Abstract
Immunoglobulin G (IgG) possesses numerous health benefits, it is a part of whey proteins which are mostly lost in whey and cheese curd virtually contains no IgG. In this study, bovine IgG was supplemented to the cheddar cheese curd at salting phase at four levels, i.e. 5, 10, 15, 20, and 0 mg/kg and labeled as T1, T2, T3, T4, and control, respectively. After vacuum packaging, all types of samples were stored at 5–6 ºC for three months for ripening. IgG supplementation at all four concentrations did not affect cheddar cheese composition. After the completion of ripening phase, total viable count of control, T1, T2, T3 and T4 were 7.91, 7.84, 7.75 and 6.18 log CFU/mL (p < 0.05), respectively. Three months old cheddar cheese samples showed that retention/ stability of IgG in all treatments. In T4, magnitudes of citric acid, lactic acid, acetic acid and propionic acid were 2577, 17,974, 192, and 288 ppm, respectively. Supplementation of cheddar cheese with IgG from 5–20 mg/kg did not change the fatty acid composition at the stages of cheddaring, pressing and ripening; however, ripening of cheddar cheese for three months considerably altered the fatty acid composition. After the completion of ripening phase, peroxide value of control, T1, T2, T3, and T4 was 0.29, 0.30, 0.28, 0.32, and 0.31 (meq O2/kg), respectively, with no variation in cholesterol contents of all variants of IgG supplemented cheese. Flavor scores of three months old control and all the treatments were more than 90% of the total scores (9).
Graphical Abstract
Keywords: Cheddar Cheese, Immunoglobulin G, Fatty Acid Profile, Organic Acids, Lipid Oxidation, Sensory Evaluation
Introduction
Dairy products are excellent carriers of added bioactive compounds to the body [46]. Immunoglobulins (Igs) are glycoproteins synthesized by plasma having150kDa molecular weight. They are comprised of two different polypeptides (90% glycoproteins and 10% carbohydrate), the first one being referred as heavy chain (50 kDa) and the second one having 25 kDa molecular weight is referred as light chain [15]. Igs are the main component of humoral immune response and contribute to 20% in total plasma proteins. Concentrations of IgG1, IgG2, IgM and IgA in bovine milk are 0.6, 0.12, 0.4 and 0.2 g/liter. Several components of bovine milk possess immunomodulatory and antimicrobial functions, especially IgG, which has been extensively studied since last fifty years for the potential role in immunity and infection. IgG can bind large number of bacteria, viruses and allergens [13]. IgG can help to maintain veracity of gastrointestinal tract by binding to toxins and detrimental bacteria in order to prevent them to cross the gut barrier and cause inflammation and decrease the permeability of intestine and improve symptoms in inflammatory bowel disease with a concentration of 200–500 μg/mL [40]. IgG has been found to lower cholesterol value, a major risk factor of cardiovascular ailments. In a placebo-controlled study, 5 g Ig/day was administered for 6 weeks, Ig supplementation reduced the cholesterol level from 6.33 mmol/L to 5.98 mmol/L [14]. To use IgG as a bioactive compound in foods, it must be stable during processing storage and gastrointestinal tract [22]. With the advancement of membrane technologies, whey proteins can be efficiently recovered. In an investigation, about90% IgG was recovered from cheese whey [47]. In another study, microfiltration and ultrafiltration were used to recover approximately 80% IgG from bovine, caprine and equine colostrum [38]. Impact of orthodox HTST pasteurization and UHT treatment of milk on the stability of IgG was studied, about 2% IgG was lost in pasteurization process while, entire IgG was denatured in UHT treatment (138 °C/4 s) [33]. The effect of freeze-drying, vacuum assisted microwave evaporation and spray drying on stability of IgG was evaluated. Results showed that vacuum assisted microwave evaporation, freeze-drying and spray drying caused 20%, 10% and 5% losses of IgG, respectively [29]. Yoghurt fermentation, blast freezing and subsequent chilling had no effect on IgG concentration [49]. Isolated IgG (> 50 tons) from bovine milk is extensively used in the formulation of baby foods and functional dairy products [41]. Discovery of new avenues for the fortification of IgG will uplift the production volume with sustainable methods. Nowadays, consumers have more knowledge of disease and nutrition and well familiar with the association of metabolic disorders with conventional foods. Therefore, a huge demand of functional foods has grown up in the world. Food industries are discovering bioactive compounds that can be used to raise therapeutic value of foods. Changes in life styles have led to a lower consumption of traditional dairy products for example, about 25–30% decrease in the consumption of milk and its products has been reported in USA. In cheese production, whey and curd are separated from each other, both phases have different composition in terms of macro and micronutrients. Most of the water-soluble fractions such whey proteins move towards the whey including IgG rendering cheese curd an extremely poor source of IgG [2]. Therefore, health advantages linked to the intake of IgG can be achieved by supplementing the cheese with IgG [18]. Novelty of this work is that the impact of traditional cheddar cheese production method and ripening phase on the stability of added IgG is not reported in literature. The current study was executed to find out the stability of added IgG in cheese matrix at different stages of cheese production and its impact on fatty acid profile, organic acids production, lipolysis development and sensory characteristics of cheddar cheese.
Materials and methods
Experimental plan
Holstein–Friesian cow milk was obtained from Dairy Animals Training and Research Center, University of Veterinary and Animal Sciences, Lahore, Punjab, Pakistan, pasteurized at (65 °C/30 min) and in the downstream side, cooled to 31 ºC. At this stage, DVS starter culture (DVS-RST-900) consisting of L. lactis spp. lactis and L. lactis spp. cremoris were used for the pre-acidification of cheese milk (45 min), each rennet and0.02% CaCl2, mixed (coagulation time, 30 min) and then cutting, cooking and cheddaring, till 5.2 pH, and milling was performed. IgG was blended with the curd at 5 mg/kg (T1), 10 mg/kg (T2), 15 mg/kg (T3) and 20 mg/kg (T4) w/w by NaCl (1.5%) using tumble mixer for uniform mixing followed by hydraulic pressing (3 bar/15 h), vacuum packaging, ripening for three months at 5–6 ºC. Samples were analyzed at preset intervals of 0, 45 and 90 days.
Composition analysis
Samples of cheese were analyzed for fat, protein, moisture contents following the methods 989.05; 981.10 & 950.46) and pH of 10% cheese solution in distilled water [3].
Total viable Count (TVC)
TV Count was tested at preset frequencies of 0, 45 and 90 days on TVC Agar using 104 dilutions and incubation 30 ºC/48 h [44].
Determination of IgG by HPLC
Samples (30 g) were dissolved in deionized H2O (300 mL), pH was set to 4.6 by 2 M HCl, kept for 20 min, followed by centrifugation at4000xg for 20 min at 4 ºC and precipitation of IgG (supernatant) with 40% NH2SO4 and re-centrifuged at 4000xg for 20 min at 4 ºC. The precipitate was dissolved in phosphate buffer (50 mM) and injected to HPLC (20μL) having model 500 absorbance detector and Hi-Trap protein column, keeping flow rate at 0.4 mL/min. For gradient elution, 0.05 M sodium phosphate (pH 6.5) and 0.05 M glycine–HCl buffer (pH 2.5) were used at 280 nm using six-point standard curve [24].
Fatty acid profile
Sample 10 g was mixed with 20 mL (CHCl3 and C2H5OH in 2:3 ratio) for 30 min, filtered and evaporated by rotary evaporator at 40 ºC till 2–3 mL residue in flask, 50 mg of the fat and 2 mL 15% HCl were taken in a test tube and reacted at 100ºC/60 min till no turbidity, test tubes were cooled to 25–30 ºC. Each n-hexane and deionized H2O were mixed by vortex (1500 rpm) for 120 s, upper layer was harvested and dehydrated over Na2SO4 and 1µL was inserted by ALS in GC–MS (7890-B) having a capillary column (L × I.D. 75 m × 0.18 mm, df 0.14 μm). Split ratio was kept at 1:100, temperatures of both injector and FID were set at 250 ºC, total run time for the method was 47 min usingFAME-37 standard [39].
Organic acids by HPLC
Organic acids were extracted from cheese samples by mixing sample and H2PO4 in the ratio of 1:4 (2000 rpm/2 min) followed by heating in a water bath at 63 ºC/10 min, centrifugation at 6000xg/10 min. Upper layer was then filtered and injected to HPLC Perkin Elmer, Germany (Series 200) by ion exchange organic column (8 mm × 300 mm internal diameter) using UV/Vis detector at 214 nm and four internal standard of citric acid, lactic acid, acetic acid and propionic acid abbreviated as CA, LA, AA and PA, respectively [23].
Determination of lipid oxidation
Peroxide value (POV), Free fatty acids (FFA) and cholesterol formation were determined at 0, 45 and 90 days of ripening in agreement with previous methodology [4].
Sensory evaluation
Color, flavor and texture of cheddar cheese samples were evaluated on a 9-point scale at 45 and 90 Days. Sensory evaluation was performed at 20 °C in separate booths by a trained panel of 10 evaluators (age24–45 years; 6 females and 4 males). Cheddar cheese samples (3 g) were cooled to 12 °C assigned with three-digit codes and offered in random manner in triplicate using FIZZ software for the statistical analysis of sensory data [28].
Statistical analysis
Two-way ANOVA technique was used to analyze the data collected in CRD. Data were represented in Mean ± SD of triplicate treatments and triplicate analysis of samples. Tukey’s HSD Test was used for determining significant difference among the means with SAS 9.1.
Results and discussion
Composition of IgG supplemented cheese
Supplementation of cheddar cheese with IgG from 5–20 mg/kg did not influence compositional characteristics (Table 1). Supplementation was dosed at 5, 10, 15 and 20 mg/kg of cheddar cheese curd, this dose was found too low to affect the cheese composition. Further, milk, additives, cheese making facilities, cheese maker and testing facilities were identical for the production and analysis of all treatments of cheese samples. This fact can also be connected to the non-variation of compositional features of control and IgG supplemented cheese samples. Effect of adding bioactive compounds on compositional features of cheddar cheese is documented in literature. Addition of Rutin from 60–240 ppm did not cause any variation in compositional characteristics of cheddar cheese [37]. Lactoferrin was used for the bio preservation of paneer at 20 mg/100 g level, fortification of lactoferrin did not cause any variation in paneer composition [42]. Effect of IgG supplementation on food composition has been studied in a limited way. For cheddar production, pH is an extremely important parameter, variations in pH can have a great effect on cheese texture, flavor and overall quality. In cheddaring, a definite delay in acid production was recorded in experimental and control cheese samples. To reach a 5.2 pH, IgG supplemented samples took more time in cheddaring than control due to antibacterial activity of IgG. Cheddaring times of control, T1, T2, T3 and T4were46 ± 0.87, 52.45 ± 1.26, 57 ± 0.62, 66.29 ± 0.53 and 72.40 ± 1.35 min, respectively. Testing of pH in 45 and 90-day old control and experimental samples showed no variation at both testing intervals (p > 0.05).In a relevant investigation, it was reported that addition of lactoferrin at a 20 mg/100 g concentration did not affect pH and composition of cheese in the ripening phase; however, a delay in cheddaring process was reported by Adnan et al. [2].To increase IgG in yoghurt, bovine colostrum was added to yoghurt milk at 5–10% levels, fermentation time of colostrum containing yoghurt was significantly higher than control to reach a 4.6-pH [30]. Fat and protein contents significantly declined in control, T1, T2, T3 and T4. The decline in fat and protein contents was not due to the supplementation of IgG but it was due to the lipolysis and proteolysis of cheese samples in the phase of ripening. During ripening of cheddar cheese, proteins are converted to peptides and free amino acids, leading to reduction in proteins while, nitrogen contents remained same determined by Kjeldahl method. Levels of fat and protein in 120 days old cheddar cheese were different from young cheese [34].
Table 1.
Composition of IgG supplemented cheddar cheese
| Treatments | Storage time (days) | Moisture (%) | Fat (%) | Protein (%) | pH |
|---|---|---|---|---|---|
| Control |
0 45 90 |
40.25 ± 0.37a 40.21 ± 0.28a 40.18 ± 0.23a |
30.53 ± 0.29a 30.42 ± 0.21a 28.73 ± 0.32b |
26.78 ± 0.12a 26.59 ± 0.17a 24.43 ± 0.28b |
5.29 ± 0.03a 5.22 ± 0.17a 5.11 ± 0.26b |
| T1 |
0 45 90 |
39.88 ± 0.18a 39.97 ± 0.44a 40.11 ± 0.67a |
30.64 ± 0.41a 60.59 ± 0.38a 28.64 ± 0.26b |
26.36 ± 0.23a 26.49 ± 0.05a 24.31 ± 0.07b |
5.28 ± 0.30a 5.24 ± 0.14a 5.10 ± 0.11b |
| T2 |
0 45 90 |
40.56 ± 0.37a 40.79 ± 0.29a 40.51 ± 0.48a |
30.38 ± 0.19a 30.32 ± 0.16a 28.15 ± 0.28b |
26.65 ± 0.14a 26.43 ± 0.26a 24.29 ± 0.31 |
5.33 ± 0.15a 5.28 ± 0.22a 5.09 ± 0.16b |
| T3 |
0 45 90 |
39.74 ± 0.27a 39.36 ± 0.19a 39.68 ± 0.16a |
30.84 ± 0.34a 30.65 ± 0.30a 28.37 ± 0.25b |
26.76 ± 0.24a 26.43 ± 0.15a 24.33 ± 0.10b |
5.27 ± 0.13a 5.25 ± 0.04a 5.13 ± 0.07b |
| T4 |
0 45 90 |
40.16 ± 0.35a 40.22 ± 0.34a 40.38 ± 0.18a |
30.52 ± 0.14a 30.51 ± 0.57a 28.23 ± 0.47b |
26.49 ± 0.12a 26.22 ± 0.06a 24.37 ± 0.07b |
5.30 ± 0.17a 5.25 ± 0.03a 5.12 ± 0.28b |
When means in every column bear a different letter, it gives significant variation (p < 0.05)
T1: 5 mg IgG/kg
T2: 10 mg IgG/kg
T3: 15 mg IgG/kg
T4: 20 mg IgG/kg
Impact of IgG on TVC of cheddar cheese
In cheese production, starter bacteria dominate the overall microflora; on production day, their count is normally 109/mL. In cheese ripening, pH, water activity and salt content changes that lead to about one percent reduction in population of starter bacteria takes place in first 30 days of cheese ripening and population of adventitious bacteria increases. A secondary microflora develops in cheddar cheese from environment, milk and manufacturing facility; even then, cheese is produced from pasteurized milk [17]. IgG belongs to whey proteins, most of these are lost in aqueous phase and do not migrate to the cheese curd. To achieve the health benefits of IgG, direct addition to curd at salting phase was carried out in present study. In ripened cheddar cheese TVC is mainly comprised of Lacticaseibacillus rhamnosus, Lactobacillus curvatus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Streptococcus thermophilus etc. [16]. Impact of IgG on TVC of cheddar cheese during processing and ripening is not previously reported. In present study, effect of IgG fortification on TVC was analyzed at the stages of cheddaring, pressing, ripening (after 45 and 90 days; Table 2). Fortification of curd with IgG at all four concentrations did not show significant inhibitory effect on TVC (p > 0.05). After cheddaring, total viable count of control and all the treatments ranged from 9.26–9.42 log CFU/mL. After pressing, non-significant variation in total viable count of control and all other treatments was recorded. However, effect of ripening time and treatments was found significant. Total viable count of 45-day old T3 and T4 was significantly lower than control, T1 and T2. After the completion of ripening phase, total viable count of control, T1, T2, T3 and T4 were 7.91, 7.84, 7.75 and 6.18 log CFU/mL, respectively. It was noticed that fortification of curd at T3 and T4 levels inhibited TVC to some extent; however, inhibition level did not reach the extent that could impact microbiological and biochemical processes in cheese matrix however, at T1 and T2 no significant impact was caused by IgG on TVC. Abdel-Ghany et al. [1] studied the effect of supplementing yoghurt with 30 mg/100 IgG on total plate count for 12 days, total plate count decreased in the whole storage phase. Total plate count of yoghurt produced by blending milk with colostrum powder from 100–200 mg/100 g decreased during storage period of 35 days [5].
Table 2.
Total viable count of cheddar cheese supplemented with IgG
| Treatments | TVC (log CFU/mL) at various stages | |||
|---|---|---|---|---|
| After Cheddaring | After Pressing | After 45-Days of ripening | After-90 Days of ripening | |
| Control | 9.42 ± 0.06a | 9.35 ± 0.07a | 8.72 ± 0.13a | 7.91 ± 0.09b |
| T1 | 9.37 ± 0.13a | 9.31 ± 0.05a | 8.66 ± 0.09a | 7.84 ± 0.16b |
| T2 | 9.44 ± 0.11a | 9.39 ± 0.02a | 8.54 ± 0.04a | 7.75 ± 0.11b |
| T3 | 9.32 ± 0.04a | 9.28 ± 0.22a | 7.91 ± 0.07b | 6.72 ± 0.07c |
| T4 | 9.26 ± 0.16a | 9.24 ± 0.17a | 7.63 ± 0.03b | 6.18 ± 0.02c |
When means in rows and column bear a different letter, it gives significant variation (p < 0.05)
Stability of IgG during production and ripening of cheddar cheese
Impact of processing and ripening phase on retention of IgG is described in Table 3. After the completion of cheddaring, IgG contents of control, T1, T2, T3 and T4 were 0.08, 4.92, 9.77, 14.95 and 19.92 mg/100 g; however, IgG contents of all types of cheese significantly decreased during pressing (Figs. 1, 2, 3, 4 and 5). After 15 h of pressing, IgG contents of control, T1, T2, T3 and T4 decreased by 2.5, 6.09, 2.70, 5.62 and 3.60%. Loss of IgG during pressing was due to its solubilization in whey. No loss of IgG was recorded in the entire ripening phase of 90 days. After 45 days, loss of IgG in control, T1, T2, T3 and T4 was 0.66, 0.64, 0.52, 0.36 and 0.32%, respectively. After the completion of ripening phase, IgG contents control, T1, T2, T3 and T4 were 0.72, 1.94, 0.94, 0.56 and 0.36%, respectively. Fully mature cheddar cheese samples showed that retention/stability of IgG in control, T1, T2, T3 and T4 was 99.28, 98.06, 99.10, 99.44 and 99.64%, respectively. Stability of IgG in yoghurt is reported in literature, yoghurts were prepared by fortifying the milk with IgG at 40 mg and 80 mg concentrations and stability of IgG was evaluated for 4 weeks. It was reported that stability of IgG in yoghurt samples (stored at 4–5 ºC) was more than 98% [1]. IgG is more stable at lower temperature; additionally, higher stability of IgG in cheddar cheese can be connected to the lower ripening temperature (5–6 ºC). IgG is stable in the range of 6–7 pH; stability is considerably decreased at 3.5–4.5 pH in high temperature environments [19]. IgG are heat sensitive proteins, heating to 75 ºC/5 min and 95 ºC/15 s can decrease IgG presence by 40 and 100%, respectively [19]. In present study, cheddar cheeses were fortified with IgG at the stage of salting to avoid thermal degradation and losses during whey draining. Pulse electric field can be used for decreasing the microbial load of colostrum used for the extraction and purification of IgG [27].
Table 3.
Retention/Stability of IgG in cheddar cheese (mg/kg)
| Treatments | After Cheddaring | After Pressing | After 45-Days of Ripening | After-90 Days of Ripening |
|---|---|---|---|---|
| Control | 0.08 ± 0.01i | 0.07 ± 0.01j | 0.06 ± 0.01j | 0.05 ± 0.01j |
| T1 | 4.92 ± 0.04 g | 4.62 ± 0.02 h | 4.59 ± 0.16 h | 4.53 ± 0.05 h |
| T2 | 9.77 ± 0.12e | 9.51 ± 0.15f | 9.46 ± 0.09f | 9.42 ± 0.17f |
| T3 | 14.95 ± 0.17c | 14.27 ± 0.08d | 14.22 ± 0.03d | 14.19 ± 0.24d |
| T4 | 19.92 ± 0.06a | 19.22 ± 0.21b | 19.16 ± 0.18b | 19.15 ± 0.07b |
When means in rows and column bear a different letter, it gives significant variation (p < 0.05) in three variables (IgG, Manufacturing Stages and Storage Period)
Fig. 1.
Retention/Stability of IgG in milk and control
Fig. 2.
Retention/Stability of IgG in T1
Fig. 3.
Retention/Stability of IgG in T2
Fig. 4.
Retention/Stability of IgG in T3
Fig. 5.
Retention/Stability of IgG in T4
Organic acids of IgG supplemented cheddar cheese
Breakdown of lactose and fat and microbiological and biochemical processes lead to the production of organic acids. Ketones, sulfur, alcohols and organic acids are responsible for the optimum flavor of cheddar cheese [45]. Mechanism of organic acids production has been documented in literature,PUFAs are transformed to hydroperoxides by biochemical pathways, hydroperoxides are eventually transformed to alcohols and organic acids via several biochemical pathways [25]. Organic acids also possess strong antibacterial activities in cheese and do not allow undesirable bacteria to grow in ripened cheeses. Several investigations on ripened cheeses have shown that cheese matrix is not a good host for pathogenic bacteria. The hostile environment in cheese is the outcome of production of organic acids, antibacterial compound (nisin), changes in pH due to production of organic acids, water activity, refrigeration temperature, etc. Cheese production is a classic example of food preservation by using multiple hurdle technology in which above mentioned hurdles are synchronized with each other to produce unfavorable/hostile environment for undesirable bacteria [11, 31]. Limited research work has been done on the supplementation of semi-hard cheeses with IgG. In the present study, the effect of IgG supplementation 5, 10, 15 and 20 mg/kg on the production of organic acids in cheese was investigated. Results showed that IgG supplementation at all concentrations did not inhibit the formation of organic acids during the three months of ripening (Table 4). In ripening phase of three months, magnitudes of CA, LA, AA and PA consistently increased and both estimation frequencies indicated a rising trend (p < 0.05). After 45 days, amounts of CA, LA, AA and PA in control were 2239, 11,375, 114 and 82 ppm, respectively. After 90 days, amounts of CA, LA, AA and PAA in control were 2569, 17,489, 178 and 291 ppm (p < 0.05) (Table 4). After 45 days, amounts of CA, LA, AA and PAA were 2224, 11,654, 105 and 86 ppm, respectively, in T4. After 90 days, amounts of CA, LA, AA and PAA were 2577, 17,974, 192 and 288 ppm, respectively, in T4. In cheese ripening, primary function of starter bacteria is to produce lactic acid which has strong antibacterial activity against coliforms further, role of organic acids in improving texture and flavor is scientifically established [12, 43]. By microbiological and biochemical processes, LA is converted to acetaldehyde and diacetyl, which are the main flavoring compounds in cheese. Lactate and citrate act as substrates for the production of AA, a major flavoring compound in ripened cheddar cheese [12]. Statistical indicated no statistically sound connection between the extents of organic acids and IgG. Effect of lactoferrin addition on production of LA was determined, lactoferrin addition had no influence on LA production [18]. Production of organic acids buffalo cheddar were assessed for six months, extents of oganic acids steadily increased [34]. Ullah et al. [48] reported that during three months of ripening of cheddar cheese the presence of organic acids increased. Further studies should be conducted to find a scientific connection between IgG and lipolysis of cheddar cheese. Batool et al. [9, 10] monitored the generation of organic acids in cow and buffalo milk-based cheddar cheese for three months, level of organic acids increased consistently during the entire ripening phase.
Table 4.
Effect of IgG supplementation on organic acids of cheddar cheese (ppm)
| Treatments | Ripening Days | CA | LA | AA | PA |
|---|---|---|---|---|---|
| Control |
45 90 |
2239 ± 1.19b 2569 ± 1.33a |
11,357 ± 1.77b 17,489 ± 2.83a |
114 ± 0.05b 178 ± 0.03a |
82 ± 0.01b 291 ± 0.16a |
| T1 |
45 90 |
2266 ± 0.85b 2541 ± 0.27a |
11,437 ± 0.67b 17,671 ± 1.33a |
109 ± 0.01b 181 ± 0.22a |
79 ± 0.07b 285 ± 0.02a |
| T2 |
45 90 |
2275 ± 1.43b 2519 ± 0.97a |
11,518 ± 2.44b 17,791 ± 1.69a |
106 ± 0.04b 193 ± 0.11a |
89 ± 0.14b 275 ± 0.03a |
| T3 |
45 090 |
2234 ± 0.54b 2597 ± 0.44a |
11,389 ± 1.60b 17,789 ± 0.95a |
119 ± 0.02b 190 ± 0.13a |
91 ± 0.02b 280 ± 0.07a |
| T4 |
45 90 |
2224 ± 0.73b 2577 ± 0.91a |
11,654 ± 0.76b 17,974 ± 1.13a |
105 ± 0.24b 192 ± 0.36a |
86 ± 0.01b 288 ± 0.12a |
When means in every column bear a different letter, it gives significant variation (p < 0.05)
Fatty acid profile of IgG supplemented cheddar cheese
Fatty acid profile (FAP) of milk fat and vegetables oils and fats are significantly different from each other; thus, diversified groups of fatty acids are present in the former fat, i.e. short, medium and long chains (SCFAs, MCFAs and LCFAs, respectively). SCFAs include C4:0, C6:0, C8:0 and C10:0, MCFAs have C12:0, C14:0 and C16:0. C18:0 (saturated), C18:1, C182 and C18:3 (unsaturated) is the part of LCFAs. Each fatty acid group performs its specific function for example SCFAs contribute to a characteristic flavor to dairy foods and MCFAs improve functional properties [36]. Conjugated linoleic acid is a group of nutritionally significant isomers. Nutritional improvement/fortification of dairy products should not cause any modification in their FAP. While improving the nutritional value of cheese by the addition of a functional ingredient, it is enormously imperative to study its effect on FAP. If fatty acid profile is affected by the added ingredient/bioactive compounds or processing conditions, it can have a great effect on sensory and functional properties of dairy products [26]. From FAP, oxidation status of dairy products can also be adjudged. In the current study, it was found that supplementation of cheddar cheese with IgG from 5–20 mg/100 did not change the FAP during the processing and ripening of three months. The non-significant of IgG supplementation (5–20 mg/kg) on FAP of cheddar cheese was due to lower concentrations and non-association between IgG (a fraction of whey proteins) and triglycerides (lipid). At 0-day storage, amounts of SCFAs in control, T1, T2, T3 and T4 were 9.66, 9.64, 9.54, 9.44 and 9.37 mg/100 g, respectively. However, ripening phase of three months considerably changed the FAP of all the experimental samples and control. The changes of control and IgG supplemented cheddar cheese samples were mainly due to the lipolysis. At zero day and after 90 days, amounts of SCFAs in control, T1, T2, T3 and T4 were 12.36 and 11.62, 12.34 and 11.20, 12.42 and 11.10, 12.24 and 11.36, 12.22 and 11.28, respectively. At 0-day time, the presence of C18:1 in control, T1, T2, T3 and T4 was 23.79, 23.91, 23.95, 23.76 and 23.72 mg/100 g, respectively. After 90 days, the presence of C18:1 in control, T1, T2, T3 and T4 was 21.19, 22.38, 22.14, 21.96 and 21.88 mg/100 g, respectively. At 0-day, the levels of C18:2 of control, T1, T2, T3 and T4 were 1.98, 1.95, 1.99, 1.97 and 1.94 mg/100 g, respectively. After 90 days, the contents of C18:2 in control, T1, T2, T3 and T4 were 1.56, 1.64, 1.59, 1.58 and 1.55 mg/100 g, respectively (Table 5). Effect of lactoferrin supplementation (a fraction of whey proteins) has been documented in literature, supplementation of lactoferrin (5–20 mg/kg did not affect the FAP of cheddar cheese [2]. FAP of cheddar cheese was not influenced by the supplementation of vitamin A form 3500–5000 IU/100 g,however, ripening induced major changes in FAP [21]. FAP of young and mature Gouda cheese were different [24]. Supplementation of whey protein concentrate did not affect the FAP of yoghurt [50]. Effect of whey protein addition on fatty acid profile of yoghurt was examined, FAP did not change as a function of addition of whey protein addition [6]. Fortification of polymerized whey protein did not have any significant impact on FAP of yoghurt [8].
Table 5.
Fatty acid profile (mg/100 g) of IgG supplemented cheddar cheese
| Fatty Acid | Control | T1 | T2 | T3 | T4 | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| 0-Day | 90-Days | 0-Day | 90-Days | 0-Day | 90-Days | 0-Day | 90-Days | 0-Day | 90-Days | |
| C4:0 | 2.91 ± 0.02a | 2.75 ± 0.04b | 2.84 ± 0.03a | 2.71 ± 0.12b | 2.88 ± 0.04a | 2.68 ± 0.02b | 2.84 ± 0.04a | 2.67 ± 0.06b | 2.83 ± 0.03a | 2.62 ± 0.01b |
| C6:0 | 3.25 ± 0.07a | 3.11 ± 0.08b | 3.24 ± 0.05a | 2.97 ± 0.09b | 3.21 ± 0.07a | 2.95 ± 0.08b | 3.20 ± 0.06a | 2.95 ± 0.09b | 3.18 ± 0.01a | 2.89 ± 0.06b |
| C8:0 | 3.16 ± 0.09a | 3.01 ± 0.01b | 3.19 ± 0.01a | 3.03 ± 0.16b | 3.14 ± 0.02a | 2.94 ± 0.05b | 3.12 ± 0.02a | 2.94 ± 0.01b | 3.16 ± 0.08a | 2.84 ± 0.17b |
| C10:0 | 3.04 ± 0.24a | 2.75 ± 0.07b | 3.07 ± 0.09a | 2.49 ± 0.15b | 3.11 ± 0.06a | 2.53 ± 0.09b | 3.08 ± 0.19a | 2.80 ± 0.14b | 3.05 ± 0.10a | 2.93 ± 0.23b |
| C12:0 | 1.97 ± 0.13a | 1.48 ± 0.03b | 2.98 ± 0.19a | 2.61 ± 0.06b | 2.96 ± 0.13a | 2.56 ± 0.12b | 2.95 ± 0.14a | 2.49 ± 0.17b | 2.87 ± 0.16a | 2.47 ± 0.31b |
| C14:0 | 13.56 ± 0.29a | 11.05 ± 0.74b | 13.58 ± 0.26a | 11.37 ± 0.23b | 13.53 ± 0.16a | 11.18 ± 0.28b | 12.51 ± 0.37a | 11.13 ± 0.25b | 12.33 ± 0.19a | 11.06 ± 0.45b |
| C16:0 | 29.37 ± 0.34a | 26.94 ± 0.56b | 29.31 ± 0.34a | 27.10 ± 0.17b | 29.17 ± 0.35a | 26.95 ± 0.52b | 29.16 ± 0.62a | 26.62 ± 0.46b | 28.79 ± 0.12a | 26.55 ± 0.79b |
| C18:0 | 8.12 ± 0.04a | 7.23 ± 0.35b | 8.14 ± 0.12a | 7.44 ± 0.16b | 8.15 ± 0.18a | 8.11 ± 0.26b | 8.04 ± 0.79a | 8.07 ± 0.27b | 7.99 ± 0.25a | 7.97 ± 0.018b |
| C18:1 | 23.79 ± 0.42a | 21.19 ± 0.13b | 23.91 ± 0.46a | 22.38 ± 0.31b | 23.95 ± 0.57a | 22.14 ± 0.65b | 23.76 ± 0.91a | 21.96 ± 0.39b | 23.72 ± 0.67a | 21.88 ± 0.46b |
| C18:2 | 1.98 ± 0.03a | 1.56 ± 0.06b | 1.95 ± 0.01a | 1.64 ± 0.02b | 1.99 ± 0.01a | 1.59 ± 0.02b | 1.97 ± 0.03a | 1.58 ± 0.04b | 1.94 ± 0.02a | 1.55 ± 0.05b |
| C18:3 | 0.62 ± 0.02a | 0.42 ± 0.02b | 0.64 ± 0.03b | 0.42 ± 0.01b | 0.64 ± 0.02a | 0.39 ± 0.01b | 0.63 ± 0.01a | 0.62 ± 0.02b | 0.62 ± 0.01a | 0.61 ± 0.01b |
When means in every row bear a different letter, it gives significant variation (p < 0.05)
Free fatty acids of IgG supplemented cheddar cheese
For the development of optimum flavor in cheese in ripening, milk fat is an essential substrate. Lipolysis is an extremely significant biochemical process taking place in cheese ripening for the development of typical flavor of cheese. Extents of lipolysis varies among diverse categories of cheeses; thus, blue cheese and certain hard Italian cheeses undergo extensive lipolysis; however, in Cheddar and Gouda cheeses, moderate level of lipolysis is present [32]. The most significant event of lipolysis is the liberation of FFAs from triacylglycerol molecules by the activities of endogenous and exogenous lipases. Milk fat contains about 10–12% SCFAs. FFAs of SCFAs possess lower flavor and significantly contribute in cheese flavor [31]. IgG fortified cheese may have different behavior in lipolysis. Therefore, the impact of IgG fortification on generation of FFAs was monitored for three months in the present study. Supplementation of cheddar cheese with IgG from 5–20 mg/100 g concentrations did not affect FFAs content. FFAs in freshly produced experimental samples were almost similar to each other (p > 0.05). FFAs of substrate milk were 0.08% (oleic acid); therefore, FFAs of control and treatments were dissimilar to each other (p > 0.05). FFAs of control, T1, T2, T3 and T4 consistently increased in the ripening phase of three months and analysis on 45 and 90 days showed increasing trends (Table 6). The rise of FFAs in control and all the treatments was primarily due to biochemical events taking place in fat moiety of cheese and no statistical correlation was found FFAs and IgG. FFA in vitamin E and selenium supplemented cheese were similar to the normal cheddar cheese [9, 10]. Gouda cheese was supplemented with mango kernel oil,the impact of three months ripening time on FFA was investigated; as a result, FFA presence in all samples increased in refrigerated storage of three months [26].
Table 6.
FFA (%), POV (meqO2/kg) and cholesterol of igg supplemented cheddar cheese
| Treatments | Ripeningtime (days) | FFA (%) | POV (meqO2/kg) | Cholesterol (mg/100 g) |
|---|---|---|---|---|
| Control |
0 45 90 |
0.08 ± 0.02c 0.16 ± 0.02b 0.38 ± 0.03a |
0.24 ± 0.04a 0.25 ± 0.02a 0.29 ± 0.01a |
219.44 ± 0.32a 195.73 ± 0.65b 172.28 ± 0.52c |
| T1 |
0 45 90 |
0.09 ± 0.01c 0.17 ± 0.02b 0.36 ± 0.04a |
0.22 ± 0.03a 0.26 ± 0.06a 0.30 ± 0.05a |
217.23 ± 0.38a 194.41 ± 0.89b 175.68 ± 0.27c |
| T2 |
0 45 90 |
0.08 ± 0.02c 0.19 ± 0.03b 0.36 ± 0.05a |
0.23 ± 0.02a 0.25 ± 0.03a 0.28 ± 0.04a |
220.54 ± 1.29a 190.46 ± 0.31b 169.79 ± 0.82c |
| T3 |
0 45 90 |
0.09 ± 0.02c 0.18 ± 0.03b 0.41 ± 0.04a |
0.26 ± 0.01a 0.29 ± 0.01a 0.32 ± 0.03a |
216.49 ± 0.97a 193.16 ± 0.19b 170.26 ± 0.46c |
| T4 |
0 45 90 |
0.09 ± 0.03c 0.15 ± 0.02b 0.35 ± 0.02a |
0.27 ± 0.08a 0.28 ± 0.02a 0.31 ± 0.01a |
218.69 ± 0.94a 188.62 ± 0.75b 174.13 ± 0.49c |
When means in every column bear a different letter, it gives significant variation (p < 0.05)
Lipid oxidation of IgG supplemented cheddar cheese
Milk fat contains about 30% unsaturated fatty acids (MUFA and PUFA); notably, lipid oxidation rate in PUFA is tremendously higher than MUFA (10:1) [7]. It is not advisable to store milk fat for a longer time due to severe problem of auto-oxidation,however, due to lower Redox potential (250 mV) in cheese, auto-oxidation is not a threat in ripened cheeses (McSweeney and Fox, 2004). In the present study, in order to analyze the association of IgG with lipid oxidation, POV in experimental and control cheeses were investigated at the intervals of 0, 45 and 90 days of ripening. No statistical correlation between IgG and POV was established, i.e. IgG did not show pro-oxidant effect at any concentration. After the completion of three months ripening, POV of three months old control, T1, T2, T3 and T4 was 0.29, 0.30, 0.28, 0.32 and 0.31 (meqO2/kg), respectively (Table 6). With the advancement of analytical chemistry, several methods of lipid oxidation have been introduced. However, POV is universally used to determine lipid oxidation in food and dairy products [35]. Production of peroxides in auto-oxidative degradation of lipids can be analyzed by POV, scientific connections between storage stability and POV are scientifically proved [36]. European Union guidelines indicate that POV of foods should not be more than 10 (meq O2/kg). POV of IgG augmented cheese samples were within the granted limit of EU. Concentration of cholesterol significantly decreased during ripening phase of three months, analysis of cholesterol after 45 and 90 days indicated a significant decline in all treatments and control. In three months old control, T1, T2, T3 and T4 concentrations of cholesterol were 172.28, 175.68, 169.79, 170.26 and 174.13 mg/100 g, respectively. In three months old IgG supplemented T1, T2, T3 and T4 and control, reduction in cholesterol content was 19.2, 23.1, 21.4, 21.2 and 21.5% (p > 0.05), respectively. IgG supplemented cheddar cheese with lower cholesterol may be regarded as functional food and it may be highly suitable for the consumers seeking low cholesterol functional cheese in their diet. Influence of ripening on cholesterol content of cheese was monitored for three months, mature cheddar cheese showed considerably higher cholesterol levels than fresh cheese [48].
Sensory characteristics of IgG supplemented cheddar cheese
Supplementation of cheddar cheese with IgG from 5–20 mg caused no effect on color of T1, T2, T3 and T4 as observed (Fig. 6). Such non-variation of color of control and experimental cheeses was due to milky color of IgG and concentrations were too low to affect the color of cheese samples. All the measurement intervals revealed that color of all samples and control remains unchanged in the entire experiment. Flavor and texture scores increased as the ripening processes proceeded. At the end of this experiment, color and flavor scores of all treatments and control were similar to each other showing that IgG fortification did not hinder in biochemical events of cheese ripening. Texture and flavor score of cheddar cheese enhanced in the ripening phase [20]. Flavor scores of three months old control, T2 and T3 were more than 90% of the total score (9). Impact of whey protein supplementation (lactoferrin) on sensory characteristics of cheddar cheese has been described in literature. Adnan et al. [2] reported that supplementation of cheddar cheese with lactoferrin at 5, 10, 15 and 20 mg/kg did not cause any effect on color, flavor and texture scores. Batool et al. [9, 10] reported that supplementation of cheddar cheese with selenium and vitamin E did not have any impact on sensory features of three months old cheddar cheese.
Fig. 6.
Effect of IgG supplementation on sensory score of cheddar cheese as influenced by ripening period
Conclusion
Supplementation of cheddar cheese from 5–20 mg/100 g did not affect cheddar cheese composition and alter the microbial processes with more than 99% stability of IgG in three months old cheese samples. Sensory characteristics of control and IgG supplemented cheddar cheese were identical to each other. This technically sound data suggest that cheddar cheese may be supplemented with IgG from 5–20 mg/kg. However, IgG supplemented cheddar cheese may have higher prices than normal cheddar cheese.
Acknowledgements
The authors acknowledge support from the Department of Dairy Technology, University of Veterinary and Animal Sciences, Lahore, Punjab, Pakistan where all research activities were carried out. The authors are also grateful to Times University, Multan, Pakistan, for helping in providing resources for literature. The authors would like to extend their gratitude to King Saud University (Riyadh, Saudi Arabia) for funding this research through Ongoing Research Funding Program (ORF-2026-406).
Authors’ contributions
Conceptualization, M.N. and S.B.; methodology, M.N. and M.A.R.; software, M.N.; validation, M.N.; formal analysis, M.N. and S.B.; investigation, M.N.; resources, M.N.; data curation, M.N.; writing—original draft preparation, M.N. and S.B.; writing—review and editing, M.A.R. E.Z. M.H.M. and S.P.A.; visualization, M.A.R..; supervision, M.N.; project administration, M.A.R..; funding acquisition, M.H.M. E.Z. and S.P.A. All authors have read and agreed to the published version of the manuscript.
Funding
The authors would like to extend their gratitude to King Saud University (Riyadh, Saudi Arabia) for funding this research through Ongoing Research Funding Program (ORF-2026–406).
Data availability
All the data used in the manuscript are available in the tables and figures.
Declarations
Ethics approval and consent to participate
All authors have given their informed consent to participate.
Consent for publication
Not applicable.
Competing interest
The authors declare no competing interests.
Footnotes
Publisher’s Note
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Contributor Information
Muhammad Nadeem, Email: muhammad.nadeem@uvas.edu.pk.
Muhammad Abdul Rahim, Email: mabdulrahim@t.edu.pk.
Mohamed H. Mahmoud, Email: mmahmoud2@ksu.edu.sa
Eliasse Zongo, Email: zongoeliasse@gmail.com.
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Data Availability Statement
All the data used in the manuscript are available in the tables and figures.