MATERIALS AND METHODS

Study area: Two independent experimental trials were performed at separate times to explore the sensorial acceptability and the viability of E. coli with the total and partial substitution of NaCl by KCl salt in a different ratio in meat and dairy food models (beef burger and Tallaga cheese).

Meat model (beef burger): Ten kilograms of frozen beef and two kilograms of fresh fat were purchased from local butcher shops in Giza market, Egypt, they were transported in frozen condition to the laboratory and stored frozen -18°C. Frozen meat and fat were minced at a suitable temperature by 8 and 4 mm plate for meat and fat, respectively, in electrical mincer (Seydelmann NW 114 E, Stuttgart, Deutschland, Germany).

Beef burger manufacture was carried out following Egyptian standard requirements for the beef burger (EOS 1688/2005) as follows: Beef meat 70%, fat 15%, spice mix 0.35%, salt 1.5% and ice 10%. The beef burger ingredients were divided into five equal groups with NaCl salt replacement 0, 25, 50, 75 and 100% with KCl salt. Each group then was subdivided into two subgroups in which one group was prepared by addition of fresh onion and garlic 5% (control BT, BT1, BT2, BT3 and BT4), respectively and the second was left without (control B, B1, B2, B3 and B4). All ingredient of each group was thoroughly mixed by buddle type mixer for five minutes. The obtained pastes were manually formed into 50 g burger patties by using a patty former and packed in foam plates and stored in the freezer at -18°C for further investigation. The burger patties were grilled for 75°C core temperature then, it was cut into portions and directly subjected for examination. Flavour, taste, texture and overall acceptability of cooked beef burger samples were assessed on sensory hedonic with a 5-points scale¹⁷.

Dairy model: Whites soft cheese “Tallaga cheese’’ was prepared according to El-Kholy et al.¹⁸. The calculated amount of normal fresh cow’s milk was pasteurized at 72.3°C/15 sec. then cooled. The microbial rennet (Reniplus^® 2000 IMCU g^–1) was added at a rate of 0.4 mg/10 kg milk/65°C (manufacturer’s instruction), followed by CaCl₂ of 0.02%. Milk was divided into two main groups with salt concentrations of 3 and 5%. Each group was subdivided into five different subgroups with NaCl replacement of 0, 25, 50, 75 and 100% KCl salt. The prepared cheese groups with 3% salt content were symbolled by control C₃, C₃1, C₃2, C₃3 and C₃4, respectively. While the cheese samples with 5% salt were represented by control C₅, C₅1, C₅2, C₅3 and C₅4, respectively. Each panellist asked for the most frequently cited flavour (saltiness, sourness and bitterness), texture and overall acceptability that were scored on the hedonic scale¹⁹. Scale classifies the cheese quality from 1 to 5 into extremely like (5), good (4), fair (3) & poor (2) and (1) extremely dislike.

The sensory panellists’ team consists of 34 members (12 males, 22 females, age range 21-57 years old) who were administrative staff and students at Food Hygiene and Control Department, Faculty of Veterinary Medicine, Cairo University. Tape water and cups were available for mouth wash between samples evaluation. All samples were coded with three-digit numbers and presented at random for judging sensorial attributes.

Experimental E. coli contamination: A new production of experimental burger patties and cheese with different NaCl/KCl salts replacement were prepared separately for the viability study. Three based independent replicate experiments at separate times were done.

Strain preparation: Escherichia coli (ATCC 25922) strain was obtained from the Animal Health Research Institute, Department of Food Hygiene. The cultures were activated by passed twice at 9 mL of tryptic soy broth, (TSB, Oxoid). Then, incubated at 37°C/18 hrs. One milliliter of the incubated inoculum was added to 100 mL of TSB and incubated with shaking at 37°C/18-24 hrs to have a final concentration of approximately 10⁸ CFU mL^–1 (determined by plating serial dilutions on EMB agar, Oxoid).

Decontamination study: The prepared beef burgers groups with salt treatments (B1, B2, B3) and (BT1, BT2, BT3) as well as control groups (control B and control BT) were inoculated with the prepared E. coli strain by dipping the burger patties for 1 min, then were dried in laminar airflow for 20 min²⁰. Ten grams of the inoculated examined burgers were stomached with 90 mL of peptone water and serially diluted for determining the count of the inoculated E. coli on selective medium (EMB) in duplicate plates. Screening for E. coli count in the used minced meat in this experiment was also done to ensure that raw materials used are free from E. coli.

Regarding the cheese trail, the NaCl/KCl salt groups (C₃1, C₃2, C₃3), (C₅1, C₅2, C₅3) and control groups (control C₃ and control C₅) were prepared. The E. coli strain was inoculated before cheese curd formed as previously mentioned to have a final concentration of E. coli about 10⁸ CFU mL^–1. All the preparations were left undisturbed till the curd constituted and whey was drained off E. coli was counted as previously mentioned.

Statistical analysis: Statistical data analysis from three experimental replicates was carried out using SPSS statistics 19.0 for windows. The difference between treatments was determined using a one-way analysis of variance (ANOVA). Differences were considered significant at (p<0.05) and comparisons of means were done using post hoc (LSD) procedure to show differences among treatments.

RESULTS

Sensory evaluation results: Sensory perception is highly appreciated from consumers' and food industries' points of view to determine the food quality. The mean values of sensory scores (flavour, taste, texture and overall acceptability) of different NaCl/KCl salt substitutions and treatment groups of both food models (burger and cheese) are recorded in Table 1 and 2.

Substantially, the treated groups of beef burgers with onion and garlic received higher score values for the detected sensory parameters than untreated groups even in control groups (control B and control BT). Both flavour and taste results went in the same pattern as the mean score values were significantly low at p-value (<0.05) in untreated burger samples in groups of B2 (2.56, 2.50), B3 (2.30, 2.00) and B4 (1.00, 1.00) for flavour and taste, respectively, than control B (4.10, 4.50) and B1 groups (3.80, 4.30) for flavour and taste, respectively. On the other hand, the treated groups with onion and garlic showed significantly low results for flavour and taste in only 75 and 100% KCl groups in BT3 (2.90, 2.50) and BT4 (1.00, 1.00) for flavour and taste, respectively. While the groups with 25% and 50% KCl in BT1 (4.90, 5.00) and BT2 (4.60, 4.43) showed high results close to control BT (5.00, 5.00) for flavour and taste, respectively. Concerning the texture, the untreated burger patties revealed lower findings than those of treated groups but, the differences were not significant (p<0.05). Consequently, the highest values of overall acceptability were gone to groups with a low percentage of KCl (25%) (B1, BT1 groups) which were judged as like and extremely like as control groups (Control B and Control BT), respectively. The significantly low results (p<0.05) of overall acceptability of untreated groups were detected in B2, B3 and B4 which were judged as poor, poor and extremely dislike, respectively as well as BT3 and BT4 of treated groups which were judged as fair and extremely dislike, respectively in Table 1.

Regarding the cheese samples in Table 2, the significant low scores (p<0.05) for flavour and taste were recorded in C₃2 (3.14, 3.21), C₃3 (1.14, 2.14) and C₃4 (1.00, 1.00), as well as in C₅3 (2.00, 2.17) and C₅4 (1.07, 1.87) for flavour and taste, respectively. While higher findings were reported in groups C₃1 (4.85, 4.28), C₅1 (4.14, 4.57) and C₅2 (4.57, 4.21) which were close to the control groups control C₃ (4.92, 4.78) and control C₅ (4.35, 4.00) for flavour and taste, respectively. The texture of all groups was not significantly affected by substitution between NaCl and KCl salt in both salt concentrations (3 and 5%). However, by completely replacing NaCl salt with KCl, the groups C₃4 and C₅4 showed the lowest results for texture (3.78 and 3.17, respectively). Higher values for overall acceptability were observed in cheese groups with 3% salt concentration in control C₃ (4.66) and C₃1 (4.47) groups than cheese with 5% salt concentration within groups control C₅ (4.22) and C₅1 (4.33).

Fig. 1:

Optimum reduction in E. coli log count CFU g–1 with NaCl/KCl substitutions in burger

Fig. 2:

Optimum reduction in E. coli log count CFU g–1 with NaCl/KCl substitutions in Tallaga cheese samples

However, the groups with 3% salt concentration received significantly lower values (p<0.05) by replacing 50% of NaCl with KCl salt and up to 100% in groups C₃2, C₃3 and C₃4 with mean values 3.52, 2.52 and 1.92. While in groups with 5% salt concentration, significant low values (p<0.05) were observed in C₅3 and C₅4 (2.77 and 2.04, respectively). the completely rejected samples (extremely dislike) were recorded in groups C₃4 (100% KCl replacement).

Effect of NaCl substitution by KCl on E. coli viability: Depending upon sensorial scores, the results revealed that the total replacement of NaCl by KCl (100% KCl) in burger patties (treated/untreated) and cheese (3 and 5% salt) was unaccepted to the panellists consequently, the consumers. So, the groups B4, BT4, C₃4 and C₅4 (100% KCl) were excluded from the viability study. The viability of E. coli is determined in groups with partial substitution of NaCl/ KCl in untreated/treated beef burger samples and cheese (3 and 5% salt concentrations) was shown in Table 3 and 4.

The E. coli counts showed higher values in both treated and untreated groups of beef burger samples than the cheese groups (3 and 5% salt concentrations), but there were no significant differences in results between groups in both food models. However, the mean log values of burger groups exhibited lower values in B2, BT2 groups than the control groups (Control B and Control BT). It was observed that there was about one log reduction CFU g^–1 in group BT 2 than that occurred in B 1 in Fig. 1 compared to the control groups which decreased from 8.15 (control BT) to 7.30 (BT 2) of the treated beef burger and from 8.20 (control B) to 7.78 (B1) of the untreated beef burger groups in Table 3. The highest reduction level in E. coli counts was detected in burger groups with 50% NaCl replacement by 50% KCl.

The cheese groups (3% salt concentration) in Table 4 showed that the lowest mean values for E. coli counts were in C₃2 (6.04) than that occurred in control C₃ (6.62), C₃1 (6.88) and C₃3 (6.47), likewise cheese groups with 5% salt concentration recorded the lowest mean values for E. coli counts in C₅2 (6.00) than that were detected in control C₅ (6.85), C₅1 (6.30) and C₅3 (6.28). However, the optimum reduction level of E. coli counts that were detected in cheese groups with 50% NaCl replacement by 50% KCl as shown in Fig. 2 in both salt concentrations.

DISCUSSION

Beef burger formulation contains about 1.5-1.8% of NaCl salt which plays a significant role in improving burger patties binding through dissociation into ions and cause increasing of myofibrillar proteins solubilization²¹. The results of flavour, taste and overall acceptability in groups B1 and BT (25% KCl) were high and nearly close to the results of control groups (100% NaCl). On increasing KCl salt replacement up to 50% in untreated burger groups, a significant reduction in results were detected (p<0.05) of the sensory parameters than control groups as well as 25% KCl groups (B1 and BT1) due to a slightly unpleasant meaty flavour with an astringent bitter taste that could be detected in the burger patties. While using natural flavouring agents like onion and garlic verified a successful strategy in compensating flavour that lost due to the reduction in NaCl salt²², so the BT 2 group (50% KCl) of the treated burger group with onion and garlic was accepted and judged good. Although groups with 50% KCl were received lower score sensory results than control groups, the burger patties were still accepted. While the groups with 75 and 100% KCl of both untreated and treated groups recorded significantly reduced results in the sensory parameters (flavour, taste and overall acceptability) and were completely rejected by the panellists due to the detecting unpleasant meaty flavour and astringent bitterness off-flavour which is the main limiting factor in using potassium chloride salt in food. Those results were also confirmed by Carvalho et al.²³ on the addition of spices and herbs to beef burgers, the sensory characteristics were improved through masking the meaty off-flavour of fat detected in case of the substitution of NaCl by 25 and 50% of KCl salt. Nonetheless, the treatment with fresh onion and garlic could not overcome the adverse changes in the flavour and taste of beef burger patties by addition of high concentration of KCl salt level in groups BT3 (75% KCl) and BT4 (100% KCl) that revealed significantly lower scores (p<0.05) for flavour, taste and overall acceptability than the control groups.

Regarding the texture, the treated burger patties (with fresh onion and garlic) have high scores of texture results but are not significant at p<0.05. The changes that had been noticed in texture scores at the different substitution levels between NaCl and KCl salts (25, 50, 75 and 100%) could be attributed also to many other contributing factors which may affect the texture of beef burgers like fat content and beef grad used.

Tallaga cheese also known as "Gebnah Bayda". It is a soft white cheese that has a wide range of salt proportions depending on consumer requests. Tallaga cheese has broad acceptability among consumers owing to its pleasant creamy taste, which is ready to consume as a fresh cheese within one month in refrigerated storage¹⁸.

The highest score results of sensory parameters of cheese were detected in groups with 75% NaCl (C₃1 and C₅1) which were close to the control groups (100% NaCl) of both salt concentrations (3 and 5%). On replacing NaCl with 50% KCl salt, a trace of slight bitterness and low salty taste was detected in the cheese group with a salt concentration of 3% (C₃2). However, the group with a 5% salt concentration (C₅2) received higher sensory evaluations. Those results may be attributed to the high salt content in group C₅2 (5% salt content) than that present in the C₃2 group (3% salt) which enable the NaCl salt to mask the bitterness taste of KCl salt, based on the fact that mentioned before by Gurtovenko et al.²⁴, who found that when two salts like sodium and potassium chloride are mixed in food, an interaction occurs on mouth receptors and the salty taste of sodium salt could cover the bitter taste of potassium salt inside the mouth. In general, the bitterness flavour developed clearly in cheese by decreasing NaCl salt content, as the reduction in NaCl salt content is directly correlated with the degradation of β-casein protein which increases the available sites on the hydrophobic protein portions to be split by the chymosin enzyme¹¹.

The texture results were not significantly affected at p-value (<0.05) by the replacement of NaCl salt by KCl salt in most groups (75, 50 and 25% NaCl/KCl) as well as control groups (100% NaCl). Nevertheless, cheese samples in groups with 100% KCl (C₃4, C₅4) showed a slight difference in body texture. The body became slightly softer and grainy. The differences in cheese texture due to the complete absence of sodium chloride salt ensure the considerable role of sodium chloride salt in the protein solubility and curd synaereses during cheese manufacture²⁵. Similar findings were also, reported in other cheese types as São João cheese²⁶, Nabulsi cheese²⁷ and Turkish white soft cheese²⁸ due to the replacing of NaCl with KCl salt.

The main reason for choosing Escherichia coli in the current study is its ability to be an indicator of product safety. Although most E. coli strains are non-pathogenic, some strains are Shiga toxin-producer, which causes a severe food poisoning illness. Contaminated dairy and improperly cooked meat products are the most food products that are incriminated in E. coli outbreaks²⁹.

Substantially, the reduction of E. coli counts was not significant at p-value (<0.05) in all groups. However, the highest reduction level in E. coli counts was detected in groups with 50% NaCl replacement by 50% KCl (B2, BT2, C₃2 & C₅2) in both food models (Fig. 1 and 2). Those findings could be confirmed by that found by Gandhi et al.³⁰, who detected that the increasing of KCl salt concentration led to a reduction in E. coli counts and the optimum reduction level in E. coli counts occurred by replacing 25 and 50% between NaCl/KCl salt, however, the reduction was less than one log reduction in E. coli counts compared to the control groups.

The reduction observed in group BT 2 of the treated burger than that occurred in B1 of the untreated burger (Fig. 1) could indicate the beneficial antimicrobial effect of using onion and garlic in the processing of the beef burger. The results of Yadav et al.³¹ confirmed that Escherichia coli is highly sensitive to fresh garlic. It has maximum lethal concentration (MLC) levels that range from 4-32% v/v³². While the fresh onion extract has a lesser antimicrobial effect against E. coli than garlic³⁰.

It is worth mentioning that the counts of E. coli reduced in the cheese groups with both salt concentrations (3 and 5%) than in beef burger patties (treated and untreated groups) owing to the higher salt content that used in cheese processing compared to the burger (1.5% wt/wt) which is below the effective lethal reduction level of the E. coli organism⁷. Those results were emphasized by Gandhi et al.³⁰, who found that the viability of E. coli reduced significantly with increasing the sodium chloride level from 2-2.5%. While, on continuously increasing NaCl salt up to 3%, the reduction was not significant as in 2.5% NaCl salt concentration.

The Na⁺ and K⁺ cations are monovalent cations that have an equivalent antimicrobial effect on bacterial cells by the destruction of their plasma membrane. However, sodium is more potent than potassium. Several other investigations in different meat and dairy products clarified that there are no additional static or lethal effects by Na⁺/K⁺ replacement^23,26,32 so, the product safety remains unaffected by replacing sodium salt with potassium salt.

CONCLUSION

Sodium chloride salt could be reduced in the beef burger and Tallaga cheese by 25% without any adverse effect on their sensorial quality. However, the substitution of NaCl by 50% KCl salt recorded low sensory attributes scores in beef burgers due to bitter off-flavour taste that could be masked by the addition of 5% fresh onion and garlic to burger formula. While cheese samples with 50:50 NaCl/KCl in the high salt concentration (5%), saltiness could overcome the bitter taste detected in cheese samples with salt level (3%). In both food models, the replacement of NaCl by 75 and 100% KCl has adversely affected the overall acceptability with rejected bitter off-flavour taste. There was a minimal positive effect of substitution 50% NaCl by 50%KCl on controlling the E. coli growth in both food models (beef burger and Tallaga cheese) were detected. Further studies should be done in other food models to obtain a reduced-sodium salt diet with a maximum effect on the product safety and a minimum alteration in the quality.

SIGNIFICANCE STATEMENT

This study can be beneficial for food technology development regarding the determination of the critical limits of substitution of the NaCl by KCl salt in both beef burgers and Tallaga cheese. Also, the study will be helpful for the manufacturers to produce the two products without affecting the sensory and safety of the examined products.