Color measurements were performed with a portable Only cheeses that obtained the best results in the sensory spectrocolorimeter Hunter Lab (MiniscanTM XE, Hunter evaluation were tested. Therefore, for the evaluation of Associates Laboratory, Reston, VA, USA), with a cool white acceptability for semi-hard cheeses, CR, CS, and CS0.8 were fluorescent (Fcw) illuminant and observation angle of 10◦ . tested. For hard cheeses, consumers tasted CR, CR0.1, and CS. Measurements were made directly upon the samples on four To know the level of acceptability of cheeses, participants different points of the surface (without rind) and the inner classified each cheese individually on a 9-point hedonic scale section of the cheeses to obtain the color space parameters, (from 1 = dislike very much to 9 = like very much). To know the namely, L∗ (lightness that ranges between 0 and 100), a∗ value level of preference, CC was also introduced and tasters classified measures from the redness to greenness (positive to negative the cheeses based on the same 9-point hedonic scale. values, respectively), and b∗ value measures from the yellowness to blueness (positive to negative values, respectively), according Statistical Analysis to the CIELAB scale. Measurements were made at days 30 and One-way analysis of variance (ANOVA) of three independent 60 of ripening. batches was performed using SPSS Statistic version 22.0 (IBM SPSS, Chicago, IL) to test differences among cheeses on each Texture Analysis day of ripening and differences with time, with a significant Texture profile analysis (TPA) was used to evaluate the textural level of P < 0.05. Mean comparisons were carried out using the properties of cheese, using a TA-TX2 Texture Analyzer (Stable Student–Newman–Keuls test. Micro System Ltd., Surrey, UK) with a 245N load cell using a 35-mm diameter cylinder probe. Cube-shaped samples (1.5 RESULTS AND DISCUSSION cm3 ) from the internal and medium area of each cheese at each stage of ripening were cut with a device consisting of 1.5 cm Determination of Salting Conditions parallel blades. Samples were equilibrated at room temperature According to the European Commission regulation, to be labeled (20◦ C) for 2 h before measurements. TPA was based on two as reduced in Na, cheese should have a reduction of the regular cyclical compressions at room temperature with a test speed of Na content of at least 25% (31). As can be seen in Table 2, 5.0 mm/s, a compression ratio of 50%, and time of 5 s between to obtain reduced-salt cheeses with a reduction of Na, it was compressions. Cheeses were characterized in terms of hardness, necessary for a brining time between 2 and 2.50 h. To elaborate friability, adhesiveness, springiness, cohesiveness, gumminess, the reduced-salt cheese with partial substitution of Na by K, and chewiness. Six replicates for each product were performed. the three tested conditions produced the intended reduction of Na > 25%. However, to avoid undesirable sensory modification Sensory Evaluation described by several authors with a replacement of ≥ 50%, the Sensory evaluation was performed by ten panelists of the brine with 60% NaCl:40% KCl ratio was selected. Universitat Autònoma de Barcelona who were experienced in the sensory assessment of cheeses. Each kind of cheese was Composition of Cheeses compared with the CN, using a 7-point negative to positive All cheeses could be described as reduced in salt, showing >25% scale (0: no differences with CN; ± 1: minimal differences with of Na reduction compared to CC (Tables 3, 4). respect to the CN; ± 2: moderate differences with respect to the A slight pH reduction was observed with ripening time, due to CN; ± 3: big differences with respect to the CN, i.e., negative the fermentation process. The reduction of salt by the reduction Frontiers in Nutrition | www.frontiersin.org 4 May 2022 | Volume 9 | Article 861383 Juan et al. Cheeses Reduced in Salt TABLE 2 | Sodium and potassium concentration of cheese. Cheese Brining conditions Brine Time Na+ (mg g−1 cheese) K+ (mg g−1 cheese) % Na+ reduction CN 100% NaCl 4.5 h 6.7 CR 100% NaCl 3.5 h 6.5 3 CR 100% NaCl 2.5 h 5.2 22.5 CR 100% NaCl 2h 4.4 33.9 CR 100% NaCl 1.5 h 4.05 39.6 CS 60%NaCl:40%KCl 4.5 h 3.3 4.2 50.7 CS 50%NaCl:50%KCl 4.5 h 3.1 5.3 53.7 CS 25%NaCl:75%KCl 4.5 h 1.7 6.7 74.6 CN, control cheese; CR, cheeses made with reduced-sodium treatment; CS, cheeses made with substituted-sodium treatment. TABLE 3 | Composition of reduced-sodium cheeses (CR) at 30 and 60 days of ripening. Cheeses* Days of ripening pH Dry matter (%) Na+ (mg g−1 cheese) % Na reduction FAA (mg Leu 100 g−1 cheese) CN 30 4.87 ± 0.04Ab 53.84 ± 0.67Aa 7.21 ± 0.02A 2.06 ± 1.00Aa CR 30 4.81 ± 0.02Ab 53.85 ± 0.67Aa 5.01 ± 0.03B 30.5 2.27 ± 1.60Aa CR0.1 30 4.81 ± 0.03Ab 54.33 ± 1.01Aa 5.09 ± 0.04B 29.4 2.60 ± 1.56Aa CR0.2 30 4.84 ± 0.01Ab 53.65 ± 0.25Aa 5.07 ± 0.04B 29,6 2.65 ± 1.20Aa CN 60 4.75 ± 0.03Aa 58.27 ± 0.83Ab 8.53 ± 0.02A 3.46 ± 0.21Ab CR 60 4.74 ± 0.04Aa 58.91 ± 0.28ABb 5.97 ± 0.02B 30 3.41 ± 0.46Ab CR0.1 60 4.77 ± 0.02Aa 59.48 ± 0.52BCb 5.76 ± 0.05B 32.5 4.20 ± 0.34Bb CR0.2 60 4.73 ± 0.03Aa 60.05 ± 0.85Cb 5.71 ± 0.04B 33 4.19 ± 0.28Bb AB Means for the same parameter and day of ripening with different superscripts differ (P ≤ 0.05). ab Means for the same cheese and different day of ripening with different superscripts differ (P ≤ 0.05). * CN, Control cheeses; CR, reduced-sodium cheeses by reduced-sodium treatment; CR0.1, reduced-sodium cheeses by reduced-sodium treatment with 0.1% of flavor enhancer; CR0.2, reduced-sodium cheeses by reduced-sodium treatment with 0.2% of flavor enhancer. of brining time treatment did not affect the pH of cheeses days of ripening, a slight increase in dry matter was observed for (Table 3). These results are in agreement with other authors who reduced-NaCl cheeses, only significant in CR cheeses containing did not find a significant effect of salting conditions on the pH of flavor enhancer. Possibly, the addition of enhancer as powder Cheddar (9) or Mozzarella cheeses (9, 40) with reductions of up could produce the increase of dry matter in cheeses. to 60% of salt. Proteolysis is the most important biochemical event that The partial replacement of NaCl by KCl produced a slight occurs during the ripening of cheeses (46), and it plays a increase in the pH levels in cheeses at 30 days of ripening direct role on cheese flavor and texture development in most (Table 4). Similar results were described in Halloumi (19, 20, cheese varieties (47). Salt, along with other parameters, influences 41, 42) and Mozzarella cheeses (43), and it was attributed to the rate of proteolysis, so it is important to study it in salt- the higher pH of KCl-NaCl brines. Fitzgerald and Buckley (15) reduced cheeses. According to the literature, it has been generally also described higher pH in Cheddar cheeses salted by KCl informed that an increase in proteolysis in cheeses with the compared to those salted by NaCl, CaCl2 or MgCl2 . At 60 reduction of salt concentration (48) attributed to the increase days of ripening, no differences in pH values were observed, in water activity and the reduction in NaCl bacteriostatic effect. neither with the ripening time, showing that at 30 days of In CR, the levels of FAA, an indicator of secondary proteolysis, ripening, the transformation of sugars into lactic acid was were higher in cheeses containing flavor enhancer, although practically complete. differences were only statistically significant in cheeses at 60 Dry matter content was not affected by the reduction of salt days of ripening (Table 3). However, no significant differences or by the partial substitution of NaCl by KCl in cheeses with 30 were found between CR and CN. Baptista et al. (44) also days of ripening (Tables 3, 4). Baptista et al. (44) also found no found no differences in the development of proteolysis due to differences in dry matter content in Prato cheese with a reduction a reduction of salt in Prato cheese. Contrarily, Dugat-Bony et of 25% and 50% of salt at 15 days of ripening. Neither differences al. (49) described an increased secondary proteolysis in semi- were observed in Halloumi (41) or Mozzarella cheese (45) with hard cheeses reduced in salt, and Murtaza et al. (13) also found partial substitution of NaCl for KCl. Ripening time produced an an increase in the FAA in buffalo milk Cheddar cheese with increase in dry matter content due to water evaporation. At 60 the reduction of salt content. However, Møller et al. (50) and Frontiers in Nutrition | www.frontiersin.org 5 May 2022 | Volume 9 | Article 861383 Juan et al. Cheeses Reduced in Salt TABLE 4 | Composition of substituted-sodium cheeses (CS) at 30 and 60 of ripening. Cheeses Days of ripening pH Dry matter (%) Na+ (mg g−1 cheese) % Na reduction FAA (mg Leu 100 g−1 cheese) CN 30 4.83 ± 0.03Aa 53.09 ± 2.03Aa 7.33 ± 0.03A 4.10 ± 0.10Aa CS 30 4.90 ± 0.06Ba 52.32 ± 0.94Aa 5.53 ± 0.04B 24.6 4.60± 0.03Ba CS0.8 30 4.86 ± 0.01ABa 53.02 ± 1.63Aa 5.4 ± 0.04B 26.3 4.83 ± 0.06Ca CS1.3 30 4.86 ± 0.02ABa 52.43 ± 0.26Aa 5.28 ± 0.04B 28 4.87 ± 0.27Ca Aa Ab A CN 60 4.82 ± 0.01 57.05 ± 0.67 8.66 ± 0.02 5.15 ± 1.16Ab Aa Ab B CS 60 4.85 ± 0.03 57.19 ± 0.72 5.37 ± 0.04 38 5.74 ± 0.80ABb Aa Ab B CS0.8 60 4.87 ± 0.06 57.22 ± 0.72 5.97 ± 0.05 31.1 6.52 ± 1.17BCbc Aa Ab B CS1.3 60 4.88 ± 0.09 57.79 ± 1.05 5.69 ± 0.06 31.7 7.18 ± 0.48Cb AB Means for the same parameter and day of ripening with different superscripts differ (P ≤ 0.05). ab Means for the same cheese and different day of ripening with different superscripts differ (P ≤ 0.05). * CN, Control cheeses; CS, reduced-sodium cheeses by substituted-sodium treatment; CS0.8, reduced-sodium cheeses by substituted-sodium treatment with 0.8% of flavor enhancer; CS1.3, reduced-sodium cheeses by substituted-sodium treatment with 1.3% of flavor enhancer. TABLE 5 | Microbiological results (log cfu g−1 cheese) of reduced-sodium cheeses by reduced-sodium treatment (CR) at 30 and 60 days of ripening. Cheeses* Days of ripening Total counts Lactococci Lactobacilli CN 30 8.60 ± 0.11BCb 8.55 ± 0.03ABb 8.63 ± 0.23Bb Cb Bb CR 30 9.01 ± 0.51 9.04 ± 0.39 9.11 ± 0.37Cb ABb Ab CR0.1 30 8.26 ± 0.39 8.37 ± 0.47 8.16 ± 0.12Ab CR0.2 30 7.74 ± 0.06Ab 8.04 ± 0.06Ab 8.00 ± 0.00Ab CN 60 6.95 ± 0.07Bca 7.76 ± 0.59Ba 7.26 ± 0.21Ba CR 60 7.09 ± 0.12Ca 7.82 ± 0.01Ba 7.23 ± 0.07Ba CR0.1 60 6.81 ± 0.05Ba 6.99 ± 0.12ABa 7.07 ± 0.10Ba CR0.2 60 6.00 ± 0.00Aa 6.75 ± 0.21Aa 6.59 ± 0.16Aa AB Means for the same parameter and day of ripening with different superscripts differ (P ≤ 0.05). ab Means for the same cheese and different day of ripening with different superscripts differ (P ≤ 0.05). * CN, Control cheeses; CR, reduced-sodium cheeses by reduced-sodium treatment; CR0.1, reduced-sodium cheeses by reduced-sodium treatment with 0.1% of flavor enhancer; CR0.2, reduced-sodium cheeses by reduced-sodium treatment with 0.2% of flavor enhancer. McCarthy et al. (51) described more FAA in high-salt Cheddar enhancer presented the highest level of FFA, since a mixture of cheeses than in reduced cheeses. Gandhi and Shah (52) observed peptides and amino acids composes the yeast extract. an increase of amino acids during storage in Akawi cheeses with a reduction from 10 to 7.5% NaCl in brine solution; however, this did not happen with a higher reduction (up to 5% NaCl in brine Microbiology solution). These differences between cheese varieties could be Salt reduction slightly increased the lactic microbial counts in attributed to variances in cheese making, which affect differently cheeses (Table 5). An increase in the starter bacteria with the the growth of microorganisms, and several parameters influence reduction of salt was also found in Cheddar (14, 55) and Akawi the activity of their proteolytic enzymes, which are responsible (52) cheeses. Sheibani et al. (56) did not find differences in the for FAA release. growth of starter culture in hard-type cheeses reduced at 17% The partial substitution of NaCl by KCl increased the FAA and 35% of salt; however, a reduction at 50% of salt increased levels, being higher in cheeses containing flavor enhancer their growth. Similarly, the reduction of salt did not modify (Table 4). These results are in agreement with Silva et al. (35) the total bacteria counts in Sao Joao cheeses (25). The addition who found higher proteolysis values in reduced-Na Prato cheeses of flavor enhancer significantly decreased the microorganism (50% NaCl:50% KCl) than in conventional cheeses, showing the counts in CR, observing the lowest values in CR0.2 at 60 days of highest values of the reduced cheeses with flavor enhancers. ripening. These results are not consistent with those of Shakeel- In contrast, no significant differences in secondary proteolysis Ur-Rehmnan et al. (30) who observed an increase in non- have been reported with the partial substitution of NaCl by starter lactic bacteria (NSLAB) at the early stages of ripening in KCl in Halloumi (19), Prato (24), semi-hard (49), Feta (53), Cheddar cheese with the incorporation of yeast extract, nor by Cheddar (22), or Akawi cheeses (54). As expected, all cheeses the Silva et al. (35) who described significantly lower lactic acid increased the levels of FAA with ripening time because of bacteria counts in reduced-Na Prato cheese, with and without the activity of proteolytic enzymes. Cheeses containing flavor flavor enhancer. Frontiers in Nutrition | www.frontiersin.org 6 May 2022 | Volume 9 | Article 861383 Juan et al. Cheeses Reduced in Salt Microbial counts decreased with time from 30 to 60 days of Color and Texture ripening because of the appearance of unfavorable conditions Texture of cheese is one of the most important parameters for for the development of microorganisms throughout the ripening consumers, which could be altered by the reduction of Na or the time. No Enterobacteriaceae was observed in any cheese at 30 or replacement of Na by K, thus affecting its acceptability. 60 days of ripening. No significant differences were found in texture parameters The partial substitution of NaCl by KCl did not produce among cheeses reduced in salt at the same storage day. Hardness, significant differences in total and lactic microbial growth fracturability, gumminess, and chewiness increased from 30 to 60 (Table 6). These results are in agreement with those of Dugat- days of ripening, whereas cohesiveness decreased. No differences Bony et al. (49) in semi-hard cheese and Ayyash & Shah (41) were observed in adhesiveness or springiness over time (results and Kamleh et al. (20) in Halloumi cheese. The addition of flavor not shown). enhancer did not cause any effect on lactic microbial growth. Sheibani et al. (56) described that the reduction of salt Silva et al. (35) described a slight decrease of the starter bacteria decreases hardness and gumminess of hard-type cheeses. in Prato cheeses elaborated with a ratio of NaCl:KCl (1:1) at Murtaza et al. (13) and Ganesan et al. (9) also described a 30 and 60 days of ripening. However, the addition of yeast reduction of hardness and cohesiveness in Cheddar cheese extract did not produce any effect in salt-reduced Prato cheeses. reduced in salt. Microbial counts decreased from 30 to 60 days of ripening. An The reduction of Na did not affect the color of cheeses average of 2.65 log cfu g−1 of Enterobacteriaceae was found (Table 7). Only the higher percentage of flavor enhancer in cheeses with 30 days of ripening, due to the environmental produced an increase in lightness and a decrease in the b∗ contamination and the manipulation during cheese making. No parameter, giving less yellowish cheeses. The flavor enhancer significant differences were observed between cheeses. These used had a strong brown color that could have changed the numbers decreased until non-detectable levels at 60 days. color of cheeses. Nevertheless, the panelists did not detect these TABLE 6 | Microbiological results (log cfu g−1 cheese) of reduced-sodium cheeses by substituted-sodium treatment (CS) at 30 and 60 days of ripening. Cheeses* Days of ripening Total counts Lactococci Lactobacilli Enterobacteriaceae CN 30 8.53 ± 0.38Ab 8.65 ± 0.29Ab 8.60 ± 0.35Ab 2.75 ± 0.17 Ab Ab CS 30 8.40 ± 0.31 8.40 ± 0.37 8.50 ± 0.35Ab 2.68 ± 0.10 CS0.8 30 8.20 ± 0.37Ab 8.33 ± 0.72Ab 8.48 ± 0.49Ab 2.70 ± 0.33 CS1.3 30 8.18 ± 0.44Ab 8.23 ± 0.24Ab 8.48 ± 0.37Ab 2.48 ± 0.17 CN 60 7.05 ± 0.47Aa 7.13 ± 0.32Aa 7.23 ± 0.38Aa ND CS 60 7.30 ± 0.27Aa 7.08 ± 0.59Aa 7.13 ± 0.42Aa ND CS0.8 60 7.15 ± 0.21Aa 7.40 ± 0.24Aa 7.48 ± 0.29Aa ND Aa Aa CS1.3 60 7.53 ± 0.40 7.73 ± 0.84 7.60 ± 0.58Aa ND AB Means for the same parameter and day of ripening with different superscripts differ (P ≤ 0.05). ab Means for the same cheese and different day of ripening with different superscripts differ (P ≤ 0.05). * CN, Control cheeses; CS, reduced-sodium cheeses by substituted-sodium treatment; CS0.8, reduced-sodium cheeses by substituted-sodium treatment with 0.8% of flavor enhancer; CS1.3, reduced-sodium cheeses by substituted-sodium treatment with 1.3% of flavor enhancer. ND, non detected. TABLE 7 | Color results of reduced-sodium cheeses by reduced-sodium treatment (CR) at 30 and 60 days of ripening. Cheeses* Days of ripening L* a* b* CN 30 91.47 ± 0.62Ab −2.10 ± 0.28Aa 15.57 ± 0.44Aa CR 30 91.72 ± 0.58ABb −2.00 ± 0.10Aa 15.39 ± 0.41Aa CR0.1 30 91.20 ± 0.73Ab −2.16 ± 0.32Aa 15.68 ± 0.35Aa Bb Aa CR0.2 30 92.26 ± 0.35 −2.15 ± 0.02 14.36 ± 0.39Ba Aa Ab CN 60 89.85 ± 1.27 −1.74 ± 0.19 16.93 ± 0.41ABb ABa Ab CR 60 89.98 ± 0.31 −1.70 ± 0.14 17.12 ± 0.63Bb Aa Ab CR0.1 60 89.58 ± 1.06 −1.23 ± 1.06 16.50 ± 0.64ABb Ba Ab CR0.2 60 90.86 ± 0.43 −1.68 ± 0.34 16.27 ± 1.02Ab AB Means for the same parameter and day of ripening with different superscripts differ (P ≤ 0.05). ab Means for the same cheese and different day of ripening with different superscripts differ (P ≤ 0.05). * CN, Control cheeses, CR, reduced-sodium cheeses by reduced-sodium treatment, CR0.1, reduced-sodium cheeses by reduced-sodium treatment with 0.1% of flavor enhancer; CR0.2, reduced-sodium cheeses by reduced-sodium treatment with 0.2% of flavor enhancer. Frontiers in Nutrition | www.frontiersin.org 7 May 2022 | Volume 9 | Article 861383 Juan et al. Cheeses Reduced in Salt differences (Figure 1). The lightness decreased, and a∗ and b∗ not appreciate these instrumental color differences (Figure 2). parameters increased with time. Lightness and a∗ parameter decreased with time, meanwhile b∗ The partial substitution of NaCl by KCl did not affect parameter increased from 30 to 60 days of ripening, coinciding the texture profile of cheeses. Only hardness increased in all with the results of Duffose et al. (57), Pinho et al. (58), Juan cheeses with time, while the rest of the texture parameters et al. (59), Gheisari et al. (60), and Alvarez & Fresno (61) who remained unchanged from 30 to 60 days of ripening (results not described the effects of ripening time in the color parameters shown). These results are consistent with other studies with NaCl of cheeses. reduction using KCl in Kefalograviera (53), Nabulsi (17), Akawi (54), Halloumi (19), and Prato cheeses (24). Sensory Evaluation Substituted cheeses presented a slight increase of lightness Figure 1 shows the sensory results of CR at 30 and 60 days at 30 days of ripening, only statistically significant in cheeses of ripening. We must take into consideration that CR were with a higher amount of flavor enhancer (CP1.3). No differences compared to CCs, with minimal, moderate, and high differences were found for a∗ and b∗ parameters (Table 8). At 60 days of being valued in the scale of 1–3, respectively. The reduction of salt ripening, cheeses were similar to color parameters, and only had no significant difference in the color or texture perception cheeses containing flavor enhancer presented higher and lower of cheeses. The aroma was higher in cheese containing 0.2% a∗ and b∗ parameters, respectively, leading to redder and less of flavor enhancer, with minimal differences with respect to yellow cheeses. This trend could be explained by the strong CC. The reduction of salt caused less sensation of saltiness in brown color of the flavor enhancer. However, the panelists did cheeses, becoming CR the least valued, followed by C0.1 and FIGURE 1 | Results of sensorial analysis of reduced-sodium (Na) cheeses by reduced-Na treatment (CR) at 30 days (A) and 60 days (B) of ripening. Frontiers in Nutrition | www.frontiersin.org 8 May 2022 | Volume 9 | Article 861383 Juan et al. Cheeses Reduced in Salt TABLE 8 | Color results of reduced-sodium cheeses by substituted-sodium treatment (CS) at 30 and 60 days of ripening. Cheeses* Days of ripening L* a* b* CN 30 90.67 ± 1.14Aa 1.76 ± 0.18Ab 14.86 ± 0.72 Aa CS 30 90.87 ± 0.58ABb 1.75 ± 0.16Ab 15.01 ± 0.53 Aa CS0.8 30 91.19 ± 1.07ABb 1.70 ± 0.26Aa 14.38 ± 0.70 Aa Bb Aa CS1.3 30 91.54 ± 0.55 1.74 ± 0.25 14.73 ± 0.77 Aa Aa Aa CN 60 90.01 ± 0.81 1.42 ± 0.10 15.86 ± 0.54 Bb Aa Aa CS 60 89.77 ± 1.24 1.42 ± 0.06 15.50 ± 0.68 ABa Aa Ba CS0.8 60 90.38 ± 0.92 1.53 ± 0.13 15.13 ± 0.87 Ab Aa Ba CS1.3 60 90.04 ± 0.88 1.56 ± 0.15 15.18 ± 0.42 Aa AB Means for the same parameter and day of ripening with different superscripts differ (P ≤ 0.05). ab Means for the same cheese and different day of ripening with different superscripts differ (P ≤ 0.05). * CN, Control cheeses; CS, reduced-sodium cheeses by substituted-sodium treatment; CS0.8, reduced-sodium cheeses by substituted-sodium treatment with 0.8% of flavor enhancer; CS1.3, reduced-sodium cheeses by substituted-sodium treatment with 1.3% of flavor enhancer. FIGURE 2 | Results of sensorial analysis of reduced-Na cheeses by substituted-Na treatment (CS) at 30 days (A) and 60 days (B) of ripening. Frontiers in Nutrition | www.frontiersin.org 9 May 2022 | Volume 9 | Article 861383 Juan et al. Cheeses Reduced in Salt C0.2. However, at 60 days of ripening, cheeses containing 0.2% not find differences between CN and Halloumi cheeses with a of flavor enhancer were reported as the saltiest. These cheeses mixture of NaCl/KCl; however, bitterness was higher in the latest. were also valued as the most bitter with the greatest aftertaste. Differences, although statistically significant, were always <1, Consumers’ Acceptability being classified as minimal. To know the general acceptability of consumers, untrained At 30 and 60 days of ripening, CCs were preferred by the panelists tasted the cheeses with the best sensorial scores in panelists, showing scores of 5.73 and 6.75, respectively. At 30 the sensorial analysis. In this sense, for semi-hard cheeses, days of ripening, salt-reduced cheese without flavor enhancer consumers evaluated CR, CS, and CS0.8 cheeses. There were was preferred to cheeses containing this compound, with values no significant differences between cheeses reduced in salt by of 5.07, 4.67, and 4.87 in CR, CR0.1, and CR0.2, respectively. the reduction of NaCl or by KCl substitution, showing the However, at 60 days of ripening, C0.1 (6.06) was preferred to CR viability of both strategies to produce cheeses reduced in salt (5.75), showing that the addition of 0.1% of this flavor enhancer (Table 9). Consumers showed a similar preference for both could improve the sensorial characteristics of CR. CN and CR0.1 reduced cheeses and conventional cheeses. However, the addition were valued with a score of >6, described as a “slightly like them.” of flavor enhancer decreased the acceptability and preference In contrast, the addition of 0.2% of flavor enhancer reduced the of cheeses, concluding that it would not be appropriate to add preferences of cheeses (5.38), possibly due to the increase in salty, it under these conditions. Baptista et al. (44) did not observe bitter, and aftertaste. These results indicate that the addition of differences in the acceptability of Prato cheeses with a reduction 0.1% of flavor enhancer improves the sensorial perception of hard of 25% and 50% of salt with 30 days of ripening. Costa et al. CR. However, it would not be a good alternative to semi-hard CR. (24) did not find differences in the acceptability of Prato cheese The partial substitution of NaCl by KCl had no significant with a replacement of 40% of Na by KCl stored for 30 days. The difference in color, texture, or aroma of cheeses (Figure 2). The replacement of up to 50% salt substitution also did not alter the reduction of salt caused less sensation of saltiness in cheeses at 30 acceptability of Coalho cheeses stored for 30 days (23). days of ripening. However, at 60 days, cheeses containing flavor For cheeses with 60 days of ripening, consumers evaluated enhancer presented a higher salty taste than CN or CS cheeses. CR, CR0.1, and CS cheeses. The acceptability of CR was The addition of flavor enhancer also slightly increased the acid higher than CS (Table 9). Consumer comments highlighted the taste at 60 days of ripening. pleasant flavor of CR cheeses. Lindsay et al. (62) also described At 30 days of ripening, panelists preferred CCs, with a a lower preference score for reduced Cheddar cheeses made medium value of 6.54. The substitution of NaCl by KCl reduced with a ratio of NaCl:KCl (1:1) than those produced with a the preferences of cheeses, with values between 5 and 6, which reduction of NaCl. In our study, the preference of both was means that “neither like nor dislike”, having the higher value lower than CC. As in the previous evaluation, the addition of CS0.8 cheese (5.95) followed by CS1.3 (5.80) and CS (5.70). of flavor enhancer decreased the acceptability and preference The addition of 0.8% of flavor enhancer appears to slightly of cheeses. improve cheese’s preference. It seems that sensory differences due The addition of flavor enhancers in cheeses elaborated with to the substitution of NaCl by KCl decreased with ripening time, mixtures of 40% NaCl:60% KCl ratio was studied by Grummer et observing better scores in cheeses with 60 days. The mean of the al. (21), describing that some seem to affect positively, whereas preference values of CN (6.63), CS (6.31), CS0.8 (6.05), and CS1.3 others have a negative effect on consumers’ acceptability of (5.99) was not statistically different. No significant differences in Cheddar cheeses. Silva et al. (28) described that the addition of 1% the sensory attributes score between CN and cheeses made with a of yeast extract in substituted-Na Prato cheese increased the salty mixture of NaCl/KCl were found in Akawi (54), Kefalograviera taste, reduced the typical flavor, and minimized the perception of (53), and semi-hard cheeses (49). Kamleh et al. (20) also did unpleasant taste. TABLE 9 | Consumer acceptance and preference of cheeses with 30 and 60 days of ripening. Cheeses* Acceptance Preference 30 days 60 days 30 days 60 days CN 6.49 ± 1.66 a 7.22 ± 1.31 a CR 7.04 ± 1.07 a 7.19 ± 1.17 a 6.28 ± 1.57 a 6.61 ± 1.18 b CS 6.84 ± 1.28 a 6.46 ± 1.44 b 6.09 ± 1.73 ab 6.22 ± 1.31 b CR0.1 5.82 ± 1.36 c 5.60 ± 1.59 c b b CS0.8 5.90 ± 1.62 5.56 ± 1.65 ab Means for the same day of ripening with different superscripts differ (P ≤ 0.05). Based on a 9-point hedonic scale (1, dislike extremely; 5, neither like nor dislike; 9, like extremely). CN, control cheeses; CR, reduced-sodium cheeses by reduced-sodium treatment; CS, reduced-sodium cheeses by substituted-sodium treatment; CR0.1, reduced-sodium cheeses by reduced-sodium treatment with 0.1% of flavor enhancer; CS0.8, reduced-sodium cheeses by substituted-sodium treatment with 0.8% of flavor enhancer. Frontiers in Nutrition | www.frontiersin.org 10 May 2022 | Volume 9 | Article 861383 Juan et al. Cheeses Reduced in Salt A possible hypothesis to explain the lower acceptability found flavor enhancer could improve the sensorial and preferential in our cheeses added with yeast extract could be associated results obtained. with the development of flavor compounds from the amino acids generated by the NSLAB. Cheeses with flavor enhancer DATA AVAILABILITY STATEMENT had higher FAA levels than the rest of the cheeses (Tables 3, 4); however, further studies are needed to evaluate free fatty The original contributions presented in the study are included acids and FAA profiles and the volatile compounds of these in the article/supplementary material, further inquiries can be cheeses. Future research should be carried out by testing directed to the corresponding author. different percentages of yeast extract and/or with different flavor enhancers, in order to improve the obtained results. AUTHOR CONTRIBUTIONS BJ analyzed the data and wrote the manuscript. A-JT and VF CONCLUSION critically reviewed the article. All authors contribute to the study The reduction of NaCl concentration or partial substitution by concept and supervision of cheese production and analysis. All KCl (a ratio of 60 NaCl:40 KCl) could be a good strategy to authors gave final approval for all aspects of the work, agreed to produce hard and semi-hard lactose-free cow’s milk cheeses be fully accountable for ensuring the integrity and accuracy of the reduced in salt. Both methodologies allow the production of work, and read and approved the final manuscript. healthier and sensorially acceptable cheeses, without significant modification of pH, dry matter, proteolysis, microbial growth, ACKNOWLEDGMENTS texture, and color of cheeses. The addition of the yeast extract as a flavor enhancer in the tested conditions increased FAAs We would like to thank Diana Coello, Tina Mazaheri, Dayana levels and the salty, bitter, and aftertaste of cheeses, reducing Palacios, and Adrian Lezcano for the assistance in cheese their acceptability. A larger study with different percentages of production and laboratory analysis. REFERENCES 11. Madadlou A, Khosrowshahi asl A, Mousavi ME, Farmani J. 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Chemical and Publisher’s Note: All claims expressed in this article are solely those of the authors microbiological changes during ripening of Iranian salt-substituted probiotic and do not necessarily represent those of their affiliated organizations, or those of white cheese. Carpathian J Food Sci and Technol. (2014) 6:102–15. the publisher, the editors and the reviewers. Any product that may be evaluated in 61. Álvarez S, Fresno M. Effect of the ripening period and intravarietal this article, or claim that may be made by its manufacturer, is not guaranteed or comparison on chemical, textural and sensorial characteristics of endorsed by the publisher. palmero (PDO) goat cheese. Animals. (2021) 11:58. doi: 10.3390/ani110 10058 62. Lindsay RC, Hargett SM, Bush CS. Effect of sodium, potassium (1:1) chloride Copyright © 2022 Juan, Trujillo and Ferragut. This is an open-access article and low sodium chloride concentration on quality of Cheddar cheese. J Dairy distributed under the terms of the Creative Commons Attribution License (CC BY). Sci. (1982) 65:360–70. doi: 10.3168/jds.S0022-030(82)82200-5 The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original Conflict of Interest: The authors declare that the research was conducted in the publication in this journal is cited, in accordance with accepted academic practice. absence of any commercial or financial relationships that could be construed as a No use, distribution or reproduction is permitted which does not comply with these potential conflict of interest. terms. Frontiers in Nutrition | www.frontiersin.org 13 May 2022 | Volume 9 | Article 861383