INTRODUCTION:

Fungal infections, both caused by mold and yeast, are one of the common abandoned health problems without adequate therapy.¹ Fungal infections are a very broad-spectrum infection, starting from non-fatal infections affecting the mucocutaneous layer to invasive infections that infiltrate the internal organs. Mucocutaneous fungal infections are often found in babies’ oral cavity, vagina, skin fold, and traumatized skin. In addition, patients with diabetes mellitus are also prone to this disease.^2,3 Systemic fungal infections spread through the bloodstream to form microabscesses in organs.³ Studies estimated that 1.6 million people die from fungal infections annually.^1,4

In health care, one of the leading causes of death and morbidity is candidiasis. Candida species that belong to the yeast type Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei, and Candida parapsilosis can cause this infectious condition. Candida albicans is found extensively in humans, and also the most common etiology of candidiasis. On the other hand, C. krusei becomes one of the most challenging type of Candida species to treat, due to intrinsic resistance against fluconazole.^2,5 The mold family, which includes Aspergillus species, including A. flavus, A. fumigatus, A. niger, and A. terreus, can also cause fungal infections. Clinically, A. terreus can cause infections ranging from superficial infections such as onychomycosis to invasive aspergillosis in immunocompromised patients. A. terreus is frequently associated with disseminated infection and poses a significant role in mortality and morbidity.⁶

Recently, resistance to antifungal agents has become a challenge for health workers worldwide. Mostly, azole antifungal agents were used to manage fungal infections. However, its extensive use in various countries has provoked resistance to this antifungal. Fluconazole, as one of the azole antifungal agents, is often used practically because of its affordability (inexpensive), availability, and safety (rarely causes side effects). Along with C. albicans as the most common etiology of candidiasis, some C. albicans has developed resistance towards fluconazole.⁷ More than 15% of Candida non-albicans are resistant to fluconazole, especially C. glabrata and C. krusei.⁸ Therefore, voriconazole, which is the first-line therapy for invasive aspergillosis, has a 52.9% failure rate against A. terreus infection.⁹

Lactoferrin (Lf), a constituent of the transferrin family with a molecular weight of 80 kDa, is a basic protein that facilitates iron transport and executes multiple physiological activities. Lactoferrin demonstrates considerable promise in the therapeutic domain due to its unique antifungal, antibacterial, and anti-inflammatory properties. It has been employed as a ligand for the targeting of various nanocarriers.¹⁰ The organisms utilized in this investigation are predominantly implicated in various fungal diseases.¹¹ Lactoferrin is a glycoprotein (also known as "red protein") present in various types of milk such as human breast milk, cow's milk, goat's milk, and others. This glycoprotein are known to have antifungal and antibacterial effects. Several studies have also shown the antifungal effect of lactoferrin in milk, especially against C. albicans and C. krusei. The inhibition of Candida spp. growth by lactoferrin from human milk was reported to be fungistatic rather than fungicidal, as shown with the previous viability test.^12,13 Lactoferrin can reduce the growth rate of C. albicans and C. krusei by various mechanisms. The antifungal mechanisms possessed by lactoferrin include disrupting the conformation of the fungal cell walls, resulting in ultrastructural changes and cell injury. In addition, lactoferrin also shows antifungal potential by competing with iron ions, with C. albicans and C. krusei that require iron ions to survive.¹²

Lactoferrin’s antifungal activity has a dual mechanistic pathway. First, its iron-sequestering capability creates an iron-starved environment that is essential for fungal growth, resulting in a primarily fungistatic effect. Second, its positively charged domains interact electrostatically with the anionic components of the fungal cell wall or membrane. This electrostatic attraction disrupts membrane integrity, leading to increased permeability or cell death. These combined effects, particularly when lactoferrin is used alongside azoles or polyenes, may progress from fungistatic inhibition toward synergistic fungicidal activity.^14,15

The combination of lactoferrin with antifungal gold standard such as fluconazole and amphotericin-B was also reported to have synergistic effects against Candida species.¹³Other studies have also shown that lactoferrin can inhibit the growth of mold such as Aspergillus fumigatus.¹⁶ Moreover, lactoferrin exhibits antifungal activity against other fungal species such as C. glabrata, and Saccharomyces cerevisiae^..¹⁷

To our knowledge, there were no studies have examined and compared the antifungal effects of lactoferrin in breast milk, cow's milk, goat's milk, and infant formula milk with fluconazole against C. albicans and itraconazole against C. krusei and A. terreus by using disk diffusion and in vitro microdilution methods. Furthermore, the synergistic effect of lactoferrin and fluconazole on C. albicans and lactoferrin and itraconazole on C. krusei and A. terreus has not been studied. Therefore, we aim to investigate the antifungal and synergistic activity of these lactoferrins. This provides a new insight into finding the natural therapies for patients with fungal infections who require long-term therapy in order to avoid the side effects of antifungal drugs.

MATERIALS AND METHODS:

Isolation and Identification of Lactoferrin:

This study used purified lactoferrin from infant formula milk (fLf), goat's milk (gLf), cow's milk (bLf), and breast milk (hLf). The source of breast milk is a 39-year-old multiparous woman (P7A0) who has recently given birth, in the postpartum period (6 months after delivery)—is still breastfeeding her child, does not smoke, and does not drink alcohol. During anamnesis, the donor stated that she did not have any history of chronic illnesses or low milk supply. Before receiving the breast milk, the study's goal was explained verbally, and informed consent was acquired. For a maximum of three months, 50 mL of donor breast milk was placed in breast milk containers and kept in a freezer at -20 degrees Celsius. Milk from cows and goats that were purchased from East Jakarta livestock farms was kept in a freezer at -20°C for a maximum of three months. To minimize bias, bovine and goat’s milk were provided by reputable local farms where livestock were reported to be in good health and raised under standard feeding practices.¹⁸ Additionally, 50 mg of lactoferrin per 100 grams of milk was utilized in formula milk for infants ages 0 to 6 months. Before the lactoferrin separation process, frozen goat, cow, and breast milk were thawed by submerging the milk container in warm water. In the meantime, 50 mL of hot water at 70°C was used to make formula milk.

The modified Hassan Abdalla method was used to isolate and purify lactoferrin.¹⁹ The procedures involved include alkalizing the milk, exposing the solution to air, and adding organic solvents. Milk alkalization was carried out by mixing 50 mL of milk with 3 mL of 40% NaOH, resulting in 2.4 g/L NaOH concentration. For one night, the alkalized milk was left out in the open. Two layers will appear in the solution after 24 hours: a bottom, crimson layer and an upper, white layer that is fat from the saponification reaction. While the fat was being removed, the crimson layer was obtained. Whatman paper number 1 was then used to filter the red solution. The filtrated solutions were combined with acetone up to twice the filtration volume in order to precipitate the lactoferrin. The precipitate was repeatedly washed with acetone to completely precipitate the lactoferrin. By employing a low-temperature centrifugation in the Hassan Abdalla method and putting the precipitates into a vacuum desiccator jar for 24 hours, the process of acetone evaporation from lactoferrin was altered. After drying the following day, the lactoferrin precipitates were placed in a cuvette tube and preserved in the freezer at -20°C until they were needed.¹⁹

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to measure the protein molecular weight of lactoferrin isolates, and the Bradford test was used to determine if protein content was present or absent. The Bradford test involved vortexing 800 µL of material with 200 µL of Bradford reagent. When a blue hue is present in the solution, it indicates a positive outcome. The material was diluted four times for this test.^20,21

SDS-PAGE gel preparation, running buffer preparation, reducing sample buffer preparation, sample preparation, and SDS-PAGE running were among the processes that comprised the SDS-PAGE procedure. SDS-PAGE was done by using 13% separating gel and 5% stacking gel. The running buffer was prepared by mixing 3.1 g Tris BASE, 14.1 g glycine, and 1000 mL ultrapure water (Milli-Q, Merck). The reducing sample buffer (RSB) preparation was made with a mixture of 125 µL Tris-HCl 1M pH 6.8, 100 µL 50% glycerol, 200 µL SDS 10%, 50 µL 2-mercaptoethanol, 25 µL bromophenol blue, and 500 µL deionized water. A sample of 1 mg was added to 1 ml of ultrapure water (Milli-Q, Merck). Samples were processed to gain a final concentration of 6,975 mg/mL fLf, 75 mg/mL hLf, 63,375 mg/mL bLf, and 66.25 mg/mL gLf. Samples were made in various concentrations because of the samples’ thick consistency, which may cause disruption in the SDS-PAGE glycosylation band appearance. A sample of 20 µL was mixed with 5 µL of RSB and heated for 10 minutes. Ten microliters of the sample were inserted into the electrophoresis gel. Electrophoresis was carried out at 90 volts for 100 minutes. After that, the gel was taken and stained with 150 ml Coomassie Brilliant Blue R-250, 50 ml glacial acetic acid, and 300 ml ultrapure water for 90 minutes. The gel is then destained in boiling water until the background is clear. By examining the bands that form and comparing them to existing markers, one can determine the molecular weight of lactoferrin.²¹According to a previous study by Mahala et al. (2022), the molecular weight of lactoferrin was around 77-80 kDa.^10,22

Antifungal Assay:

The potential antifungal and synergistic activity of lactoferrin against C. albicans ATCC 90028 (susceptible to fluconazole), C. albicans ATCC 10231 (resistant to fluconazole), C. krusei (wild-type), and wild-type A. terreus were examined using the disk diffusion and microdilution method. Each species was sub-cultured on CHROMAgar (Oxoid, United Kingdom) to confirm C. albicans species showed a green colony and a pink colony for C. krusei. In contrast, A. terreus does not show any reliable color-based differentiation on CHROMAgar; therefore, we identify this fungi based on macroscopic and microscopic morphology, including cinnamon-brown pigmentation, biseriate conidial heads, and smooth-walled hyaline conidiophores. The disk diffusion method used refers to the Clinical Laboratory Standard Institute (CLSI) M44 with modifications. Sabouraud Dextrose Agar (SDA, Oxoid, United Kingdom) was used as a medium instead of Mueller-Hinton agar. A blank disk (Oxoid, United Kingdom) was used as a tool.²³ The microdilution method refers to CLSI M27 with modification. Briefly, we used Sabouraud Dextrose Broth (SDB, Oxoid, United Kingdom) rather than Mueller-Hinton broth as the medium, and a 96-well microplate (Biologix, USA) as the tool. The fungi can grow optimally on the Sabouraud Dextrose medium.²⁴

In this study, we used non-albicans which resistant to fluconazole. The microdilution test was carried out on all of the above species and strains to determine the equivalent dose of inhibition with the existing standard antifungal. The test for the synergistic activity of lactoferrin and antifungal was done by using the disk diffusion and microdilution method, resulting in a combined inhibition zone of lactoferrin and antifungal and fractional inhibitory concentration (FIC). Both disk diffusion and microdilution used different antifungals as positive controls for C. albicans, C. krusei, and A. terreus. Fluconazole was used as a positive control for C. albicans, and itraconazole was used as a positive control for C. krusei and A. terreus. This is due to the nature of C. krusei, which showed intrinsic resistance to fluconazole.^25,26 Meanwhile, the negative control used for the three species is distilled water. Inhibition zone and minimum inhibitory concentration (MIC) were then compared after incubation for 24 hours at 35^oC. Lactoferrin MIC results are expressed in the smallest inhibition % against fungal growth.

The potential synergistic activity between lactoferrin and the azole was examined using disk diffusion and microdilution methods, in which both methods were performed by combining fluconazole and lactoferrin against C. albicans, and itraconazole with lactoferrin against C. krusei and A. terreus. This combination is carried out in the disk diffusion test by dipping the antifungal disc into lactoferrin and then setting it on agar media that has been infected with Candida species. The addition of inhibition zone is documented and categorized into: 1) antagonist if there is a decrease in the zone of inhibition, 2) additive if there is an addition in the zone of inhibition but does not exceed the zone of antifungal plus lactoferrin inhibition, 3) synergistic if there is an increase in the zone of inhibition greater than the zone of antifungal plus lactoferrin inhibition.²⁷

In microdilution test, the result of potential synergistic activity is called fractional FIC. The FIC can be calculated using the following formula:

FIC Index = FIC (A) + FIC (B)

Where

MIC (A) in combination

FIC (A) = -----------------------------

MIC (A) clone

MIC (B) in combination

dan FIC (B) = -----------------------------

MIC (B) clone

Where FIC value ≤0.5 indicates synergistic activity, FIC >4 indicates antagonistic activity, and FIC 0.5 to 1 indicates additive activity, and FIC between 1 and 4 is categorized as indifference.²⁸ In the microdilution test, each well was filled with 50 µL of Sabouraud Dextrose Broth (SDB, Oxoid, United Kingdom). In the first column, 50 µL of lactoferrin and 50 µL of fluconazole (Hangzhou Hyper Chemicals Limited) against C. albicans, and itraconazole (Murli Krishna Pharma Private Ltd.) against C. krusei and A. terreus were added. After that, a two-fold dilution was performed from the first column to the 10^th column. 50 µL Candida spp were added into column 1 to column 11. Column 11, which does not contain lactoferrin and antifungal, acts as a negative control, and column 12 acts as a parameter if the microdilution process is contaminated. Meanwhile for positive control, a separate microdilution test was performed to determine the MIC of the tested antifungal. The result of the lactoferrin MIC was the percent concentration of total solution in the well. Meanwhile, the antifungal MIC was the minimum antifungal concentration (µg) contained in 1 whole well solution.

Descriptive statistics were used to summarize the data. All inhibition zone diameters and MIC values were measured in triplicate, and results are presented as mean values (mm or %). This descriptive approach allowed comparison between groups and highlighted the differences in antifungal compounds and their synergistic activity, while avoiding over-interpretation of the findings.

RESULT:

A red paste-lactoferrin isolate was easily dissolved in water (Figure 1). Blue color in protein identification test by using the Bradford method (Figure 2) indicates positive results. These indicate the presence of protein in infant formula milk, goat's milk, cow's milk, and breast milk.

Figure 1. Lactoferrin in the form of red paste.

Figure 2. Lactoferrin Bradford Assay.

The molecular weight of the proteins in each sample is shown by a band that lies between the 72 and 95 kDa marker bands, according to SDS-PAGE analysis. The data above clearly show that lactoferrin was present in the isolate (Figure 3).

Figure 3. Lactoferrin SDS-PAGE.

Our study demonstrated that lactoferrin from cow’s milk had the strongest antifungal activity in disk diffusion, as shown with the largest inhibition zones against C. albicans, C. krusei, and A. terreus. In contrast, human lactoferrin showed the lowest MIC values in the microdilution assay, indicating superior inhibitory effect at lower concentrations. In terms of synergistic activity, human lactoferrin exhibited the strongest synergy with fluconazole against resistant C. albicans (ATCC 10231), while bLf provided the strongest additive effect when combined with itraconazole against C. krusei. Goat lactoferrin demonstrated intermediate activity. While the formula milk lactoferrin consistently produced the weakest antifungal and synergistic effects.

Lactoferrin isolated from breast milk, cow's milk, goat's milk, and formula milk in the disk diffusion test showed antifungal activity (Table 1). The largest zone of inhibition in C. albicans ATCC 90028, C. albicans ATCC 10231, and wild-type C. krusei produced by cow's milk lactoferrin is 32 mm, 23 mm, and 34.33 mm, respectively. Meanwhile, lactoferrin in formula milk created the smallest zone of inhibition. The antifungal potential of cow's milk lactoferrin and breast milk was greater than fluconazole against fluconazole sensitive-C. albicans (bLf = 32 mm; hLf = 30 mm; fluconazole = 28 mm). In addition, cow's milk lactoferrin also provides a greater zone of inhibition than itraconazole against C. krusei (bLf = 34.44 mm; itraconazole = 31 mm). In fluconazole-resistant C. albicans, cow's milk lactoferrin and goat's milk showed a potential inhibitory equivalent to fluconazole. The addition of lactoferrin to the fluconazole disc against C. albicans ATCC 90028 gave additional zones of 32% (hLf), 39% (bLf), 30% (gLf), and 24% (fLf). In C. albicans ATCC 10231, there were additional zones of 29% (hLf), 34% (bLf), 31% (gLf), and 20% (fLf). In wild-type C. krusei, the addition of lactoferrin to the itraconazole disk resulted in additions of 10% (hLf), 20% (bLf), 15% (gLf), and 4% (fLf). These results indicate that lactoferrin could increase the antifungal (additive) activity of fluconazole and itraconazole.

Table 1. Candida spp. inhibition zone in disk diffusion

Antifungal	*Inhibition zone in Candida albicans* ATCC 90028 (mm)**					*Inhibition zone in Candida albicans* ATCC 10231 (mm)**						*Inhibition zone in Candida krusei* wild-type (mm)**
Antifungal	I	II	III	Mean	Note	I	II	III	Mean	Note	I		II	III	Mean	Note
Azole	28	-	-	28	-	23	-	-	23	-	31		-	-	31	-
Aquadest	0	0	0	0	-	0	0	0	0	-	0		0	0	0	-
hLf	30	28	32	30	-	22	20	21	21	-	25		23	22	23.33	-
Azole + hLf	43	42	40	41.67	Additive	31	32	34	32.33	Additive	34		35	36	35	Additive
bLf	33	33	30	32	-	25	23	21	23	-	35		34	34	34.33	-
Azole + bLf	47	45	46	46	Additive	35	36	34	35	Additive	39		38	39	38.67	Additive
gLf	21	18	22	20.33	-	25	21	23	23	-	26		25	27	26	-
Azole + gLf	42	39	39	40	Additive	33	34	33	33.33	Additive	35		37	38	36.67	Additive
fLf	25	27	23	25	-	17	17	16	16.67	-	21		20	21	20.67	-
Azole + fLf	37	38	35	36.67	Additive	29	28	29	28.67	Additive	32		33	32	32.33	Additive

hLf: human lactoferrin; bLf: bovine lactoferrin; gLf: goat lactoferrin; fLf: formula milk lactoferrin

In the microdilution test, lactoferrin also showed antifungal activity (Table 2). Breast milk lactoferrin provided the smallest MICs in wild-type C. krusei, C. albicans ATCC 90028, and C. albicans ATCC 10231. hLf demonstrated the best activity against the three fungal strains, with MICs of 0.78% for wild-type Candida krusei, 0.39% for C. albicans ATCC 90028, and 1.56% for C. albicans ATCC 10231. In combination test (table 4), there was synergistic activity between fluconazole and lactoferrin against C. albicans ATCC 10231. Meanwhile, lactoferrin also increased (additive) the antifungal effect of fluconazole in combination test between fluconazole and lactoferrin against C. albicans ATCC 90028. This is also shown in the combination test results between itraconazole and lactoferrin against wild-type C. krusei.

Table 2. Candida spp. minimum inhibitory concentration in microdilution

Antifungal	*Minimum inhibitory concentration*
Antifungal	*Candida albicans* ATCC 90028	*Candida albicans* ATCC 10231	*Candida krusei* *(wild-type)*
Fluconazole	1 µg/mL	32 µg/mL	-
Itraconazole	-	-	0.125 µg/mL
hLf	0.39%	1.56%	0.78%
bLf	0.39%	3.13%	3.13%
gLf	0.78%	3.13%	3.13%
fLf	0.78%	3.13%	3.13%

hLf: human lactoferrin; bLf: bovine lactoferrin; gLf: goat lactoferrin; fLf: formula milk lactoferrin; MIC: minimum inhibitory concentration

In the disk diffusion test, lactoferrin that was separated from formula, goat's milk, cow's milk, and breast milk exhibited antifungal activity against A. terreus (table 3). With an average inhibition diameter of 24.67 mm, bLf generated the biggest zone of inhibition for A. terreus. On the other hand, fLf generated the lowest zone of inhibition, with an average inhibition diameter of 21 mm. Lactoferrin also shown antifungal efficacy in the microdilution test (Table 2). hLf produces the highest MIC against A. terreus.

Table 3. Aspergillus terreus inhibition zone in disk diffusion

Lactoferrin	*Aspergillus terreus* wild-type
	Inhibition zone (mm)			Mean	MIC
	I	II	III	Mean	MIC
Itraconazole	35	33	34	34	1 µg/mL
Aquadest	0	0	0	0	0 µg/mL
hLf	23	22	25	23.33	1.5625%
bLf	28	24	22	24.67	0.78125%
gLf	22	24	22	22.67	0.78125%
fLf	21	20	22	21	0.78125%

hLf – human lactoferrin, bLf – bovine lactoferrin, gLf – goat lactoferrin, fLf – formula milk lactoferrin

In the synergistic activity test, the results varied between Candida species (Table 1 and Table 4). Breast milk lactoferrin and fluconazole demonstrated the strongest synergistic efficacy against C. albicans ATCC 10231, with a range of 0.127 to 0.129. However, there was a discrepancy in the synergistic test utilizing the disk diffusion method, where lactoferrin produced additive results. Also, a superior result was seen in cow's milk lactoferrin. Both disk diffusion and microdilution methods gave varying results from additives to indifference against C. albicans ATCC 90028 and wild-type C. krusei, where cow's milk lactoferrin was the most superior. In all synergistic tests against C. albicans ATCC 90028, C. albicans 10231, and wild-type C. krusei, formula milk lactoferrin produced the lowest inhibitory activity.

In combination test, there was an additive effect between itraconazole and hLf against wild-type A. terreus. Meanwhile, the combination test between itraconazole with bLf and gLf showed indifference results. However, fLf with antagonist action displayed the lowest combination test results (table 5).

Table 4. Candida spp. fractional inhibitory concentration in microdilution

Lactoferrin	Combination	*Candida albicans* ATCC 90028			*Candida albicans* ATCC 10231			*Candida krusei* Wild-Type
		MIC	FIC	Note	MIC	FIC	Note	MIC	FIC	Note
Human	Fluconazole	0.5 µg/mL	2.5	Indifference	0.125 µg/mL	0.129	Synergistic	0.0625 µg/mL	1	Additive
	hLf	0.78125%			0.1953125%			0.390625%
Bovine	Fluconazole	0.5 µg/mL	2.5	Indifference	0.25 µg/mL	0.127	Synergistic	0.0625 µg/mL	0.624	Additive
	bLf	0.78125%			0.390625%			0.390625%
Goat	Fluconazole	0.5 µg/mL	1.5	Indifference	0.25 µg/mL	0.127	Synergistic	0.125 µg/mL	1.249	Indifference
	gLf	0.78125%			0.390625%			0.78125%
Formula	Fluconazole	0.5 µg/mL	1.5	Indifference	0.25	0.127	Synergistic	0.125 µg/mL	1.249	Indifference
	fLf	0.78125%			0.390625%			0.78125%

hLf: human lactoferrin; bLf: bovine lactoferrin; gLf: goat lactoferrin; fLf: formula milk lactoferrin; MIC: minimum inhibitory concentration; FIC: fractional inhibitory concentration

Table 5. Aspergillus terreus fractional inhibitory concentration in microdilution

Lactoferrin (Lf)	*Aspergillus terreus wild-type*
	Minimum Inhibitory Concentration (MIC) with Azole Combination		FIC	Note
Human	Itraconazole	0.125 µg/mL	0.625	Additive
	hLf	0.78125%
Bovine	Itraconazole	0.125 µg/mL	1.125	Indifference
	bLf	0.78125%
Goat	Itraconazole	0.25 µg/mL	2.25	Indifference
	gLf	1.5625%
Formula	Itraconazole	0.5 µg/mL	4.5	Antagonist
	fLf	3.125%

hLf: human lactoferrin; bLf: bovine lactoferrin; gLf: goat lactoferrin; fLf: formula milk lactoferrin; MIC: minimum inhibitory concentration; FIC: fractional inhibitory concentration

DISCUSSION:

Our study showed that lactoferrin derived from different types of milk sources could exhibit different antifungal effects against C. albicans, C. krusei, and A. terreus. Among all of the tested lactoferrins, bovine lactoferrin showed the strongest antifungal activity. Meanwhile, human lactoferrin had the best synergistic effect with fluconazole against resistant C. albicans. Goat lactoferrin demonstrated moderate antifungal activity, while infant formula milk lactoferrin produced the weakest antifungal and synergistic effects. These results indicate that the antifungal activity of lactoferrin is source-dependent, with bovine and human lactoferrin showing the greatest potential as therapeutic adjuvants. Despite employing the same test substance and method, this experimental study produced varied outcomes. These findings suggest that the test type, assay media composition, and isolate resistance profile all had an impact on antifungal potential inhibition. This may also explain why some possible antifungals may produce different outcomes in in vitro research compared to in vivo and clinical trials.²⁹

In studies using hLf, it appeared that the inhibitory effect varies depending on the species and strain used. The MIC results of C. krusei were better than C. albicans ATCC 10231, but hLf showed synergistic activity with fluconazole against C. albicans ATCC 12031. This is similar with Valenti, et al. which showed that hLf can inhibit C. albicans better than C. krusei. In his study, lactoferrin exposed to the surface of Candida cells and inhibited the growth of C. albicans, while C. krusei did not.³⁰ Another explanation is that C. krusei has now changed its name to Pichia kudriavzevii, which is phylogenetically located in a different clade to the Candida genus. Fungi in different genera such as Pichia sp. and Aspergillus sp. gives a different response to the combination of antifungal and lactoferrin, but the location of this difference still needs further investigation.

Lactoferrin isolated from cow's milk gave the best results compared to hLf, gLf, and fLf. This might be due to the structural differences between bLf and the other three types of lactoferrin. There are two types of N-glycans found in bLf but not in gLf or hLf. The glycosylation process can protect proteins from pathogenic proteases so that the antimicrobial effectiveness of lactoferrin does not decrease. Therefore, a decrease in the glycosylation process in lactoferrin could make these proteoglycans susceptible to protease digestion. gLf gave similar results to hLf in most tests. This may be due to the similarity in the structure of lactoferrin and the significant level of homology of lactoferrin N-glycans between gLf and hLf, related as previously mentioned, that lactoferrin is a proteoglycan.³¹ Several mechanisms of fungal growth inhibition by lactoferrin make this glycoprotein a promising component for a therapeutic combination. The synergistic activity of this antifungal combination was shown when the antifungal used has a different mechanism of action.^15,32

Lactoferrin from breast milk, cow's milk, goat's milk, and formula milk showed synergistic activity in the microdilution test against C. albicans, which had resistance to fluconazole, where hLf was the most superior (FIC = 0.129; MIC hLf = 0.1953125%; MIC of fluconazole = 0.125 µg / mL). This result is similar with Lupetti, et al. where fluconazole resistant-C. albicans can be inhibited by using a combination of breast milk lactoferrin and fluconazole. In the fluconazole alone, there was no visible disruption of the membrane, while the combination of fluconazole and hLf showed a disruption of fungal membrane integrity.³³ Another study suggested that the synergistic effect of lactoferrin was not due to changes in fluconazole uptake.³⁴ In a study by Lupetti, et al., lactoferrin was highly active against C. albicans, which was resistant to fluconazole at non-candidacidal concentrations, and showed synergistic activity with fluconazole against C. albicans and C. krusei. Candidacidal activity is initiated by these peptides, while fluconazole is required during the effector phase.³³

Although hLf gives inferior results to A. terreus, however, when combined with itraconazole, hLf can give an additive effect, whereas other lactoferrin gives indifferent (bLf; gLf), even antagonist (fLf) effects. Previous studies by using a combination of iron chelators with several antifungals against A. fumigatus found a synergistic relationship between lactoferrin and amphotericin B. Also, an antagonistic relationship between lactoferrin and azoles was present.³⁵ Synergistic activity of lactoferrin and azole is still being investigated, data obtained from radiolabeled fluconazole assays indicate that the presence of lactoferrin does not affect the intracellular concentration of azoles, so it is hypothesized that this synergistic relationship is not caused by alteration of intracellular uptake. The addition of exogenous iron is also reported to affect the action of lactoferrin in combination and disrupt the synergistic activity, so it is hypothesized that this synergistic mechanism of action is influenced by the function of lactoferrin as an iron chelator. In addition, lactoferrin which experiences direct interaction on the surface of fungal cells, can cause damage and leakage from the cell membrane.^1,35

In this study, lactoferrin also showed better inhibitory potential against fluconazole susceptible-C. albicans ATCC 90028 compared to other tested subjects. This is thought to be related to the surface of the fungal cell walls and the mechanism of lactoferrin antifungal activity, which attacks the proteins contained in the fungal cell membrane. Study by Nogueira et al. using the flow-cytometry method showed that C. albicans and C. krusei have different cell wall structure compensatory activities under stressful conditions. In C. albicans, due to antifungal administration, there was a significant increase in mannans. However, in C krusei, there was a very high increase in chitin.³⁶

The difference in the results of the inhibition test and the synergistic activity of lactoferrin between Candida species is thought to be related to differences in cell wall structure changes between Candida species and the lactoferrin antifungal mechanism. A study states that the antifungal mechanism of this cationic peptide is thought to be through adhesion to the negatively charged plasma membrane, which causes the formation of a transmembrane pore-like structure or ion channel-like structure. In this case, permeabilization of the cell membrane causes leakage of intracellular cell organelles, which results in cell death. However, there has been no further research on their relationship³⁵. The variations in the antifungal effects of the species are unsurprising, as they may result from changes in phytochemical features among the species.³⁷

The disc diffusion test result and the microdilution test result in this study are believed to differ due to a number of technical factors.^38,39 The antifungal potential of different samples could give varying test results; usually, this is due to differences in the physical properties of a substance, for example, solubility, volatility, and the diffusion coefficient in agar. A substance with a high diffusion coefficient or solubility can maximally penetrate, so that even though the substance has a low antifungal activity in a small amount, it can give the appearance of a zone of inhibition similar to a substance with high antifungal activity and high penetration activity. Furthermore, the amount of the test material that can be appropriately absorbed into the disc affects the zone of inhibition in the diffusion disc test.^40,41The difference between the two methods left many puzzles among researchers in determining the conclusions of the experiments. Therefore, the standardized microdilution method remains superior to disk diffusion.⁴² Apart from differences in the absorption of a substance into the product in disk diffusion test, the microdilution method is more sensitive for susceptibility testing because it can determine MIC, which is the lowest concentration required to inhibit fungal growth, that crucial for developing antifungal drugs.^43–46

Our study is the first to compare and analyze the lactoferrin derived from various milk sources (human, bovine, goat, and infant formula milk) against both azole-susceptible and azole-resistant C. albicans, C. krusei, and. A. terreus. Using two different antifungal susceptibility test (disk diffusion and microdilution), our study provides a more complete picture of antifungal activity that resulted in inhibitory zones and MIC values. In addition, the inclusion of synergistic testing with fluconazole and itraconazole highlights the potential of lactoferrin as an adjuvant to existing antifungal therapies. Our study also has several limitations. First, due to in vitro study design, our study may not fully reflect clinical outcomes. Second, even though we have designed the donor and livestock selection criteria, we did not perform biochemical analysis of milk quality after storage to minimize bias. Future studies with larger sample sizes, multiple donors, and in vivo models will be important to confirm these findings in clinical settings.

CONCLUSION:

Lactoferrin isolated from breast milk, cow's milk, goat's milk, and formula showed antifungal activity in disk diffusion and microdilution methods against fluconazole-sensitive C. albicans, fluconazole-resistant C. albicans, C. krusei, and A. terreus. Bovine and human lactoferrin showed the strongest inhibition compared with other milk lactoferrin, with bovine lactoferrin exceeding fluconazole against fluconazole-sensitive C. albicans and itraconazole against C. krusei. Human lactoferrin exhibited the lowest MICs across species and synergized with fluconazole against resistant C. albicans. These findings suggest that specific milk lactoferrin, particularly bovine and human, may serve as promising antifungal adjuvants.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this research.

ACKNOWLEDGMENTS:

We would like to thank the Faculty of Medicine and Health Sciences, University of Atma Jaya for facilitating this study and dr. Irene, SpA who has provided a lot of input and guidance in the completion of this study. We are also grateful to the Indonesian Institute of Sciences (LIPI), laboratory assistants (Linda Widyawati A.Md. Keb., Ariska Verri Marantika, Ida Afiyah, Sri Handayani, M. Yashir A.Md. Kes., Agus Siswanto), and photographers (Davin Alviandi, Bachelor of Business), as well as other colleagues who has devoted their time to this study.

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Received on 30.11.2024 Revised on 20.06.2025

Accepted on 10.10.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):223-232.

DOI: 10.52711/0974-360X.2026.00032

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