Special Issue:

Veterinary Medicine between Sustainable Development and Public Health to Confront Global Changes

Evaluation of Spirulina platensis and Propolis as Natural Promising Preservatives for Controlling of Candida albicans in Minced Meat

Elham M. Hosny1*, Saad Mahmoud Saad1, Marionette Zaghloul Nassif2, Amani Mohamed Salem1

1Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Benha University, Qaluobia 13736, Egypt; 2Food Hygiene Department, Animal Health Research Institute (AHRI), Benha branch. Agriculture Research Center (ARC), Egypt.

Received | June 07, 2024; Accepted | August 04, 2024; Published | August 31, 2024

*Correspondence | Elham M. Hosny, Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Benha University, Qaluobia 13736, Egypt; Email: [email protected]

Citation | Hosny EM, Saad SM, Nassif MZ, Salem AM (2024). Evaluation of Spirulina platensis and propolis as natural promising preservatives for controlling of Candida albicans in minced meat. Adv. Anim. Vet. Sci. 12(s1): 67-74.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.s1.67.74

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Recently, there has been a growing emphasis on improving food safety, leading to a rise in the use of natural preservatives that include antibacterial properties. Concurrently, there is an increasing inclination towards natural foods and ingredients, which are commonly perceived as being safer, healthier, and less susceptible to risks compared to those containing artificial additives. This is especially true for meat products, which are particularly susceptible to the growth of microorganisms that can cause spoilage and foodborne illnesses in humans (Abd El-Malek, 2017).

Yeast is a ubiquitous organism found throughout nature. Yeasts contribute positively to the fermentation process of numerous substances, such as wine and beer. However, they are also responsible for causing food spoilage. In addition, specific yeast species, such as Candida spp., can infiltrate the human body through the consumption of food and beverages, leading to a range of disorders (Riesute et al., 2021).

Among the yeast species representing a health issue is Candida species which are frequently occurring fungal pathogens that exploit opportunities in human hosts. Candida albicans (C. albicans) is the most widespread pathogen responsible for both mucosal and systemic fungal infections (Poulain, 2015). The pathogenicity of the C. albicans significantly contributes to host cell invasion and damage (Höfs et al., 2016).

Spirulina is a type of blue-green microalgae that has a spiral cellular structure. It belongs to the Arthrospira and Spirulina genera. Spirulina is classified as a “superfood” due to its composition, which consists of 55-70% protein and amino acids such as leucine, valine, isoleucine, tryptophan, phenylalanine, threonine, methionine, lysine, and gamma-linolenic acid (36% total PUFAs). It also contains glycolipids, sulfolipids, carbohydrates like mannose, glucose, galactose, xylose, and rhamnose, carotenoid (4000 mg/kg), 1.5–2% vitamins A, B, and E, minerals such as iron, calcium, manganese, zinc, magnesium, selenium, and potassium, as well as pigments like chlorophyll, carotenoids, xanthophylls, and the blue pigment known as phycocyanin (Pyne et al., 2017). Spirulina platensis is one of the algae that the FDA has categorized as generally regarded as safe (GRAS). The FDA classified Spirulina platensis as generally regarded as safe (GRAS) in 2017. However, the antifungal activities of Spirulina using meat products as substrates had received less attention.

Propolis is a mixture of pollen, waxes, essential oils, balsam, resins, and other substances, including enzymes and compounds related to bee metabolism. Propolis is composed of around 300 distinct chemical compounds, including flavonoids, phenolic acids, cinnamic acid, caffeic acid, terpenes, and esters. The variances in propolis originate due to differences in the plant source as well as the time and location of its collection. Ethanol is the predominant and widely employed solvent for extracting propolis. Propolis has antifungal properties because it can effectively combat many types of fungi by acting as both a fungicidal and fungistatic agent. Propolis possesses antioxidant, antibacterial, and antifungal properties, which make it an attractive option for use as a natural preservative in creative culinary uses (Shehu et al., 2016).

The antibacterial activities of propolis are attributed to its high amounts of phenolics, namely derivatives such as pinobanksin and pinocembrin. The antibacterial properties of propolis have been linked to a bioactive compound called caffeic acid phenyl ester (Gavanji et al., 2014). However, the use of propolis in the elimination of yeast contamination in meat and meat products is less investigated.

In sight of the previous information, this study was conducted to examine the impact of Spirulina platensis and propolis ethanolic extracts against the growth of C. albicans in minced meat and to improve the quality of the meat and extend its shelf life.

MATERIALS AND METHODS

The Institutional Animal Care and Use Committee of the Faculty of Veterinary Medicine at Benha University approved this research (approved number BUFVTM 03-12-23).

Sampling

Precisely, a total of 2.4 kg of beef flesh (with each piece weighing 200g) was bought from a butcher shop. The meat was promptly minced and transferred to the laboratory in pre-cooled insulated containers with ice packs, ensuring sterile conditions. Samples were grouped and inoculated with C. albicans without delay. The Spirulina platensis and propolis were acquired from the National Research Center in Dokki, Cairo, Egypt.

Preparation of Extracts

Spirulina platensis ethanolic extract (SEE): A quantity of 25g of algae powder was combined with 250mL at 1:10 ratio of 96% ethanol and placed in a Soxhlet device at a temperature of 60°C for duration of 24 hours in order to extract the active chemicals from the algae. The obtained extract was passed through a filter paper of Whatman No.1 and subsequently subjected to evaporation using a rotary vacuum evaporator (EYELA Auto jack NAJ-100, Tokyo, Japan) at a temperature of 45°C. Ultimately, the extract underwent a process of dehydration in a vacuum-drying oven at a temperature of 55°C for duration of 48 hours, as described by Saranya et al. (2014).

Propolis ethanolic extract (PEE): The 30g of raw propolis sample was crushed and dissolved in 100mL at 1:10 ratio of ethanol: water (80:20 v/v) while being stirred continuously at a temperature of 50°C for duration of 30 minutes. The suspension was passed through a Whatman no. 1 filter paper and then subjected to centrifugation at a speed of 600×g for duration of 20 minutes. The supernatant was concentrated using a rotary evaporator at 40°C under decreased pressure for three hours. The propolis powder was prepared through freeze-drying (Jonaidi Jafari et al., 2018).

In vitro Study

Minimum inhibitory concentration (MIC): The antifungal properties of extracts from Spirulina platensis and propolis were assessed using the broth microdilution assay in a 96-well microdilution plate, using 5 concentrations of the two tested antimicrobials starting from the 0 concentration as a negative control and using 64 µg/mL of fluconazole as a positive control for antifungals against C. albicans. The used concentrations of SEE were 0, 0.25, 0.5, 1, 2 mg/mL and for PEE were 0, 1.5, 3, 6, 12 mg/mL. The M27-A3 technique established by the Clinical Laboratory Standards Institute (CLSI, 2008) was followed.

Preparation of Candida albicans: Candida albicans ATCC10231 (were sub-cultured once into Sabouraud’s dextrose agar (SAD) and incubated for 24h at 37°C. Five colonies were transferred to sterile distilled water to make inocula (5 mL). After fungal growth, the cells were collected by centrifuging the conical tubes at 1,200 x g for 5 min at room temperature. Discard the supernatant and add 10 mL of PBS. Resuspend the cells and centrifuge again at 1,200 x g for 5 min at room temperature. Repeat the PBS wash and centrifugation twice more. After the third wash, resuspend the cells in 5 mL (according to the pellet size) of 2X RPMI-1640 medium. To ensure homogeneity, the suspensions were mixed for 15 seconds then adjust cell density with a spectrophotometer by adding sufficient sterile saline or sterile water to match the turbidity of a 0.5 McFarland standard.

Preparation of Spirulina platensis extract: The ethanolic extract of Spirulina platensis was dissolved in dimethyl sulfoxide (DMSO) and then weakened in peptone water using a two-fold serial dilution method. The dilution concentrations ranged from 2 mg/mL to 0.0675 mg/mL. El-Baz et al. (2013) reported that the non-toxic dose of the ethanolic extract of Spirulina platensis varied between 1.6 and 2 mg/ml.

Preparation of propolis extract: The ethanolic extract of propolis was dissolved in dimethyl sulfoxide (DMSO), and then diluted in peptone water using a serial two-fold dilution method. The concentrations ranged from 12 mg/mL to 0.1875 mg/mL.

About 100 μL of the inoculum was put into each well of the 96-well sterile culture plates. The inoculum contained varied quantities of Spirulina platensis and propolis extracts. Using 100 μL of Candida albicans, a negative control was established with 100 μL per well of DMSO, while a positive control was set up with 100 μL per well of Candida albicans. The plates were thereafter placed in an incubator set at a temperature of 37 °C for duration of 48 hours. Regarding the proliferation of Candida albicans. The entire technique was replicated three times. We found the minimum inhibitory concentration (MIC), which is the lowest concentration that stopped all fungal growth. This was done by adding propolis and Spirulina platensis to Sabouraud’s Dextrose Agar (SDA) as antifungal agents and leaving it to grow at 37 °C for 48 hours.

In vivo Study

Preparation of inoculums: The preparation of inoculums involved cultivating a strain of Candida albicans ATCC10231 to obtain a stock culture with a population of approximately 109, as reported by Krifors et al. (2023). This stock culture was then artificially introduced into all meat samples that were being examined, except for one (referred to as the blank sample), resulting in a final concentration of 105 as specified by the APHA (1985). Following inoculation, the first group (not inoculated) received no treatment (control 1), while the second group (control 2) also received no treatment. The third and fourth groups were thoroughly mixed with SPP at a concentration of 0.5 mg/ml and PEE at a concentration of 6 mg/ml (both dissolved in DMSO), respectively. Treatment groups were preserved at 4°C for 12 days and examined on days 0, 3, 6, 9, and 12 (n = 4 for each examination point).

Preparation of samples: Each sample, weighing twenty-five grams, was thoroughly mixed with 225mL of 1% peptone water using a stomacher for duration of 1 minute. Subsequently, serial dilutions were made. Multiple replicates of one milliliter of selected dilutions were placed on petri dishes, and Sabauroud’s dextrose agar was poured onto the dishes. The plates were then incubated at a temperature of 30 °C for a period of 3–5 days. By following the steps outlined by Cruickshank et al. (1975), we were able to count the number of Candida albicans colonies per gram after 0, 3, 6, 9, and 12 days of storage at 4 °C. The experiment was performed in triplicate.

Sensory evaluation (Horwitz, 2020): Personnel at the Animal Health Research Institute, specifically the Benha branch, assessed the samples. Every participant was required to evaluate the degrees of odor, color, consistency (firmness or succulence), and visual aspect on days 0, 3, 6, 9, and 12 of refrigeration. The representative samples were assigned random numbers, and the judges were kept ignorant of the experimental method. Samples of the various treatments were presented in small porcelain dishes that were covered. Each judge was given their own separate area where distractions, noises, and odors were minimized. They were then asked to score each sample based on its overall acceptance, taking into account factors such as color, odor, texture, and appearance. Overall acceptability is assessed using a nine-point descriptive scale. The scale ranges from 9 (excellent) to 8 (very good), 7 (very good), 6 (good), 5 (medium), and S (spoiled).

Statistical Analysis

The collected data were subjected to analysis of variance (one-way ANOVA) using SPSS software, following the method described by Sabine and Brian (2014). A one-way analysis of variance (ANOVA) was conducted using Dunnett’s multiple comparisons test to compare treatment groups with the control. Individual variances were estimated for each comparison, and the significance level was set at p < 0.05. The mean values were reported with their corresponding standard deviations (SD).

 

Results And Discussion

In vitro Study

The antifungal properties of Spirulina platensis and propolis extracts were demonstrated by the reduction in the growth rate of the tested yeast. Higher concentrations of the extracts improved their effectiveness, as shown in Figure 1. The findings demonstrated a noteworthy suppressive impact at various concentrations of SEE (0.25, 0.5, 1, 2 mg/mL) and PEE (1.5, 3, 6, 12 mg/mL). It was found that 0.5 mg/mL of Spirulina platensis and 6 mg/mL of propolis extracts were the lowest concentrations needed to stop the growth of Candida. This concentration was the minimum amount required to completely prevent the fungus from growing.

In vivo study

Table 1 shows how Spirulina platensis and propolis extracts affect the number of Candida albicans colonies (cfu/g) in minced meat. The results revealed that the untreated samples (control 1 group) had a microbial load of 6.85 ± 0.02, which increased to 7.74 ± 0.02 on the 3rd and 6th days, and then decreased to 6.86 ± 0.02 on the 9th day and 5.63 ± 0.02 on the 12th day, respectively. Spirulina platensis caused a decrease in yeast counts to 5.86 ± 0.01, 4.25 ± 0.01, 2.78 ± 0.01, and 1.49 ± 0.01 on the zero, 3rd, 6th, and 9th days of storage, respectively. On the 12th day, yeast counts were not detectable. The application of propolis resulted in a reduction in yeast count in the samples, with counts of 6.03 ± 0.01, 3.58 ± 0.01, and 1.02 ± 0.01 on the zero, 3rd, and 6th days, respectively. Furthermore, on the 9th and 12th days, Candida albicans was not detected. The untreated sample remained free of any measurable quantities of yeast during the zero, 3rd, and 6th days of storage. However, the samples were already degraded by the sixth day.

 

Table 1: Candida albicans count (Log10) in non-treated (control) and treated samples with spirulina and propolis during storage period at 4°C (Mean ± SE).

Storage Day	0	3	6	9	12
Control 1	ND	ND	ND	ND	ND
Control 2	6.85b ± 0.02	7.74a ± 0.02	7.74a ± 0.02	6.86b ± 0.02	5.63c ± 0.02
Spirulina platensis	5.86a ± 0.01	4.25b ± 0.01	2.78c ± 0.01	1.49d ± 0.01	-
Propolis	6.03a ± 0.01	3.58b± 0.01	1.02c ± 0.01	-	-

 

Means with different letters within the same row differed significantly at (P <0.05), ND: Not detected

 

Sensory Evaluation and Overall Acceptance

The overall acceptance of minced beef samples held at 4°C revealed that both the control 1 and control 2 samples were fully ruined by the 6th day of cold storage. The inclusion of Spirulina platensis preserved the product’s overall sensory acceptability until the ninth day. Additionally, adding Spirulina platensis to the meat gave it a green hue. On the other hand, the addition of propolis maintained the product’s acceptability until the 12th day of cold storage, as shown in Table 2.

To assess the impact of Spirulina platensis and propolis on Candida albicans in minced beef, we initially conducted in vitro experiments to determine the appropriate dosage to add to the meat. To our knowledge, this study is the first to examine the antifungal properties of spirulina and propolis against Candida albicans in beef mince. We tested Spirulina platensis and propolis against the testing strain of Candida albicans and found that they were both effective antifungals, with MIC values of 0.5 mg/ml for Spirulina platensis and 6 mg/ml for propolis.

 

Table 2: Sensory evaluation of the untreated and treated samples of minced meat during cold storage at 4ºC.

Samples	0 day	3rd day	6th day	9th day	12th day
Control 1	9	6	5	S	S
Control 2	9	6	5	S	S
Spirulina platensis	8	7	6.5	5	S
Propolis	9	8.5	7	6	5

 

9: Excellent; 8: Very very good; 7: Very good; 6: Good; 5: Medium; S: spoiled.

 

The alga’s action may be attributed to the intracellular and extracellular metabolites possessing antifungal characteristics (Al-Ghanayem, 2017). Overall, research has shown that the primary antifungal chemicals found in spirulina are predominantly polyphenols and polysaccharides. These compounds are effective in inhibiting the growth of microorganisms and can also directly destroy the cellular structures of fungi (Bajpai, 2016).

Likely, Marangoni et al. (2017) conducted a study to assess the effectiveness of a water extract from Spirulina platensis in inhibiting the growth of Candida albicans in a laboratory setting. Furthermore, it was discovered that the extract derived from microalgae also has fungicidal properties. According to Marangoni et al. (2016), the researchers found that the extract from the microalga has a strong antibacterial activity against Candida albicans. They observed that the MIC of the extract was 0.25 mg/ml, which was consistent with previous findings. In a study conducted by Usharani et al. in 2015, the antimicrobial activity of solvent extracts from Spirulina platensis against pathogenic fungi was examined. The researchers found that each solvent extract had a different MIC. The lowest MIC value was 2 mg/ml for the methanol extract, while the ethanol extract had a MIC of 8 mg/ml against Candida albicans. The minor variations observed in different studies are likely due to the extraction process used and the chemical composition of the extract (Vonshak, 1997).

Several authors have conducted studies on the antifungal properties of propolis against C. albicans, yielding varying findings depending on the origin of the propolis. Sowmya et al. (2021) reported that the MIC value of Indian propolis is 0.03 mg/mL. In contrast, Yusoff et al. (2016) recorded a MIC value of 500 mg/mL for propolis from New Zealand and South Australia. In a study conducted by Joya et al. in 2016, the researchers investigated the antifungal properties of ethanol extracts of propolis from different regions (Germany, Italy, Spain, and Venezuela) against C. albicans. The study found that all propolis extracts from the four regions exhibited significant fungistatic and fungicidal effects. However, there were variations in the MIC among the different propolis extracts. 

The observed variations in chemical composition across different regions of the world are believed to be responsible for these results. It is hypothesized that the differences in minimum inhibitory concentration (MIC) are a result of the interaction between distinct propolis-specific compounds (de Souza et al., 2013; Zhang et al., 2015). The phenolics and flavonoids in propolis contribute to its antifungal activity. These compounds alter the permeability of the cytoplasmic membrane, resulting in cellular leakage and ultimately cell death.

During the 12-day storage period at 4°C, all the treated meat samples in the experimental group exhibited a notable decrease in Candida count.

The statistical analysis revealed a significant difference (p <0.05) between the control samples (non-treated) and each of the treated samples on the zero, 3rd, 6th, and 9th days of storage, respectively. Compared to Spirulina, propolis has shown greater efficacy in reducing the count. Spirulina possesses powerful antioxidants and free-radical scavengers that can hinder the growth of certain types of bacteria and yeast, including Candida albicans (Marangoni et al., 2017). The polysaccharides derived from Spirulina possess qualities that inhibit tumor growth, prevent oxidation, slow down the aging process, and combat viruses (Choi et al., 2019). Microalgae biomass or its separated components can be directly included as techno-functional additives in various processed meat products. The study conducted by de Medeiros et al. (2020) examined several meat products, such as sausages (Marti-Quijal et al., 2019), burgers (Marti-Quijal et al., 2019), pork liver pâté, turkey patties (Zamuz et al., 2019), beef patties (Zugcic et al., 2018), and chicken roti (Parniakov et al., 2018). 

Propolis exhibits antimicrobial properties that can be attributed to either its direct antimicrobial effects or its indirect effects on the immune system, which result in the elimination of a greater number of microorganisms. Research has shown that propolis has the ability to promote the production of antibodies, activate macrophages, and increase their ability to fight against microorganisms (Gavanji et al., 2014). Researchers studied propolis extracts mixed in ethanol and found that they were antibacterial (Abdullah et al., 2019), antifungal, antioxidant, and anticancer (Abubakar et al., 2014; Abdullah et al., 2019).

Pobiega et al. (2019) stated that propolis is a desirable ingredient for use in food production due to its chemical composition, namely its high concentration of bioactive compounds. Propolis is commonly used as a natural preservative in food to maintain food quality and prevent microbial growth during storage (Viera et al., 2016). Foodborne saprophytic microbiota and foodborne pathogens can be diminished or eradicated by directly administering propolis powder and its extracts to food. The study observed that the inclusion of Spirulina platensis in minced meat resulted in a green color, which may not be deemed acceptable to consumers. In contrast, the blank, control, and propolis-treated samples exhibited a desirable bright red color. These findings align with the findings of Awadalla et al. (2020), who observed that the inclusion of Spirulina platensis had a negative impact on the color of uncooked meatball samples.

The sensory attributes of the treated samples were enhanced; resulting in a longer shelf life compared to the untreated samples during the 0, 3, 6, 9, and 12 days of refrigerated storage. According to Ismet Ozturk’s 2014 study, samples treated with propolis exhibited increased firmness and brightness compared to the other samples. Propolis was found to effectively reduce the presence of yeasts and molds in sausages without causing significant changes to their color and scent attributes.

Among the practical applications of the current study is the potential use of propolis in controlling fungal growth of minced meat. While the limitations of the present study appear in using one meat product and in future using different types of meat products is highly recommended. The change in the color of meat after adding spirulina makes it difficult to use it in the meat industry and therefore finding methods to stabilize the red color of the meat while using spirulina is regarded as one of the future approaches in our research.

CONCLUSIONs And RECOMMENDATIONS

From the results it could be concluded that 0.5 mg/ml of ethanolic extracts of Spirulina platensis and 6 mg/ml of propolis effectively inhibit the growth of Candida albicans in fresh minced beef that has been stored at 4°C. Compared to Spirulina, propolis has shown superior efficacy in reducing the count. Additionally, propolis exhibited superior sensory qualities, particularly in terms of color, and prolonged the meat preservation period for an extended duration. In samples treated with propolis, the meat began to deteriorate on the 12th day, but in samples treated with Spirulina platensis, it ruined on the 10th day. Propolis is suggested as a natural antifungal agent for the preservation of minced meat. Finding alternatives to stabilize the red color of the meat while using spirulina could be a challenge to get the highest benefit of this alga and will be one of our future work.

ACKNOWLEDGMENTS

We thank our colleagues at the Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Benha University, for their contributions to the study.

AUTHOR’S CONTRIBUTION

All authors contributed equally to the manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

REFERENCES

Abd El-Malek, A.M. 2017. Bacteriological status and detection of adulteration with donkey meat by PCR in frozen beef meatballs. IOSR J. Environ. Sci. Toxicol. Food Technol., 11(10): 1-7.

Abdullah, N.A., Ja’afar, F., Yasin, H.M., Taha, H., Petalcorin, M.I.R., Mamit, M.H., Kusrini, E., Usman, A. 2019. Physicochemical analyses, antioxidant, antibacterial, and toxicity of propolis particles produced by stingless bee Heterotrigona itama found in Brunei Darussalam. Heliyon, https://doi.org/10.1016/j.heliyon.2019.e02476

Abubakar, M.B., Abdullah, W.Z., Sulaiman, S.A., Ang, B.S. 2014. Polyphenols as key players for the antileukaemic effects of propolis. Evidence-Based Complementary and Alternative Medicine, 2014(1):371730 https://doi.org/10.1155/2014/371730

Al-Ghanayem, A.A. 2017. Antimicrobial activity of Spirulina platensis extracts against certain pathogenic bacteria and fungi. Adv. Bioresour., 8: 96-101.

American Puplic Health Association (APHA). 1985. Compendium of food, 2nd Ed., American puplic Health Association, Washington, D.C.

Awadalla, K.S., Ahmed, A.M., Abdel-Daim, M.M., Elshraway, N.T., Abdel-Wahab, M.A. 2020. Evaluation of Sensory and Bacteriological Quality of Meatball Supplemented by Spirulina Platensis. J. Appl. Vet. Sci., 5(2): 13-24. https://doi.org/10.21608/javs.2020.85554

Bajpai, V.K. 2016. Antimicrobial bioactive compounds from marine algae: A mini review. Indian J. Geomarine Sci., 45: 1076-1085.

Bauer, L.M., Vieira Costa, J.A., Conteno da Rosa, A.P., Santos, L.O. 2017. Growth stimulation and synthesis of lipids, pigments and antioxidants with magnetic fields in Chlorella vulgeri cultivations. Bioresour. Technol., 244(2): 1425–1432. https://doi.org/10.1016/j.biortech.2017.06.036

Casquete, R., Castro, S.M., Jacome, S., Teixeira, P. 2016. Antimicrobial activity of ethanolic extract of propolis in “Alheira”, a fermented meat sausage. Cogental Food Agric., 2: 1125774. https://doi.org/10.1080/23311932.2015.1125774

Choi, W.Y., Sim, J.H., Lee, J.Y., Kang, D.H., Lee, H.Y. 2019. Increased anti-inflammatory effects on LPS-induced microglia cells by Spirulina maxima extract from ultrasonic process. Appl. Sci., 9: 2144. https://doi.org/10.3390/app9102144

Clinical and Laboratory Standards Institute (CLSI). 2008. M27-S3: Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Third Informational Supplement. 3rd. Wayne, Pennsylvania, 2008. http:// medicine. kaums. ac. ir/ Uploaded Files/ angal shena se/ M27-S3Thi rd International Supplement. pdf

Cruickshank, R., Duguid, J.P., Marino, B.P., Swain, R.H.A. 1975. Medical Microbiology 12th Ed. Vol., Chuurchill Livingston. London and New York.

de Medeiros, V.P.B., Pimentel, T.C., Sant’Ana, A.S., Magnani, M. 2020. Microalgae in the meat processing chain: feed for animal production or source of techno-functional ingredients, Curr. Opin. Food Sci., 2020. https://doi.org/10.1016/j.cofs.2020.10.014

de Souza, G., Pfenning, L., de Moura, F., Salgadom, M., Takahashi, J. 2013. Isolation, identification and antimicro­bial activity of propolis-associated fungi. Nat. Prod. Res., 27: 18, 1705-1707 https://doi.org/10.1080/14786419.2013.771348.

El-Baz, F.K., El-Senousy, W.M., El-Sayed, A.B., Kamel, M.M. 2013. In vitro antiviral. Science, 3: 052-056.

Gavanji, S., Larki, B., Bakhtari, A. 2014. The effect of extract of Punicagranatum var. pleniflora for treatment of minor recurrent aphthous stomatitis. Integrat. Med. Res., 3: 83-90. https://doi.org/10.1016/j.imr.2014.03.001

Höfs, S., Mogavero, S., Hube, B. 2016. Interaction of Candida albicans with host cells: Virulence factors, host defense, escape strategies, and the microbiota. J. Microbiol., 54: 149–169. https://doi.org/10.1007/s12275-016-5514-0

Horwitz, W. 2020. Pearson’s Chemical Analysis of Foods. 8th Edition. J. Assoc. Official Analitical Chem. 65: 1037-1037. https://doi.org/10.1093/jaoac/65.4.1037

Ismet Ozturk, I. 2014. Antifungal activity of propolis, thyme essential oil and hydrosol on natural mycobiota of sucuk, a turkish fermented sausage: monitoring of their effects on microbiological, color and aroma properties. J. Food Process. Preserv., 38: 1745-4549. https://doi.org/10.1111/jfpp.12329

Jonaidi Jafari, N., Kargozari, M., Ranjbar, R., Rostami, H., Hamedi, H. 2018. The effect of chitosan coating incorporated with ethanolic extract of propolis on the quality of refrigerated chicken fillet. J. Food Process. Preserv. 42(1): e13336. https://doi.org/10.1111/jfpp.13336

Krifors, A., Lignell, A., Lipcsey, M., Sjolin, J., Castegren, M. 2023. An experimental porcine model of invasive candidiasis. Intensive Care Medical Experiments, 11: 27. https://doi.org/10.1186/s40635-023-00514-6

Marangoni, A., Foschi, C., Micucci, M., Nahui Palomino, R.A., Gallina Toschi, T., Vitali, B. 2017. In vitro activity of Spirulina platensis water extract against different Candida species isolated from vulvovaginal candidiasis cases. PLoS ONE, 12(11): e0188567. https://doi.org/10.1371/journal.pone.0188567

Marti-Quijal, F.J., Zamuz, S., Tomašević, I., Gómez, B., Rocchetti, G., Lucini, L., Remize, F., Barba, F.J., Lorenzo, J.M. 2019. Influence of different sources of vegetable, whey and microalgae proteins on the physicochemical properties and amino acid profile of fresh pork sausages. LWT - Food Sci. Technol., 110: 316–323. https://doi.org/10.1016/j.lwt.2019.04.097

Marti-Quijal, F.J., Zamuz, S., Tomašević, I., Rocchetti, G., Lucini, L., Marszałek, K., Barba, F.J., Lorenzo, J.M. 2019. A chemometric approach to evaluate the impact of pulses, Chlorella and Spirulina on proximate composition, amino acid, and physicochemical properties of turkey burgers. J. Sci. Food Agric., 99: 3672–3680. https://doi.org/10.1002/jsfa.9595

Parniakov, O., Toepfl, S., Barba, F.J., Granato, D., Zamuz, S., Galvez, F., Lorenzo, J.M. 2018. Impact of the soy protein replacement by legumes and algae based proteins on the quality of chicken rotti. J. Food Sci. Technol., 55: 2552–2559. https://doi.org/10.1007/s13197-018-3175-1

Pobiega, K., Kra´sniewska, K., Gniewosz, M. 2019. Application of propolis in antimicrobial and antioxidative protection of food quality – a review. Trends Food Sci. Technol., https://doi.org/10.1016/j.tifs.2018.11.007

Poulain, D. 2015. Candida albicans, plasticity and pathogenesis. Critical Rev. Microbiol., 41: 208–217. https://doi.org/10.3109/1040841X.2013.813904

Pyne, P.K., Bhattacharjee, P., Srivastav, P.P. 2017. Microalgae (Spirulina Platensis) and Its Bioactive Molecules: Review. Indian J. Nutr., 4(2): 160.

Riesute, R., Salomskiene, J., SaezMoreno, D., Gustiene, S. 2021. Effect of yeasts on food quality and safety and possibilities of their inhibition. Sciences, 108: 1-10. https://doi.org/10.1016/j.tifs.2020.11.022

Sabin, L., Brian, S.E. 2014. A handbook of statistical analysis using CHAPMAN and HALL/CRC. A Press User manual SPSS. INC. /SPSS.Ver 20.

Saranya, C., Hemalatha, A., Parthiban, C., Anantharaman, P. 2014. Evaluation of antioxidant properties, total phenolic and carotenoid content of Chaetoceros calcitrans, Chlorella salina and Isochrysis galbana. Int. J. Curr. Microbiol. Appl. Sci., 3(8): 365–377.

Shehu, A., Ismail, S., Rohin, M.A.K., Harun, A., Aziz, A.A., Haque, M. 2016. Antifungal Properties of Malaysian Tualang Honey and Stingless Bee Propolis against Candida albicans and Cryptococcus neoformans. J. Appl. Pharmacol. Sci., 6: 044-050. https://doi.org/10.7324/JAPS.2016.60206

Shehu, A., Rohin, M.A.K., Aziz, A.A., Ismail, S. 2015. Antifungal, characteristic properties and composition of bee glue (propolis). J. Chem. Pharmacol. Res., 7(3):1992-1996.

Sowmya, S., Gujjari, A.K., Swamy, K.N.R., 2021. Antifungal effect of Hubballi Propolis on Candida albicans - an in vitro study. J. Evolut. Med. Dental Sci., 10: 1979-1983. https://doi.org/10.14260/jemds/2021/406

Usharani, G., Srinivasan, G., Sivasakthi, S., Saranraj, P. 2015. Antimicrobial activity of Spirulina platensis solvent extracts against pathogenic bacteria and fungi. Adv. Biol. Res. 9: 292-298.

Vonshak, A. 1997. Spirulina platensis (Arthrospira): Physiology, Cell-Biology and Biotechnology. Taylor and Francis Ed; 1997. https://doi.org/10.1201/9781482272970

Viera, V.B., Piovesan, N., Moro, K.I.B., Rodrigues, A.S., Scapin, G., Rosa, C.S., Kubota, E.H. 2016. Preparation and microbiological analysis of Tuscan sausage with added propolis extract. Food Sci. Technol., https://doi.org/10.1590/1678-457x.0045

Yusoff, N.Y.N., Mohamad, S., Abdullah, H.N., Ab Rahmana, N. 2016. Antifungal Activity of Malaysian Honey and Propolis Extracts against Pathogens Implicated in Denture Stomatitis, AIP Conference Proceedings, 1791, 020006. https://doi.org/10.1063/1.4968861

Zamuz, S., Purriños, L., Galvez, F., Zdolec, N., Muchenje, V., Barba, F.J., Lorenzo, J.M. 2019. Influence of the addition of different origin sources of protein on meat products sensory acceptance. J. Food Process. Preservat., 43: 1-12. https://doi.org/10.1111/jfpp.13940

Zugcic, T., Abdelkebir, R., Barba, F.J. 2018. Effects of pulses and microalgal proteins on quality traits of beef patties. J. Food Sci. Technol. 55: 4544-4553. https://doi.org/10.1007/s13197-018-3390-9

Zhang, H., Wang, G., Beta, T., Dong, J. 2015. Inhibitory properties of aqueous ethanol extracts of propolis on alpha-glucosidase. Evidence-Based Complement. Alternat. Med., https://doi.org/10.1155/2015/587383