Propolis as natural additive: A systematic review

1 School of Nutrition, Federal University of Bahia, Rua Araújo Pinho, n° 32, Canela, Cep: 40.110-160, Salvador, BA, Brazil. 2 CIMO-Mountain Research Center, Department of Biology and Biotechnology, Agricultural College of Bragança, Polytechnic Institut of Bragançą, Campus Santa Apolónia, Cep: 5300-253 Bragançą, Portugal. 3 Center of Agricultural Sciences, Environmental and Biological. Federal University of Bahia Reconcavo. Rua Rui Barbosa, n° 710, Centro, Cep: 44.380-000. Cruz das Almas, Bahia, Brazil. 4 Department of Bromatological Analysis, Pharmacy Faculty, Federal University of Bahia, Barão of Geremoabo Street, s/n, Ondina, Cep: 40.171-970, Salvador, BA, Brazil. 5 Department of Biology, Microbiology Sector, Federal University of Lavras, Cep: 37.200-000, Lavras, MG, Brazil.


INTRODUCTION
The concern for safety in the use of synthetic preservatives in foods and the preference for natural products have led to the increase in research on antioxidants and other preservatives derived from natural sources such as cocoa, rice, apple, red onion, oregano, rosemary, honey and propolis (Jiang et al., 2013). In this context, apiculture products (propolis) have been highlighted for use in foods either for therapeutic *Corresponding author. E-mail: rogeriac@ufba.br. Tel: + 55 71 3283 7719 / 3283 7717.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License purposes, or to be used as food, and their use is one of the oldest universal practices. Propolis is a resinous honeybee product and a traditional therapeutic agent in folk medicine. It is produced by bees (Apis mellifera L.) to meet some needs in the hive, such as filling in gaps, reducing input and output of the hive openings, mummification of insect bodies, covering the inner walls of the hive and the inside cells, repairing damaged honeycombs and consolidation of mobile honeycombs (Falcão et al., 2010;Moreira et al., 2011;Bankova et al., 2016;Cuesta-Rubio et al., 2017;Frozza et al., 2017;Thamnpoulos et al., 2018).
The propolis antimicrobial activity was already empirically known by the ancient Egyptian priests that used it in the embalming process to protect the mummies from fungi and bacteria attack (Gonsales et al., 2006). This antimicrobial property is mainly attributed to the presence of flavonoids with phenolic acids and esters, phenolic aldehydes and ketones (Pietta et al., 2002). Related to the antibacterial activity, the mechanism is considered complex and can be attributed to the synergism among flavonoids, hydroxy and sesquiterpenes (Krol et al., 1993). The product has a complex chemical composition, with over 300 compounds and minerals such as aluminum, calcium, strontium, iron, copper, manganese and minor amounts of vitamins B1, B2, B6, C and E (Pietta, 2002;Khalil et al., 2015). It stands out for its antimicrobial, antioxidant, antiinflammatory, immunomodulatory, hypotensive, healing and anesthetic properties (Burdock, 1998). Propolis is known to be the most concentrated source of bioflavonoids, which are the main antioxidants found in plant extracts. The activity of phenolics and other bioactive compounds in inhibiting autoxidation in some foods and biological systems has been attributed to their redox properties (Ukulo et al., 2013).
In its native application, the primary function of propolis is biocidal action against bacteria and invasive fungi, suggesting its potential for industrial application. In the foods application, propolis has been used in industry as a preservative in meat products, increasing the shelf life of frozen meat and other foods (Netikovã et al., 2013;Feás et al., 2014;Bankova et al., 2016, Vargas-Sánchez et al., 2014Casquete et al., 2016). The action mechanism is still not fully understood due to the existing synergy between the different substances in propolis and the presence of multiple targets in each affected organism (Seidel et al., 2008). The chemical properties of the propolis are highly variable and depend on the source plant and the flora at the site of collection. These different chemical compositions do not always lead to significant differences in physicochemical and pharmacological activities. Nevertheless, some difference between the diterpene-rich Mediterranean propolis that has weak antioxidant activity and the poly-phenol-rich types (mainly Brazilian green and poplar type) has been verified, while its antibacterial properties are comparable to those of poplar type (Bankova et al., 2016). Mechanisms to enrich food products with beneficial bioactive compounds have been developed by the food industry. To ensure minimal impact on the organoleptic and qualitative properties of developed products, the use of encapsulation technology is a powerful tool, since it enables the protection of a wide range of compounds by their embedding into a protective matrix (Dordevic et al., 2014). The use of this technology, since the 1950s, has increased in food industry, because the encapsulated material can protect food from heat, moisture, oxidation, or other chemical reactions in extreme conditions, increasing its stability and maintaining its viability (Gouin, 2004). Based on these considerations, this study aimed to conduct a systematic review of the use of propolis as a food additive and the microencapsulation technology for incorporation of the product in food.
The analysis of the studies was carried out using the summarization of the information in articles, such as: author, year of publication, sample, methodology used, and main results found.

Incorporation of propolis in foods
Research in the mainly databases inserting the term "propolis" identified a total of 18.536 articles (Figure 1). Based on the total of identified articles, the importance of propolis to the scientific community in the fields of microbiology, chemical and pharmaceutical areas can be measured. However, when entering in the databases the subject "Propolis as a natural food additive" 16 articles were viewed and in the search for "Propolis in foods" 12 articles were identified ( Figure 2). Other databases The analysis showed that there are few reports in the literature on propolis as a natural additive in food for Records identified through data bases searching: Medline, Pubmed, Lilacs and Google Scholar related to keyword "microencapsulation", "food microencapsulation", "microencapsulation of propolis" and "propolis microencapsulation in food". human consumption and propolis in foods. Regarding the use of propolis as antioxidants natural source, a study performed by Mendiola et al. (2010) highlights the use of natural sources of antioxidants to replace synthetic

Antioxidant
Dihydroxychalcones eliminates riboflavin (Rf)photogenerated reactive oxygen species (ROS). González et al. (2015) Elimination of free radicals, inhibiting lipid peroxidation and hemolysis of human erythrocytes. Coelho et al. (2015) Sinapic acid and rutin potential alternative as a natural antioxidant. Antioxidant properties, total phenols and pollen analysis of propolis samples from Portugal Parvovirus swine inhibition (PPV) in vitro and in vivo Ma et al. (2015) antioxidants using plants, fruits, Spirulina, tubers and propolis. The authors demonstrate that propolis has a significant amount of antioxidants, indicating that they can be an alternative replacement for synthetic antioxidant (Table 1). The antimicrobial and antioxidant properties attributed to propolis are valuable for the food industry due to its effect in delaying lipid oxidation and improving the food shelf life. These characteristics are due to the presence of chemical compounds like flavonoid, pinocembrin, flavonol, galagina and caffeic acid phenylethyl ester, which exhibit-based-action mechanism probably in inhibiting bacterial RNA polymerase (Funari and Ferro, 2006). Other components such as flavonoids, caffeic acid, benzoic acid and cinnamic acid, probably act at the level of membrane or cell wall of the microorganisms, causing structural and functional damage (Funari and Ferro, 2006). Moreover, polyphenols contain an extensive range of other compounds with the ability to remove excess free radicals from our body (Santos, 2015). Table 2 shows studies related to use of propolis as a natural additive in foods. Some of these studies are the basis for future work in order to add propolis to food for human consumption, because it contains biological properties necessary for inhibition of microbial growth and consequently for food preservation.
It is important to consider that the consumption of foods containing chemicals that are not nutrients, can lead to the appearance of different effects in different degrees and with several designations such as: toxic effect, harmful, detrimental adverse or unexpected (Menezes, 2005), associated with the emergence of allergies, behavioral changes such as hyperactivity and cancer (Moutinho et al., 2007). Such prospects will obligate the food industry to eliminate the use of chemical preservatives and adoption of natural alternatives to food. In recent times, the increasing interest in the food industry to find natural additives have stimulate the efforts both in obtaining bioactive compounds in natural raw materials, and in development of stable products and functional derivatives (Silva et al., 2013). Valero et al. (2014) evaluated the effect of natural additives as propolis or essential oils on meat quality of crossbred (Aberdeen Angus vs. Nellore) bulls. Addition of natural additives as propolis extract or cashew and castor oils in the diet of bulls when they are finished in a feedlot Table 2. Studies related to use of propolis as a natural additive in foods.

References
Countries Title Description Koc et al. (2007) Turkey Antifungal activity of propolis in four different fruit juices.
This study evaluated the effect of ethanol extract of Turkish propolis treatments in juices nonpasteurized fruit (apple, orange, white grape and mandarin) against six different yeasts. The authors indicated the potential use of propolis as an alternative to chemical fruits juices preservation agents.

Özdemir et al. (2009) Turkey
The effects of ethanoldissolved propolis on the storage of grapefruit cv.

Star Ruby
Effect of propolis on the storage life of Star Ruby grapefruit was investigated. Fruits were dipped in ethanol-extracted propolis (1%, 5% and 10%). This study showed that the weight loss was significantly higher in fruits without treatment. The best concentration of propolis for a period of 5 months was 5%, demonstrating that the propolis has a strong anti-microbial effect and limits the growth of microorganisms.

Kim et al. (2013) Korea
Synergistic effect of propolis and heat treatment leading to increased injury to Escherichia coli O157:H7 in ground pork.
This study determined the thermal inactivation of Escherichia coli O157: H7 in the presence of propolis in culture and ground pork. According to the authors, E. coli O157:H7 was inhibited in culture and in ground meat by decreasing the heat resistance, demonstrating a synergistic effect. Kamel et al. (2015) Egypt The effect of propolis and sodium metabisulfite as postharvest treatments on pomegranate arils storage.
This study compared the effect of sodium metabisulfite, extract of propolis and mixed solution for treatment of aryls during storage under refrigeration. Highest effect at 25 days was achieved when combined treatment was used. The overall result showed that both sodium as propolis extract has the potential to maintain arils attributes. However, the use of higher concentration of propolis as a single treatment demonstrated negative effects.

Thamnpoulos et al. (2018) Greece
Inhibitory activity of propolis against Listeria monocytogenes in milk stored under refrigeration The objective of this study was to develop a protocol for adding propolis into milk and to determine whether the addition of propolis can confer anti-listerial activity during the storage of milk under optimal or improper refrigeration conditions. Results highlight the strong antilisterial potential of propolis in milk. did not change meat qualities. The works related to potential of the propolis as biological and chemicalpharmaceutical product highlighting the need for research on the potential of propolis as a natural additive in food for human consumption, with emphasis on how the compounds found in propolis can replace chemical additives commonly used in foods. Some research confirms that low concentrations of propolis extract can be used as antimicrobial and antioxidant substances for food protection (Mendiola et al., 2010;Bernardi et al., 2013).
However, propolis toxicological studies are required, considering the cumulative effects or synergistic protection. It is important not only to know the specific properties that convert the food additive, but also all actions and contra-indications, especially those derived from prolonged use (Salinas, 2002). Araújo et al. (2011) observed little toxicity of a hydroalcoholic extract of Brazilian propolis. Similar results were reported by Mohammadzadeh et al. (2007), who found no toxic effects after the ingestion of Iranian propolis. The application of propolis in food, however, has limitations due to the strong flavor and aroma and its difficult solubility. Because of this, usually propolis is administered in alcoholic solutions, which limits its application in food. These disadvantages result in storage problems, transport, development and management (Silva et al., 2013).

Microencapsulation technology
Related to microencapsulation technology, a total of 36.532 articles was identified in the mainly databases, using the term "Microencapsulation" (Figure 2 Microencapsulation is the packaging of solid, liquid droplets or gaseous material with fine polymer coverage. This procedure involves the incorporation of food ingredients, enzymes, cells or other materials in small caps. It is a barrier to prevent chemical reactions and allow the modified release of ingredients under specific conditions and speed, masking odors and tastes (Rosenberg et al., 1990;Gouin 2004Gouin , Ðordevic et al., 2014. The encapsulated material can be protected in extreme conditions, increasing its stability and maintaining its viability (Gouin, 2004).
By filtering the search for "food microencapsulation", 2.673 articles were found, demonstrating a widespread technique. However, research on the use of the term "Microencapsulation of propolis" are still limited, having identified only 19 articles, while for the search for the term "Propolis microencapsulation in food" two articles was found. Therefore, it can be concluded that the studies about the potential use of propolis as a food additive is very small in the scientific literature, as shown in Table 3. Favaro-Trindade et al. (2008) evaluated the effects of microencapsulation of the ethanol extract of propolis on solubility in water and the product properties in vitro to release components. To this end, encapsulation using ciclodextrins was tested and samples of propolis from Southern Greece. The authors observed increased water solubility of various bioactive components of propolis extracts and demonstrated that the net release of the compounds depends not only on the chemical properties of the product, but also on the relative abundance in the sample of propolis extract.
Work performed by Koo et al. (2002) discusses the use of spray drying method for preparing microparticles of propolis, using gelatin as the polymer. It was shown that this method besides being cheap and practical, maintains the activity of propolis extract against Staphylococcus aureus. This study represents new perspectives for microencapsulation technique of propolis. Studies related to the use of propolis as a food additive was developed by Gomes et al. (2011) (Table 3). The authors evaluated the antimicrobial efficacy of extracts of natural compounds against Gram-positive and Gram-negative bacteria, the antimicrobial activity of microencapsulation of natural compounds beta-cyclodextrins and the degree of radiosensitization of Salmonella spp. on spinach sprayed with microencapsulated compounds.
Data consisted the effectiveness of all compounds in inhibiting the growth of Gram-positive and Gram-negative; both of them were used directly as extract and microencapsulated. Still, according to the authors, the microencapsulation improved stability, solubility and masked the strong odor and off flavor. The combination of irradiation dosage levels <1 kGy and spray microencapsulated natural antimicrobial compounds ensured a reduction of 5 log cycles in the population of Salmonella spp., without detrimental effects on product quality.
According to Bernardi et al. (2013), propolis prevented lipid oxidation in salami during storage, but showed lower sensorial acceptance. To improve sensorial acceptance of fish burgers, Spinelli et al. (2014) used ingredients such as potato flakes and extra virgin olive oil and obtained a final fish product with good acceptability. Burgers showed increase of both phenolic content and antioxidant activity. Reis et al. (2017) evaluated the characteristics of the microencapsulated propolis coproduct extract (MPC) and their effects on stability and sensory quality in burger meat. The efficiency of the microencapsulation technology regarding the phenolic compounds content was high. Smell and flavor in burger meat containing MPC showed lower grades than the ideal scale. However, color, appearance, and texture demonstrated ideal grades (Table 3).

CONCLUSION AND FUTURE PERSPECTIVE
Based on the systematic review, it was verified that the scientific articles in the period studied, focused mainly on studies to investigate biological and chemicalpharmaceutical potential, with only a few studies about the incorporation of propolis in food. From this perspective, considering the potential of propolis as a natural additive and its antioxidant and antimicrobial properties, there are necessary further studies covering the incorporation of propolis in food, considering its potential as a natural additive and its antioxidant and antimicrobial properties. It also shows the need for Table 3. Studies using the microencapsulation technology for food.

References
Title Description Gomes et al. (2011) Microencapsulated antimicrobial compounds as a means to enhance electron beam irradiation treatment for inactivation of pathogens on fresh spinach leaves.
The study aimed to evaluate the effectiveness of the use of natural antimicrobials. The minimum inhibitory concentration of 5 compounds and natural extracts (trans cinnamaldehyde, eugenol, garlic extract, propolis extract, and lysozyme with EDTA were determined against Salmonella spp. and Listeria spp. The efficacy of the microencapsulated compounds was tested by spraying onto the surface of contaminated spinach. The results confirmed that the combination of microencapsulated spraying with electron beam irradiation is effective in enhancing the lethal effects of irradiation.

Nori et al. (2011) Microencapsulation of propolis extract by complex coacervation
This study aimed to obtain encapsulate propolis extract by complex coacervation using isolated soy protein and pectin as encapsulant agents. Encapsulate propolis extract was obtained in the form of powder, alcohol-free, stable, with antioxidant property, antimicrobial activity against Staphylococcus aureus and with the possibility of controlled release in foods. Spinelli et al. (2014) Microencapsulated propolis to enhance the antioxidant properties of fresh fish burgers The study aimed to evaluate the antioxidant properties of microencapsulated propolis added in fish burgers. To improve sensorial acceptance, ingredients such as potato flakes and extra virgin olive oil were tested and optimized to give a final product with good acceptability. Burgers showed increase of phenolic content and antioxidant activity. Koga et al. (2016) Consumer acceptance of bars and gummies with unencapsulated and encapsulated resveratrol.
The objective of this research was to show the application of resveratrol in microcapsules in food to be consumed. The microencapsulation in a matrix of sodium caseinate was used as a strategy to overcome the bitterness of resveratrol. The microcapsules resveratrol there was a significant lower overall taste than the control with the same protein and / or content of resveratrol. Rutz et al. (2016) Elaboration of microparticles of carotenoids from natural and synthetic sources for applications in food.
The study aimed to encapsulate the palm oil and β -carotene with chitosan tripolyphosphate/chitosan or sodium/carboxymethylcellulose and evaluate the performance of these microparticles in food systems by analyzing their release profile under gastric conditions and simulated intestinal. The encapsulation efficiency was greater than 95%. Rached et al. (2016) Ceratonia siliqua L. hydroethanolic extract obtained through ultrasonication: antioxidant activity, phenolic compounds profile and effects in yogurts functionalized with their free and microencapsulated forms.
Bioactive extracts were obtained from carob pulp powder through an ultrasonic extraction process and then evaluated in terms of antioxidant activity in yogurt.
The study confirmed the efficiency microencapsulation to stabilize the functional ingredients in food matrices almost maintaining the structural integrity of polyphenols extracted from carob pulp and improving in addition, the antioxidant potency of the final product.
Physico-chemical characteristics of microencapsulated propolis coproduct extract and its effect on storage stability of burger meat during storage at -15°C.
Characteristics of the microencapsulated propolis co-product extract (MPC) and their effects on stability and sensory quality was evaluated in burger meat. The efficiency of the microencapsulation technology regarding the phenolic compounds content, was high. Smell and flavor in burger meat containing MPC showed lower grades than the ideal scale. However, color, appearance, and texture demonstrated ideal grades. studies to enhance the technology of product microencapsulation in order to preserve the characteristics of interest and mask those that may influence the food acceptance.

CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.