Effect of lactoferrin supplementation on composition, fatty acids composition, lipolysis and sensory characteristics of cheddar cheese

ABSTRACT

Lactoferrin is the part of whey proteins, which does not become the part of curd and is lost in the whey. The objective of the current investigation was to develop lactoferrin-supplemented cheddar cheese. Cheddar cheese was supplemented with lactoferrin at 5, 10, 15, 20, and 0 mg/100 g concentrations (T₁, T₂, T₃, T₄, and control) and matured for 90 days. The supplementation of lactoferrin at all four concentrations did not affect the compositional attributes of cheddar cheese. In mature cheddar cheese, values of log-colony forming units in T₁, T₂, T₃, T₄, and control per mL were 7.82, 7.77, 6.51, 6.21, and 7.95, respectively. Lactoferrin contents of T₁, T₂, T₃, T₄, and control were 4.98, 9.89, 14.91, 19.88, and 0.11 mg/100 g cheese. In vitro total antioxidant capacity of mature T₁, T₂, T₃, T₄, and control were 32.47%, 47.68%, 65.37%, 78.82%, and 45.79%. High-performance liquid chromatography (HPLC) analysis revealed that concentrations of citric acid, lactic acid, acetic acid, and propionic acid in mature T₄ were 2389, 16,468, 126, and 141 mg/kg. Analysis of cheese samples on gas chromatography–mass spectrometry (GC-MS) showed that fatty acids composition was not influenced by the supplementation of lactoferrin. Peroxide value of matured T₁, T₂, T₃, T₄, and control was 0.31, 0.30, 0.28, 0.35, and 0.28 (meqO₂/kg) with no variation in color, flavor and texture score. The results of the current investigation proved that lactoferrin can be used for the supplementation of cheddar cheese.

KEYWORDS:

• Cheddar cheese
• Lactoferrin supplementation
• Antioxidants
• Fatty acids composition
• Lipolysis

Introduction

Milk proteins are composed of about 80% caseins and 20% whey proteins; immunoglobulins, alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin and lactoferrin are included in whey proteins. These proteins have dietetic and nutraceutical potential to improve health and inhibit diseases.^[1] Whey proteins remunerate bioactive peptides having anti-hypertensive, anti-thrombotic, antibacterial, antiviral and hypocholesterolemic activities. Lactoferrin is an iron-binding glycoprotein having 80 kDa molecular weight; it is mainly present in milk. Lactoferrin is composed of a single polypeptide chain in the form of a bilobal structure having the capacity of binding one iron atom in each lobe.^[2] Bovine lactoferrin is composed of 689 amino acids; lactoferrin having 5% iron is known as apolactoferrin; hololactoferrin is a saturated form of lactoferrin; milk contains lactoferrin in the form of apolactoferrin; intake of 10 to 20 mg lactoferrin on daily basis may prevent carcinogenesis.^[3] Lactoferrin possesses the largest number of biological activities; the most significant of which is associated with the defense system of the host as it possesses broad-spectrum antimicrobial properties against bacteria, viruses, protozoa, and virus.^[4] Antibacterial activity of lactoferrin is due to its chelating ability that impedes the use of iron and changes the conformation and stability of bacteria.^[5] The antiviral activity of lactoferrin is attributed to the interaction with the envelop protein or the interference of the attachment mechanism of virus to the host cells.^[6] Anti-inflammatory and antitumoral activities of lactoferrin has been reported in the literature; the enrichment of foods with 1 mg/mL of bovine foods with lactoferrin considerably reduced clostridium in the gut and improved the population of beneficial microorganisms.^[7] The addition of lactoferrin to soybean oil improved the oxidative stability. In viniculture, lactoferrin suppressed the yeast Dekkera bruxellensis, a major cause of wine deterioration .^[8,9] Lactoferrin is isolated from bovine cheese whey and is currently being used in a large number of food applications such as baby foods, fermented milk products, and chewing gums. For the supplementation of foods, bovine lactoferrin has been used for the last 20 years. Lactoferrin-supplemented foods have been recognized as functional foods. More than 60 tons of bovine lactoferrin is commercially prepared for the production of dietary and pet supplements and cosmetics and for the enrichment of dairy products and tablet coats for treating salvia-deficient subjects.^[10] Finding new applications of lactoferrin in functional foods may increase its recovery from whey (mostly drained in developing countries), which will decrease the cost of lactoferrin. Increased knowledge of functional foods and their association with well-being and rapid rise of metabolic diseases have led the consumers and food industries to use and develop functional foods. However, a large number of functional foods have been developed all over the world. The intake of milk and dairy products has decreased in several countries due to the lack of functional value of dairy products. Lactoferrin is the part of whey proteins, which does not become the part of curd and are lost in the whey.^[11] Chemistry of proteins of cheese curd, whey, and milk is considerably different from each other. Therefore, cheese is not a good source of lactoferrin, and health benefits associated with the intake of lactoferrin can be obtained by supplementing the cheese curd with lactoferrin. The effect of processing technology on lactoferrin has been reported in the literature: supplementation of yogurt with lactoferrin had a non-significant impact on physicochemical and microbiological characteristics of yogurt, effect of lactic acid (LA) production, and yogurt manufacturing process, and 6 days of storage had no effect on the stability of lactoferrin.^[12] The ripening of cheddar cheese is performed to develop flavoring compounds and the desired textural properties in cheddar cheese. The impact of lactoferrin supplementation on chemical characteristics of cheddar cheese has been studied in a limited way. The aim of this research work was to know the impact of lactoferrin supplementation on fatty acids composition, organic acids, lipolysis, stability of lactoferrin, and sensorial acceptability of cheddar cheese.

Materials and methods

Materials

Milk from Holstein-Friesian was used in the current investigation. Rennet, starter culture (Lactococcus lactis ssp. lactis and cremoris strains, DVS 850) were purchased from Chr. Hansen (Denmark). Lactoferrin, HPLC-grade methanol, ethanol, n-hexane, aluminum trichloride, ascorbic acid, standards of organic acids, and fatty acid methyl esters (FAME-37) standards of fatty acids were purchased from Sigma Aldrich (St. Louis MO, USA).

Manufacturing of cheddar cheese and experimental plan

Current study was planned in completely randomized design (CRD) with triplication of every treatment. Ten-liter cheese milk was subjected to batch pasteurization at 65°C for 30 min and subsequent cooling to 31°C. Bulk starter culture was added (2%) for the pre-acidification of milk (40 min) and then CaCl₂ (1.5 g) and rennet (0.02%) were added, and the given coagulation time was 30 min. Cutting was performed and temperature was raised to 39°C in 40 min with further cooking at this temperature for 45 min (pH 5.2). Curd was milled and lactoferrin was mixed with salt (1.5%) and added to the curd at the stage of salting at 5, 10, 15, and 20 mg/100 g concentrations (T₁, T₂, T₃, and T₄). The samples of cheddar cheese without the addition of lactoferrin were used as control. The samples of cheese were transferred to the molds and pressed at 3.5 bar pressure for 16 h followed by vacuum packaging. Lactoferrin-supplemented and control cheeses were matured at 4–6°C for 90 days and analyzed for chemical and sensorial prospects at 0, 45, and 90 days. Cheddaring and pressing time for every kind of cheese was 30 min and 16 h under a hydraulic press at 3.5 bar pressure.

Compositional attributes of cheese

The moisture contents in lactoferrin-supplemented cheese samples were determined according to the standard method (926.08). Briefly, a 3-g grated cheese sample was dried in a flat-bottom aluminum dish at 100°C till constant weight is achieved in a hot air oven (Memmert Germany). Babcock method (926.08) was used for the analysis of fat content in cheese samples. Protein contents in all kinds of cheese samples were determined using Kjeldahl method (920.123). The 20-g grated cheese samples were blended at 25°C; pH values were measure with pH meter (InoLab WTW series 720) and calibrated with 4 and 10 pH buffers following standard protocols of AOAC .^[13]

Total viable count

Total viable count in lactoferrin-supplemented cheddar cheese samples was determined at 0, 45, and 90 days of ripening using Total Viable Count Agar, and dilutions of 10⁴ were used followed by incubation of petri dishes at 30°C for 48–72 h in aerobic conditions.^[14]

Determination of lactoferrin concentration with reverse-phase HPLC

For HPLC determination of the lactoferrin, separation was achieved using module Alliance 2695 with diode-array detector (DAD) PDA 2996 (Waters, Millford, USA). The method prescribed by Osel et al.^[15] was used for sample preparation; 50 μL of each sample was injected to reverse-phase (RP) HPLC analysis using an Agilent 1100 HPLC system fitted with DAD at 210 nm fitted with a column (C18 300 R µm, 250 × 4.6 mm, Phenomenex) linear gradient and flow rate of 0.5 mL/min. The mobile phase consisted of 0.1% trifluoroacetic acid in deionized water and acetonitrile (95:5 v/v). Temperature of column was set at 45°C with injection volume of 10 µL. Five standards of lactoferrin were prepared, and the concentrations of lactoferrin were calculated on a calibration curve.^[16]

Total antioxidant capacity

Total antioxidant capacity (TAC) of cheddar cheese was examined using the method described previously (Dasgupta and De. 2007). TAC reagent contains three ingredients, which are 0.6 M H₂SO₄, 28 mM Na₃PO₄, and 4 mM (NH₄)₆Mo₇O₂₄. These three ingredients are mixed very well and termed TAC reagent. Water-soluble cheese extract of about 0.3 mL was poured in test tube and mixed with 3000 µL of TAC reagent. The whole mixture was allowed to incubate at 85°C for 90 min. The absorbance of the samples was checked by using a spectrophotometer at 695-nm wavelength. The trolox curve was used as standard, and the results were in mg (TE)/g of the sample.^[17]

1,1-Diphenyl-2-picrylhydrazyl free radical scavenging activity

The oxidative stability of water soluble extract (WSE) of cheddar cheese, which was accelerated, ripened, and checked, was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging procedure described previously (Cervato. 1999). Briefly, 1 mL of (1 mM) solar solution of methanolic DPPH was allowed to mix with 1 mL WSE of cheddar cheese. The whole mixture was placed in a dark place at 37°C for 30 min for incubation. The wavelength of 517 nm was used to check %inhibition using a spectrophotometer and the following formula:^[18]

DPPH FRSA (%) = [(A blank – A sample)/A blank] × 100

Antioxidant activity in linoleic acid

The sample was dissolved by mixing in 1.5 mL of 0.1 M phosphate buffer whose pH was 7.0. These contents were properly mixed with 1.0 mL of 50 mM linoleic acid, which is prepared in ethanol (99.5%). Overall, 5 mL of this sample mixture was transferred to a test tube tightened with silicone rubber caps and then placed at 40°C in the dark. Overall, 50 µl aliquots of the reaction mixture were mixed with 2.35 mL of 75% ethanol, 50 µL of 30% ammonium thiocyanate, and 5 µL of 20 mM ferrous chloride solution, which were prepared in 3.5% HCl. Aliquots of the reaction mixtures were withdrawn from the test tube with a micro-syringe and transferred to cuvettes for measuring the oxidation. After a 3-min stay time, the antioxidant activity of colored solution was checked at 500 nm by spectrophotometer and expressed in %.^[19]

Fatty acid profile

Fat from cheese samples was removed as given in AOAC.^[13] The extracted fat (50 mg) was placed in a test tube, followed by the addition 2 mL each of n-hexane and 0.5 N CH₃NaO (HPLC methanol) and vortexing at 2000 rpm for 2 min; a stay time of 15 min was given; the upper layer was extracted and transferred to GC vials; and 1 µL of the mixture was injected (split ratio 50:50) through auto liquid sampler in a GC-MS (7890-B, Agilent Technologies) in a fused silica capillary column SP-2560 (100 m × 0.25 mm, Supleco) using helium as a carrier gas at the flow rate of 2 mL/min, while the flow rate of hydrogen and oxygen was 4 mL and 40 mL/min, respectively. Temperatures of inlet and flame ionization detector were set at 200°C and 250°C using FAME-37 standards.^[20]

Characterization of organic acids by HPLC

For the measurement of organic acids, a 10-g grated sample of cheese was added with 40 mL of H₂PO₄ followed by vortexing at 2000 rpm for 1 min and incubation at 63°C in a water bath. Samples were centrifuged at 6000 rpm for 10 min, the supernatant was filtered on a filter paper (Whatman no. 1), and the filtrate was transferred to GC vials (Agilent). The measurement was performed on HPLC, Perkin Elmer, Germany (series 200), attached with the Shodex RSpak KC‐118 model ion exchange organic column (8 mm × 300 mm) using a UV absorbance detector at 214 nm. Concentrations of organic acids in samples of cheese were calculated in a calibration curve using five standards of each citric acid (CA), LA, propionic acid (PA), and acetic acid (AA) .^[21]

Lipolysis

Lipolysis in the cheese sample was assessed by measuring free fatty acids (FFAs), PV, and cholesterol using standard methods.^[22]

Sensory evaluation

For sensory analysis, a panel of ten judges was verified, selected, and trained according to the standard method.^[23] For the standardization of pre-drafted terminology (color, flavor, and texture), four training meetings (each for 2 h) were conducted. The quantitative and qualitative calibrations of the panel were attained by familiarization with reference materials and scale definitions. The samples were tested in individual sensory evaluation booths in a well-lit and ventilated laboratory at 20°C on a 9-point scale by FIZZ software, version 2.46B.

Statistical analysis

This research work was planned in a CRD with every treatment performed in triplicate. For the measurement of the effect of lactoferrin, storage duration, and their interaction and significant difference among the treatments, two-way analysis of variance and Duncan's multiple range (DMR) test were used with the aid of SAS 9.4 statistical software.

Results and discussion

Composition of cheddar cheese

Supplementation of lactoferrin at all four concentrations did not affect compositional traits of cheese (Table 1). The non-significant variation in the composition of cheese may be assigned to the non-variation in ingredients, formulation and processing conditions.^[24] supplemented the cheese with 20 ppm lactoferrin, compositional characteristics of lactoferrin-supplemented cheese were similar to standard cheese. The evolution of pH in cheddar cheese was monitored at the stage of cheddaring and 0, 45, and 90 days of ripening. In cheddaring, there was a certain delay in the decline of pH in lactoferrin-supplemented samples. pH of the control sample decreased more rapidly as compared to the experimental samples, and delay in pH decrease was dependent upon the concentration of lactoferrin. Cheddaring time of control, T₁, T₂, T₃, and T₄ was 45 ± 1.22, 51 ± 1.53, 58 ± 1.88, 68 ± 2.15, and 71 ± 1.65 min (p < 0.05). However, subsequent determination frequencies indicated no difference in pH of T₁ and T₂ and control (p > .05). After 45 days of ripening, non-significant decrease in pH was recorded at T₃ and T₄ concentrations. Lactoferrin at T₃ and T₄ concentrations significantly inhibited the bacterial activities that led to a less decrease in pH of T₃ and T₄ as compared to the control. The effect of lactoferrin supplementation on pH of yogurt is documented in the literature, and the supplementation of lactoferrin delayed pH drop.^[12] The supplementation of lactoferrin did not affect pH and composition of fresh carrot juice.^[25] Vitamin A from 3500 to 5000 IU/kg did not affect cheese composition.^[26] Moisture, fat, and protein contents in cheddar cheese were 37%, 33%, and 25%, respectively.^[27] The effect of storage on compositional traits of cheese was monitored for 90 days. It was found that 90-day ripening significantly affected the chemical composition of cheese. During ripening, proteolysis and lipolysis occur, which cause modifications in the composition of cheese. Compositions of 120 days old cheddar cheese were different from those of fresh cheese.^[28,29]

Table 1. Effect of Lactoferrin Supplementation on Composition of Cheddar Cheese.

Treatments	Ripening days	Moisture%	Fat%	Protein%	pH
T1	0 45 90	39.74 ± 0.22^a 39.71 ± 0.26^a 39.65 ± 0.14^a	30.42 ± 0.34^a 30.41 ± 0.31^a 29.36 ± 0.27^b	24.62 ± 0.17^a 24.58 ± 0.05^a 23.19 ± 0.09^b	5.29 ± 0.02^b 5.22 ± 0.04^c 5.08 ± 0.09^d
T2	0 45 90	39.76 ± 0.18^a 39.73 ± 0.13^a 39.69 ± 0.24^a	30.45 ± 0.31^a 30.42 ± 0.52^a 29.39 ± 0.47^b	24.68 ± 0.12^a 24.61 ± 0.18^a 23.11 ± 0.14^b	5.25 ± 0.06^b 5.20 ± 0.02^c 5.10 ± 0.01^d
T3	0 45 90	39.81 ± 0.14^a 39.80 ± 0.08^a 39.76 ± 0.21^a	30.49 ± 0.28^a 29.14 ± 0.08^b 28.22 ± 0.16^c	24.71 ± 0.04^a 23.51 ± 0.17^b 22.05 ± 0.19^c	5.37 ± 0.03^b 5.22 ± 0.01^c 5.11 ± 0.04^d
T4	0 45 90	39.85 ± 0.13^a 39.82 ± 0.16^a 39.79 ± 0.19^a	30.55 ± 0.41^a 29.31 ± 0.31^b 28.11 ± 0.26^c	24.78 ± 0.13^a 23.44 ± 0.08^b 22.97 ± 0.03^c	5.39 ± 0.06^a 5.37 ± 0.02^a 5.31 ± 0.01^b
Control	0 45 90	39.62 ± 0.23^a 39.60 ± 0.12^a 39.55 ± 0.20^a	30.67 ± 0.37^a 30.51 ± 0.22^a 28.78 ± 0.15^c	24.81 ± 0.18^a 24.36 ± 0.23^a 23.87 ± 0.15^b	5.43 ± 0.07^a 5.39 ± 0.05^a 5.28 ± 0.02^b

If in one column, means are expressed with a different letter; this indicates significant difference (p < 0.05)

T₁: Lactoferrin 5 mg/100 g

T₂: Lactoferrin 10 mg/100 g

T₃: Lactoferrin 15 mg/100 g

T₄: Lactoferrin 20 mg/100 g

Effect of lactoferrin supplementation on total viable count of cheddar cheese

During cheese manufacturing process, the dominant microflora is starter bacteria; on the day of production their count is usually 109 cfu/mL. However, during ripening the population of starter bacteria declines due to low pH, temperature, moisture content, and high salt content; the decline in bacterial population depends upon the strain, in most ripened varieties of cheese, usually 1% decline has been recorded after 1 month of ripening. With the decline of starter bacteria in cheese, an increase in secondary adventurous tarter bacteria occurs.^[30] At the industrial scale, cheddar cheese is manufactured from pasteurized milk using the standard procedure under hygienic conditions. However, in spite of dedicated precautions during cheese making, a secondary microflora develops in the curd from milk, processing facility, and cheese making environment. Water, salt, and pH can have a little impact and the production of organic acids and inhibitory substances can have a significant impact on the growth and final population of nonstarter LA bacteria.^[31] The impact of lactoferrin supplementation on the growth of bacteria was determined during the time of cheese manufacturing (Table 2). Lactoferrin is a part of whey proteins. Most of it does not become the part of curd; therefore, in the current investigation, lactoferrin supplementation was made to the curd and not to the milk to retain its maximum concentration. After pressing and cheddaring, lactoferrin had no effect on total viable count of cheddar cheese. After pressing, total viable count in all treatments of lactoferrin-supplemented cheddar cheese and control ranged from 9.21 to 9.35 (log cfu/mL). After 45 days of ripening, values of log-colony forming units per mL ranged from 5.21 to 6.95 in experimental samples and control. After 45 days of ripening, values of log-colony forming units in T₁, T₂, T₃, T₄, and control per mL were 8.84, 8.22, 7.91, 7.51, and 7.25. The results of this investigation indicated that lactoferrin supplementation decreased the total viable count at T3 and T4 concentrations. After 90 days of ripening, values of log-colony forming units in T₁, T₂, T₃, T₄, and control per mL were 7.95, 7.82, 7.77, 6.51, and 6.21, respectively. In the current investigation, it was found that lactoferrin at T₃ and T₄ concentrations slightly inhibited the bacterial growth. After cheddaring, value of r² between total viable count and retention of lactoferrin after cheddaring was 0.0896, value of r² between total viable count and retention of lactoferrin after pressing was −0.0289, value of r² between total viable count and retention of lactoferrin after 45 days of ripening was −0.1170, and value of r² between total viable count and retention of lactoferrin after 90 days of ripening was −0.0658. The effect of lactoferrin supplementation on the growth of Lactobacillus acidophilus was investigated; it was found that lactoferrin had a slight inhibitory effect on the growth of these bacteria with no difference in sensory characteristics.^[32] Griffiths et al.^[33] reported that iron lactoferrin inhibited the growth of bifidobacteria and lactobacilli. Lactoferrin at 5-mg concentration efficiently inhibited Escherichia coli O157: H7.^[34]

Table 2. Effect of Lactoferrin Supplementation on Total Viable Count.

Treatments	Total viable count (log cfu/mL) at various stages
	After cheddaring	After pressing	After 45 days	After 90 days
T1	9.25 ± 0.08^a	9.22 ± 0.06^a	8.84 ± 0.15^b	7.95 ± 0.15^g
T2	9.34 ± 0.17^a	9.31 ± 0.02^a	8.22 ± 0.09^c	7.82 ± 0.09^g
T3	9.41 ± 0.05^a	9.35 ± 0.14^a	7.91 ± 0.18^d	7.77 ± 0.18^g
T4	9.24 ± 0.03^a	9.21 ± 0.09^a	7.51 ± 0.16^e	6.51 ± 0.16^h
Control	9.30 ± 0.21^a	9.28 ± 0.12^a	7.25 ± 0.10^f	6.21 ± 0.10^i

If in rows and column, means are expressed with a different letter, it indicates significant difference (p < 0.05)

Retention stability of lactoferrin

Lactoferrin was mixed with salt and added to the curd at the stage of salting; after cheddaring, lactoferrin contents of T₁, T₂, T₃, T₄, and control were 4.98, 9.89, 14.91, 19.88, and 0.11 mg/100 g, respectively. It was found that pressing (3.5 bar for 16 h) had a significant impact on concentrations of lactoferrin (Table 3). After pressing, the decline in concentrations of lactoferrin in T₁, T₂, T₃, T₄, and control were 4.21%, 4.34%, 2.68%, 2.56%, and 2.77%, respectively. Lactoferrin is a water-soluble protein, pressing removed some whey from the curd, and the loss of lactoferrin in allexperimental samples and control was due to this phenomenon. The ripening phase had a non-significant (p > 0.05) impact on the concentration of lactoferrin in all treatments and control. After 45 days of ripening, the decline in the concentration of lactoferrin in T₁, T₂, T₃, T₄, and control were 1.04%, 0.21%, 0.20%, 0.10%, and 0.96% from the values recorded after pressing. After 90 days of ripening, the decline in the concentration of lactoferrin in T₁, T₂, T₃, T₄, and control were 6.02%, 4.85%, 3.10%, 2.81% and 4.62%, from the initial values recorded after cheddaring. The lactoferrin stability in mature T₁, T₂, T₃, T₄, and control were 93.98%, 95.15%, 96.9%, 97.19%, and 95.38%. The stability of lactoferrin depends largely upon environmental factors, such as pH, minerals, and existence of other whey proteins.^[35] The effect of yogurt manufacturing and storage phase of 3 weeks was investigated on stability of added lactoferrin; lactoferrin was not considerably affected by the storage phase, in 21 days’ samples of yogurt, the stability of lactoferrin was 85%. Lactoferrin in supplemented yogurt and value-added dairy products were determined, the results were consistent to the current investigation.^[36] The impact of using raw and pasteurized milk on the concentration of lactoferrin in soft cheese was investigated. Pasteurization had no effect on concentration of lactoferrin; however, whey drainage considerably decreased lactoferrin in the cheese.^[37] Retention and stability of lactoferrin in supplemented yogurt samples were studied for 3 weeks; the concentration of lactoferrin was not affected by the storage period.^[12]

Table 3. Concentration of Lactoferrin at Different Stages of Processing and Storage (mg/100 g).

Treatments	After cheddaring	After pressing	After 45 days	After 90 days
T1	4.98 ± 0.03^g	4.77 ± 0.02^h	4.72 ± 0.01^h	4.68 ± 0.05^h
T2	9.89 ± 0.05^e	9.46 ± 0.11^f	9.44 ± 0.12^f	9.41 ± 0.010^f
T3	14.91 ± 0.04^c	14.52 ± 0.06^d	14.49 ± 0.18^d	14.45 ± 0.07^d
T4	19.88 ± 0.09^a	19.37 ± 0.15^b	19.35 ± 0.13^b	19.32 ± 0.23^b
Control	0.108 ± 0.01^i	0.105 ± 0.02^j	0.104 ± 0.01^j	0.103 ± 0.01^j

If in rows and columns, means are expressed with a different letter, it indicates significant difference (p < 0.05)

Total antioxidant capacity

TAC is a prestigious way of measuring the oxidative stress. TAC is directly proportional to the oxidative stability of foods. Foods having higher TAC are perceived to have superior oxidative stability and vice versa.^[38] For the estimation of antioxidant status of cheddar cheese in ripening, TAC was used as a key indicator used.^[18] The results of TAC of lactoferrin-supplemented cheddar cheese is shown in Table 4. In this investigation, the impact of lactoferrin supplementation on TAC of cheddar cheese was investigated for a period of 90 days. Lactoferrin-supplemented cheddar cheese samples had higher TAC than control (p < 0.05). At 0 day, TAC of control, T₁, T₂, T₃, and T₄ was 20.91%, 21.59%, 22.98%, 24.07%, and 27.33%. TAC of lactoferrin was 87.52%, which was the reason for higher TAC of lactoferrin-supplemented cheese samples. TAC of all treatments and control increased in the ripening phase. TAC of all treatments and control was dependent upon the antioxidant activity of lactoferrin, milk, and peptides having antioxidant activity. Earlier investigations have shown that during the ripening of cheddar cheese, certain peptides that increase the antioxidant activity of cheese are produced.^[39] Lactoferrin has antioxidant, antibacterial, immune regulation, and iron transport activities.^[40] Lactoferrin has many functional properties including iron transport, antibacterial effect, enhancement of cell growth, and promotion of the development of beneficial intestinal bacteria.^[41]

Table 4. Effect of Lactoferrin Supplementation on Antioxidant Characteristics of Cheddar Cheese.

Treatments	Ripening days	TAC%	DPPH%	ALA%
	0 45 90	20.91 ± 0.15^l 32.74 ± 0.28^g 45.97 ± 0.14^d	12.16 ± 0.06^j 13.54 ± 0.35^i 18.43 ± 0.32^g	9.79 ± 0.03^k 14.64 ± 0.08^g 12.33 ± 0.02^i
T2	0 45 90	22.98 ± 0.07^j 27.63 ± 0.17^h 47.68 ± 0.41^c	15.74 ± 0.09 20.56 ± 0.04^f 23.72 ± 0.03^e	13.19 ± 0.16^h 14.53 ± 0.06 17.87 ± 0.09^d
T3	0 45 90	24.07 ± 0.05^i 36.75 ± 0.12^f 65.37 ± 0.26^b	16.76 ± 0.11^h 25.79 ± 0.15^d 34.48 ± 0.21^b	14.55 ± 0.04^g 16.19 ± 0.10^e 22.36 ± 0.15^b
T4	0 45 90	27.33 ± 0.12^h 42.55 ± 0.16^e 78.82 ± 0.18^a	18.25 ± 0.17^g 29.35 ± 0.26^c 40.66 ± 0.43^a	15.77 ± 0.09^f 19.34 ± 0.05^c 29.36 ± 0.21^a
Control	0 45 90	21.59 ± 0.25^k 25.69 ± 0.36^i 32.47 ± 0.24^g	14.31 ± 0.17^h 15.22 ± 0.06 19.74 ± 0.013^g	11.29 ± 0.13^j 13.47 ± 0.16^h 15.92 ± 0.29^f

If in one column, means are expressed with a different letter, this indicates a significant difference (p < 0.05)

1,1-Diphenyl-2-picrylhydrazyl free radical scavenging activity

For the characterization of antioxidant status of milk and milk products, DPPH assay was used.^[42,43] For the characterization of antioxidant status of cheese, this method by Ullah et al. was used.^[44] In the current trial, DPPH assay was used to monitor the transition in antioxidant activity of lactoferrin-supplemented cheddar cheese. Lactoferrin at all four concentrations raised the DPPH of cheddar cheese. DPPH of control was lesser than that for all treatments (p < 0.05). DPPH of all treatments and control increased in 90-day ripening. After 90 days, DPPH of T₃ and T₄ was considerably higher the control (p < 0.05). DPPH of mature cheese was more than fresh cheese.^[45] Antioxidant capacity of cheese increased in the ripening phase.^[46] Cheddar cheese samples were supplemented with vitamin A at four different concentrations and ripened for the duration of 90 days. It was found that the antioxidant capacity of cheddar cheese increased in the ripening period.^[26] Out of 100% inhaled oxygen, univalent reduction of O₂ transformed only 5% or more into reactive oxygen species (ROS). These ROS can be treated using antioxidants such as vitamin C. Antioxidants are a very strong reducing agent; they have the ability to remove free oxygen, which is the main cause of oxidation. Antioxidants have the capacity to block the chain reaction of lipid peroxidation by attaching at the receptive sites to stop the production of free radical (OH) or by blocking the metallic actions. Nowadays, a new trend of using ROS in beneficiary direction is common. Molecular oxygen used by aerobic organism in their metallic processes receive electrons and generated ROS. These ROS help in killing harmful microbes and apoptosis in the defective cells of the body. They also play an important role in treating cancer, arteriosclerosis, and neuro-degenerative disorder by altering lipids, proteins, and DNA strains of the body.

Antioxidant activity in linoleic acid

Supplementation of cheddar cheese with lactoferrin increased the antioxidant activity in linoleic acid (ALA). ALA of the control was lesser than that of all treatments (p < 0.05). ALA of all the treatments and control increased in 90-day ripening (Table 2). After 45 days of ripening, DPPH free radical scavenging activity of T₃, T₄, and control was 16.19%, 19.34%, and 14.64%. In mature cheddar cheese (90 days old), ALA of T₃ and T₄ were considerably higher than the control (p < 0.05). Antioxidant characteristics of lactoferrin-supplemented cheddar cheese were strongly connected with PV. Cheese samples having strong antioxidant characteristics revealed lower PV. ALA of cheddar cheese escalated in ripening.^[18] Antioxidant capacity of cheese increased during the ripening phase.^[47]

Organic acids

During the ripening phase of cheddar cheese, due to the catabolism of lactose, fat, and microbiological activities, organic acids are produced. Organic acids play a significant role in the flavor of cheese.^[28] Flavor of the cheddar cheese is predominantly due to organic acids, methyl ketones, sulfur compounds, and alcohols.^[48] Organic acids also possess antibacterial features and play a vital role in the creation of multiple hurdles in cheese to stop the undesirable bacterial activities. Ripened cheeses are generally regarded as pathogen-free substrates due to limiting factors that make cheese a hostile environment for pathogenic and spoilage bacteria.^[49] In this investigation, the influence of lactoferrin on organic acids production was determined (Table 5). It was found that lactoferrin from 5 to 20 mg/100 g had a non-significant effect on organic acids production. However, the ripening period had a significant effect on the production of organic acids; all testing intervals indicated thatconcentrations of CA, LA, AA, and PA increased in all types of samples and control. In control after 90 days, concentrations of CA, LA, AA, and PA were 2392, 16,731, 132, and 166 mg/kg. In T4, after 90 days, concentrations of CA, LA, AA, and PA were 2389, 16,468, 126, and 141 mg/kg. LA is extremely important for the quality characteristics and the appropriate ripening of cheese.^[50] LA bacteria convert citrate to LA, acetate, acetaldehyde, and diacetyl; the last two significantly contribute to the flavor of cheese.^[51] AA is generated by the fermentation of lactate and citrate by microorganisms. AA highly contributes to the flavor of ripened cheese.^[52] Organic acids content of cheese prepared from vegetable fat blend and milk fat were different.^[53] No connection has been established between the production of organic acids and lactoferrin. No connection between sensory characteristics and concentrations of organic acids was also recorded in the current investigation. The addition of lactoferrin to yogurt had no effect on the production of LA.^[12] Concentrations of organic acids increased in long-term ripening of Parmigiano cheese.^[54,55] Murtaza et al.^[28] monitored the concentration of organic acids in buffalo milk-based cheese during the long-term ripening; it was found that the magnitude of organic acids escalated in the ripening Table 5.

Table 5. Effect of Lactoferrin Supplementation on Organic Acids of Cheddar Cheese.

Treatments	Ripening days	Citric acid mg/kg	Lactic acid mg/kg	Acetic acid mg/kg	Propionic acid mg/kg
T1	45 90	2187 ± 1.55^b 2315 ± 0.66^a	12,853 ± 5.32^b 16,523 ± 3.15^a	105 ± 0.45^b 137 ± 0.39^a	73 ± 0.51^b 169 ± 0.37^a
T2	45 90	2156 ± 1.36^b 2388 ± 0.82^a	12,114 ± 2.88^b 15,638 ± 2.41^a	98 ± 0.33^b 131 ± 0.74^a	52 ± 0.14^b 155 ± 0.11^a
T3	45 90	2138 ± 1.16^b 2351 ± 1.27^a	12,966 ± 1.67^b 16,289 ± 1.22^a	94 ± 0.54^b 125 ± 0.40^a	39 ± 0.08^b 158 ± 0.05^a
T4	45 90	2167 ± 0.42^b 2389 ± 0.36^a	12,255 ± 3.56^b 16,468 ± 3.31^a	98 ± 0.30^b 126 ± 0.19^a	24 ± 0.03^b 141 ± 0.06^a
Control	45 90	2174 ± 0.65^b 2392 ± 0.94^a	12,697 ± 2.79^b 16,731 ± 1.98^a	95 ± 0.72^b 132 ± 0.13^a	84 ± 0.08^b 166 ± 0.13^a

If in one column, means are expressed with a different letter, it indicates significant difference (p < 0.05)

Fatty acid profile

To know the oxidative quality and shelf stability of functional dairy products, it is extremely important to adjudge the effect of added functional ingredients in fresh and stored form. Milk fat is composed of diversified fatty acids such as short-chain (SCFA), medium-chain (MCFA), and long-chain fatty acids (LCFA). Each category of fatty acids confers specific characteristics to milk and dairy products. For example, SCFA contribute a typical dairy aroma to milk and dairy products, MCFA are extremely important for functional properties’ view points, and LCFA are important from health and oxidative stability perspectives.^[56] If any undesirable change is caused in the fatty acids profile of dairy products by a functional additive, processing technology, and storage phase, it can seriously affect the quality and acceptability of functional dairy products.^[57] Furthermore, lipid oxidation in dairy products can also be adjudged by examining the variation in fatty acids profile during the processing and storage conditions. For the measurement of oxidative stability of dairy products, transitions in fatty acids profile were used. In this investigation, the effect of supplementing cheese with four different concentrations of lactoferrin (5, 10, 15, and 20 mg/100 g) was determined on the fatty acid profile of cheddar cheese for the duration of 90 days. Lactoferrin supplementation at all concentrations did not affect fatty acids composition of all treatments (p > 0.05). All the determination frequencies revealed a non-significant effect of lactoferrin supplementation on fatty acids composition (p > 0.05). The storage phase of 90 days created significant changes in fatty acids composition of all treatments and control of lactoferrin-supplemented cheddar cheese (p < 0.05). At 0 day, concentrations of unsaturated fatty acids (oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3)) in cheese curd were 28.45%. After 90 days of storage concentrations of UFA in T₁, T₂, T₃, T₄, and control were 26%, 27.05%, 27.61%, 26.76%, and 26.47%, respectively. The non-significant impact of functional ingredients on fatty acids profile of cheese has been reported in literature. Fatty acids profile of selenium-enriched cheddar cheese was similar to cheese; however, storage period induced major changes in fatty acids profile. Fatty acid profile of mature cheddar cheese produced from cow and buffalo milk was considerably different from young cheese.^[18]

Lipolysis

Cheese usually does not endure oxidative breakdown due to lesser oxidation/reduction potential (approximately 250 mV).^[58,59] Lipids having a higher concentration of unsaturated fatty acids undergo rapid oxidative deterioration, the rate of oxidation in linolenic acid is about 100 times greater than oleic acid.^[56] Lactoferrin supplemented cheese may have different behaviors toward lipid oxidation; therefore, in this study the effect of lactoferrin supplementation of cheddar cheese was monitored after 45 and 90 days of storage. In addition to oxidative deterioration, cheese also undergo hydrolytic degradation. Indigenous, endogenous, and exogenous lipases may cause hydrolysis of triglycerides that lead to the formation of FFA in the ripening phase. Milk fat triglycerides are rich in short-chain fatty acids, after liberation from triglycerides, due to low flavor threshold add significant flavor to several ripened cheeses. Lipolysis plays an imperative part in the flavor of semi-hard and hard varieties of cheese, low degree of lipolysis is desirable in cheddar, Swiss cheese, etc., and higher degree of lipolysis is undesirable and lead to rancidity.^[60,61] Table 6 presents the results of lipolysis of lactoferrin-supplemented cheddar cheese. In the current investigation, the magnitude of lipolysis was assessed using FFAs, peroxide value (PV) and cholesterol (total) assays as key indicators. Lactoferrin at all the four concentrations did not affect generation of FFA. Concentration of FFA in milk fat was 0.08%, which was the reason for lower amount of FFA in freshly prepared cheese samples. In ripening, FFA increased throughout the ripening duration of 90 days. Batool et al.^[18] studied the generation of FFA in cow and buffalo cheddar cheese. Concentration of FFA in fully matured cheddar cheese was 9492 mg/100 g.^[62] Higher concentrations of FFA in foods may lead to the development of off-flavors and also escalate the auto-oxidation.^[63] PV is the most commonly used test for the valuation of lipid oxidation in oils and fats.^[64] PV is directly connected to the storage stability, and food systems with lower PV can be kept for a longer period of time.^[42] According to the allowable limits of European Union, PV in foods should be less than 10 (meqO₂/kg). PV of lactoferrin-supplemented treatments and control were less than the permissible limits of the European Union. After 45 days, T₃, T₄, and control, PV were 0.27, 0.29, and 0.26 (meqO₂/kg). After 90 days, T₃, T₄, and control, PV were 0.30, 0.31, and 0.28 (meqO₂/kg). The lower PV of T₃ and T₄ may be justified by the higher total antioxidant capacity than the control. Strong correlations were recorded for PV and TAC, cheese samples having higher TAC revealed lower peroxide (R₂ = 0.989). Flavor score was strongly correlated with PV; cheese samples showing higher flavor score had lower PV. PV of immitant processed cheese was within allowable limits.^[65] PV of dairy products generally increases with the progression of storage duration.^[66] In this investigation, moderate lipolysis took place that led to the development of balance sensory characteristics (Table 7). Addition of lactoferrin to cheddar cheese at all the four concentrations did not affect the concentration of cholesterol. Ripening had a pronounced impact on cholesterol; analysis at 45 and 90 days indicated a declining trend. The concentration of cholesterol in fermented milk products was less than that of the native milk, which shows the health importance of the fermented milk products.^[67–70]

Table 6. Effect of lactoferrin supplementation on fatty acid profile of cheddar cheese.

Fatty Acid	Control			T1		T2		T3		T4
	0 Day	45 Days	90 Days	45 Days	90 Days	45 Days	90 Days	45 Days	90 Days	45 Days	90 Days
C4:0	1.85 ± 0.04^a	1.84 ± 0.02^a	1.78 ± 0.03^b	1.77 ± 0.01^a	1.62 ± 0.02^b	1.75 ± 0.03^a	1.61 ± 0.05^b	1.68 ± 0.02^a	1.16 ± 0.03^b	1.74 ± 0.02^a	1.55 ± 0.07^b
C6:0	2.17 ± 0.02^a	2.15 ± 0.01^a	2.05 ± 0.06^b	2.10 ± 0.07^a	1.98 ± 0.02^b	1.80 ± 0.06^a	1.65 ± 0.02^b	1.64 ± 0.06^a	1.51 ± 0.06^b	1.74 ± 0.05^a	1.42 ± 0.04^b
C8:0	2.37 ± 0.06^a	2.34 ± 0.07^a	2.25 ± 0.11^b	2.31 ± 0.03^a	2.21 ± 0.05^b	1.97 ± 0.02^a	1.77 ± 0.07^b	1.76 ± 0.12^a	1.71 ± 0.07^b	1.80 ± 0.15^a	1.65 ± 0.03^b
C10:0	2.51 ± 0.01^a	2.49 ± 0.06^a	2.42 ± 0.15^b	2.44 ± 0.02^a	2.32 ± 0.09^b	2.04 ± 0.09^a	1.89 ± 0.01^b	1.88 ± 0.14^a	1.75 ± 0.09^b	1.84 ± 0.07^a	1.79 ± 0.11^b
C12:0	2.72 ± 0.07^a	2.71 ± 0.05^a	2.64 ± 0.04^b	2.69 ± 0.08^a	2.44 ± 0.10^b	2.23 ± 0.05^a	2.08 ± 0.014^b	2.13 ± 0.06^a	2.04 ± 0.13^b	2.14 ± 0.13^a	1.89 ± 0.08^b
C14:0	11.24 ± 0.14^a	11.21 ± 0.15^a	10.72 ± 0.26^b	10.43 ± 0.25^a	9.87 ± 0.13^b	9.63 ± 0.12^a	7.55 ± 0.27^b	7.19 ± 0.21^a	6.98 ± 0.16^b	9.39 ± 0.06^a	8.55 ± 0.19^b
C16:0	28.71 ± 0.10^a	28.65 ± 0.28^a	27.43 ± 0.34^b	27.67 ± 0.44^a	26.37 ± 0.20^b	28.37 ± 0.26^a	23.38 ± 0.34^b	23.45 ± 0.46^a	22.18 ± 0.35^b	27.63 ± 0.67^a	25.49 ± 0.39^b
C18:0	9.55 ± 0.17^a	9.48 ± 0.21^a	8.32 ± 0.24^b	9.22 ± 0.17^a	8.65 ± 0.33^b	9.18 ± 0.11^a	6.29 ± 0.53^b	8.17 ± 0.24^a	9.83 ± 0.14^b	8.75 ± 0.41^a	8.16 ± 0.16^b
C18:1	25.63 ± 0.22^a	25.51 ± 0.18^a	24.19 ± 0.36^b	24.95 ± 0.51^a	23.44 ± 0.29^b	24.94 ± 0.36^a	24.47 ± 0.87^b	24.72 ± 0.16^a	24.48 ± 0.05^b	24.53 ± 0.33^a	24.21 ± 0.28^b
C18:2	2.27 ± 0.13^a	2.21 ± 0.09^a	1.95 ± 0.02^b	2.19 ± 0.03^a	2.17 ± 0.02^b	2.18 ± 0.06^a	2.23 ± 0.73^b	2.21 ± 0.10^a	2.79 ± 0.05^b	2.25 ± 0.02^a	2.23 ± 0.05^b
C18:3	0.55 ± 0.01^a	0.52 ± 0.02^a	0.33 ± 0.01^b	0.51 ± 0.09^a	0.39 ± 0.06^b	0.48 ± 0.07^a	0.35 ± 0.04^b	0.49 ± 0.07^a	0.34 ± 0.07^b	0.46 ± 0.04^a	0.32 ± 0.14^b

If in one row, means are expressed with a different letter, it indicates significant difference (p < 0.05)

Table 7. Effect of Lactoferrin Supplementation on Lipolysis of Cheddar Cheese in Accelerated Ripening.

Treatments	Storage days	FFA %	PV (meq.O2/kg)	Cholesterol mg/100 g
T1	0 45 90	0.09 ± 0.01^i 0.11 ± 0.02^h 0.15 ± 0.01^f	0.25 ± 0.02 0.28 ± 0.04 0.31 ± 0.01	176.56 ± 0.11 168.43 ± 0.24 145.37 ± 0.16
T2	0 45 90	0.11 ± 0.02^h 0.12 ± 0.01^g 0.16 ± 0.02	0.22 ± 0.01 0.24 ± 0.02 0.28 ± 0.04	172.74 ± 0.15 160.39 ± 0.19 137.95 ± 0.35
T3	0 45 90	0.13 ± 0.01^g 0.16 ± 0.02^f 0.21 ± 0.01^e	0.24 ± 0.02 0.27 ± 0.01 0.30 ± 0.03	169.19 ± 0.19 153.64 ± 0.14 124.38 ± 0.16
T4	0 45 90	0.16 ± 0.01 0.19 ± 0.03^f 0.24 ± 0.01^d	0.26 ± 0.03 0.29 ± 0.01 0.31 ± 0.04	117.44 ± 0.08 107.43 ± 0.23 98.77 ± 0.27
Control	0 45 90	0.08 ± 0.02 0.13 ± 0.03^g 0.17 ± 0.02^f	0.24 ± 0.05 0.26 ± 0.02 0.28 ± 0.01	216.62 ± 0.05^a 205.42 ± 0.33^b 188.91 ± 0.36

If in one column, means are expressed with a different letter, it indicates significant difference (p < 0.05)

Sensory characteristics

Supplementation of cheddar cheese with lactoferrin at all four concentrations did not affect color score of all the treatments and control. Among all treatments and control, color score ranged from 8.1–8.5 (p > .05) as evaluated by a panel of ten trained judges (age 28–42 years). Sensory evaluation of lactoferrin supplemented cheese samples after 45 days indicated a non-significant variation in flavor score. Flavor score of control, T₁, and T₂ were 7.5, 7.6, and 7.5 (p > 0.05). However, after 45 days of ripening, flavor score of T₃ and T₄ were 7.9 and 8.1 (p < 0.05). After 45 days of ripening, sensory evaluation of cheese samples showed that texture score of T1 and T2 was significantly lower than control, T₃, and T₄, similar trend was also recorded when the texture score was evaluated by a panel of 10 trained judges after 90 days of storage (Table 8). Sensory properties of cheddar cheese significantly improved in the long-term ripening.^[55] The ripening of cheddar cheese considerably increased the ripening time; the desired sensory characteristics were achieved in this process.^[18] Sensory properties are important attributes of food commodities that give an insight into the public acceptance of the food commodities Table 8.^[71,72]

Table 8. Effect of Lactoferrin Supplementation on Sensory Evaluation of Cheddar Cheese.

Treatments	Ripening days	Color	Flavor	Texture
T1	45 90	8.1 ± 0.05^a 8.2 ± 0.02^a	7.5 ± 0.02^c 7.8 ± 0.03^b	7.4 ± 0.14^c 7.8 ± 0.19^b
T2	45 90	8.2 ± 0.06^a 8.4 ± 0.09^a	7.6 ± 0.06^c 7.8 ± 0.09^b	7.5 ± 0.17^c 7.6 ± 0.13^c
T3	45 90	8.3 ± 0.04^a 8.1 ± 0.12^a	7.9 ± 0.11^a 8.1 ± 0.14^a	7.8 ± 0.12^b 8.0 ± 0.09^a
T4	45 90	8.1 ± 0.03^a 8.0 ± 0.07^a	8.1 ± 0.016^a 8.3 ± 0.02^a	7.9 ± 0.05^a 8.1 ± 0.04^a
Control	45 90	8.4 ± 0.10^a 8.5 ± 0.13^a	7.5 ± 0.13^c 8.0 ± 0.15^a	8.1 ± 0.03^a 8.3 ± 0.06^a

If in one column, means are expressed with a different letter, it indicates significant difference (p < 0.05)

Conclusion

Lactoferrin-supplemented cheddar cheese had more antioxidant capacity than the control with no difference in fatty acids composition and organic acids contents. Color, flavor, and texture scores of lactoferrin-supplemented cheese were identical to the control. These results suggested that lactoferrin can be used to produce functional cheddar cheese.

Author’s contribution

Ali Adnan performed the methods and investigation. Muhammad Nadeem performed conceptualization, funding acquisition, and writing of original draft. Muhammad Imran helped in writing this manuscript. While Muhammad Haseeb Ahmad, Muhammad Tayyab, and Muhammad Kamran Khan helped with software, Aamir Iqbal, Muhammad Abdul Rahim, and Chinaza Godswill Awuchi supported in the analysis and supervision of research work.

Consent for publication

All authors agreed for publication of this manuscript.

Code availability

Not applicable.

Acknowledgments

The authors are highly obliged to the Library Department, University of Veterinary and Animal Sciences (UVAS), and IT Department, Higher Education Commission (HEC, Islamabad), for access to journals, books, and valuable database. The study was completed after utilizing the available resources in the Department and University. The authors are also thankful to Kampala International University, Kampala, Uganda, for collaborative support.

Data availability statement

The data supporting the conclusions of this article is included within the article.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

Funding for this work was provided by Higher Education Commission Pakistan under National Research Program for Universities (Grant # NRPU/6978).