Impact of the extraction temperature on the antifungal efficiency and valorization potential of anise (Pimpinella Anisum L.) essential oil residues: A sustainable strategy

Impacto da temperatura de extração na eficiência antifúngica e potencial de valorização de resíduo de óleo essencial de anis (Pimpinella Anisum L.): Uma estratégia sustentável

ABSTRACT

The exploration of essential oils (EOs) in the pharmaceutical and food industries highlights their importance, due to the bioactive compounds they contain, which possess antioxidant, antimicrobial, and antifungal properties. This study aimed to evaluate the impact of different extraction temperatures on the yield, chemical composition, and the antifungal efficacy of anise essential oil (Pimpinella anisum L.) using hydrodistillation. Additionally, it explored the potential of producing a flour from the EO extraction residue as an innovative waste utilization strategy. Findings indicated that an optimal extraction temperature of 145ºC led to a predominance of E-anethole, as identified by gas chromatography-mass spectrometry (GC-MS), and demonstrated superior antifungal activity against Colletotrichum sp. The produced flour with a granulometry of 250 µm, stood out for its high protein content (19.81±0.09%) and high content of minerals (manganese, calcium, phosphorus, zinc, and magnesium), with iron and copper values that exceed the daily reference values set by the Ministry of Health. It also featured low water activity (0.25), medium hygroscopicity (48.02%), a pH of 5.6, and titratable acidity of 0.93% (expressed as malic acid). The analysis of bioactive compounds revealed total phenolics at 28.93 mg GAE/100g and antioxidant capacity of 5.60±0.897 µM trolox/g. This study underscores the importance of extraction temperature in essential oil quality and efficacy, proposing a sustainable approach to utilizing EO production residues making it a viable option for human consumption due to its nutritional composition and antioxidant potential.

Index terms:
Bioactive compounds; sustainability; waste valorization

RESUMO

A exploração de óleos essenciais (OEs) nas indústrias farmacêutica e alimentícia destaca sua importância, devido aos compostos bioativos que contêm, os quais possuem propriedades antioxidantes, antimicrobianas e antifúngicas. Este estudo teve como objetivo avaliar o impacto de diferentes temperaturas de extração no rendimento, na composição química e na eficácia antifúngica do óleo essencial de anis (Pimpinella anisum L.) pelo método de hidrodestilação. Além disso, explorou o potencial de produzir uma farinha a partir do resíduo da extração do OE como uma estratégia inovadora de utilização de resíduos. Os resultados indicaram que uma temperatura de extração otimizada de 145ºC levou à predominância de E-anetol, conforme identificado por cromatografia gasosa-espectrometria de massa (CG-EM), e demonstrou atividade antifúngica superior contra Colletotrichum sp. A farinha produzida, com granulometria de 250 µm, destacou-se por seu alto teor de proteína (19,81±0,09%) e alto conteúdo de minerais (manganês, cálcio, fósforo, zinco e magnésio), com valores de ferro e cobre que excedem os valores de referência diários estabelecidos pelo Ministério da Saúde. Apresentou também baixa atividade de água (0,25), higroscopicidade média (48,02%), pH de 5,6 e acidez titulável de 0,93% (expressa como ácido málico). A análise de compostos bioativos revelou fenólicos totais de 28,93 mg GAE/100g e capacidade antioxidante de 5,60±0,897 µM trolox/g. Este estudo ressalta a importância da temperatura de extração na qualidade e eficácia do óleo essencial, propondo uma abordagem sustentável para a utilização de resíduos da produção de OEs, tornando-a uma opção viável para consumo humano devido à sua composição nutricional e potencial antioxidante.

Termos para indexação:
Compostos bioativos; sustentabilidade; valorização de resíduos

Introduction

Anise (Pimpinella anisum L.), belonging to the Apiaceae family, is a plant species native to Mediterranean countries (Metwaly, 2021). Its seeds and essential oil (EO) are widely used in cooking and cosmetology, in addition to possessing therapeutic characteristics, acting as a digestive, expectorant, and carminative (Andallu & Rajeshwari, 2011).

The essential oil of anise can be extracted from various parts of the plant, including fruits, stems, and roots (Tabanca et al., 2006). Various methods can be employed, such as steam distillation, hydrodistillation, and solvent extraction, where the bioactivity and chemical composition of the extracted oil can vary due to process conditions (Busato et al., 2014; Tran et al., 2024). In hydrodistillation, the raw material used is in contact with boiling water, either immersed or floating, and in some cases, it is necessary to grind the material to facilitate the extraction of the EO (Busato et al., 2014). There is a lack of information on how different temperatures can influence the hydrodistillation process.

Anise seeds contain about 1.0-5.0% EO, with the chemical composition consisting of monoterpenes, sesquiterpenes, and phenylpropanoids, among which trans-anethole stands out, potentially representing 90% of the EO (Tabanca et al., 2005). Trans-anethole is considered “GRAS” (Generally Recognized as Safe) by the Food And Drug Administration (FDA) and the Flavor Extract Manufacturers Association (FEMA) in the United States (Rietjens et al., 2023). It has a strong and characteristic smell capable of masking unpleasant odors and, for this reason, is used as a flavoring by the food industry in beverages, sweets, and gums, as well as in industrial products such as medicines, pesticides, and synthetic flavors (Rocha & Fernandes, 2016; Cantó-Tejero, Pascual-Villalobos, & Guirao, 2022).

The degradation of food by the action of microorganisms is a recurring problem in the food industry. Microbiological control is mostly done through the use of chemicals that present undesirable aspects, such as toxicity, bioaccumulation, and unknown long-term health effects. Such points provide consumers with concern and a growing search for foods without the addition of synthetic products (Matrose et al., 2021)

Concern over the use of chemicals generates interest and a search for new natural additives that possess antimicrobial and antioxidant activity, improving the shelf life of foods (Jafarzadeh et al., 2020). In this sense, the use of essential oils becomes an option for consumers and industry demands (Silva et al., 2021).

Among the microorganisms that cause food deterioration is the fungus Colletotrichum sp., one of the main responsible for the occurrence of anthracnose in fruit trees such as banana, mango, and papaya (Sandoval-Contreras et al., 2020; Nishshankage et al., 2024; Zhu et al., 2024). For the management of this disease, chemical fungicides are traditionally used, which raise serious concerns about health effects, such as the development of congenital diseases and exposure to carcinogenic substances (Bordoh et al., 2020; Ciofini et al., 2022). Thus, the use of EO presents an interesting alternative for the management of anthracnose (Ciofini et al., 2022), especially the EO of P. anisum, which has already demonstrated an inhibitory effect on fungal pathogens (Soliman & Badeaa, 2002).

Despite the extensive use of EO in the cosmetic, food, pharmaceutical industries, and its antimicrobial and antifungal potential, the extraction process generates a significant amount of residues with potential for utilization (Santana-Méridas, González-Coloma, & Sánchez-Vioque, 2012; Gavarić et al., 2015). When these materials are not properly managed, they cause environmental problems, presenting an intense polluting effect due to the presence of hydrolates. However, they are a considerable source of biologically active phenolic compounds, with various health benefits and that can become appealing for the development of new products (Sánchez-Vioque et al., 2013; Gavarić et al., 2015; Skendi et al., 2022).

Considering the growing demand for products of natural origin, the aim of the work was to provide a broad overview of the extraction method used, addressing the effect of using different temperatures in the hydrodistillation process on the chemical composition and antifungal activity of anise essential oil against the fungus Colletotrichum sp., in addition to the production and characterization of flour made from the plant residue, presenting a new possibility for the utilization of the generated residue.

Material and Methods

Raw material

Anise seeds were sourced from suppliers of the Bem Viver Grãos - Bulk Natural Products group, a company located in Minas Gerais.

Production of anise products

The seeds underwent a hydrodistillation process using the Clevenger method to obtain essential oil. A total of 457 g of anise seed was used in a 5L volumetric flask. The seeds, immersed in distilled water, were subjected to two heating processes.

In the first test, the flask was heated at 145 °C for 4 hours. The second experiment was conducted at 97 °C for 4 hours. The essential oil obtained from the first test was named OE145C, and the one from the second experiment was named OE97C.

The crude oil samples collected from the first and second trials, separately, were centrifuged at 10000 RPM for 15 minutes. Sodium sulfate (Na2SO4) was added as a means of removing residual water, followed by filtration and storage in amber-colored bottles under refrigeration (Anastasopoulou et al., 2020). The extraction yield (mL/Kg) of the essential oil (EO) obtained in both experiments was determined by Equation 1 as described by Das et al, (2021).

$$ExtractionyieldEO=\frac{ExtractedEOsample(mL)}{ExtractedEOsample(Kg)}$$ (1)

The residue from the anise seeds used in the hydrodistillation was collected after the completion of the essential oil extraction process. The residue was dried under conditions of 60 °C for 24 hours and ground in a mill (MA 048 - Marconi). The crude flour obtained was sieved through 250 µm/60 mesh screens, achieving a granularity similar to that of wheat flour. After obtaining the flour, the sample was stored in plastic containers under room temperature conditions and protected from light. The samples of crude anise residue were frozen for comparative purposes.

Chemical characterization of anise essential oil by gas chromatography

The obtained anise essential oil product was subjected to gas chromatography-mass spectrometry (GC-MS) analysis at the Laboratory of Natural Products Chemistry of Embrapa Tropical Agroindustry in Fortaleza/CE using a Varian (Agilent) model CG-450 /MS-240 instrument. In the analysis, parameters such as electron impact at 70 eV, VF-5MS methylpolysiloxane column with dimensions of 30 m x 0.25 mm x 0.25 µm, split-flow injection mode 1:30, carrier gas helium with a flow rate of 1.00 mL.min-1 (8.7 psi) and a constant linear velocity of 36.7 cm.s-1, with the injector and transfer line temperature at 250 °C were recorded. The chromatographic oven had an initial temperature program of 70 °C with a heating ramp of 4 °C.min-1 up to 180 °C for 27.5 min, followed by a heating ramp of 10 °C.min-1 up to 250 °C, at the end of the run (34.5min). Through the analysis of fragmentation patterns displayed through the mass spectra, it was possible to identify compounds present in the anise essential oil.

Evaluation of antifungal and fungitoxic activity in vitro

The Colletotrichum sp. isolate (UFCm 0761) used in this study is part of the fungal collection of the Plant Pathology Laboratory at Embrapa Tropical Agroindustry, located in Fortaleza, CE. The phytopathogen was isolated directly from papaya fruits showing symptoms of the disease and signs of the phytopathogen, thus indicating a high likelihood of being part of the Colletotrichum gloeosporioides complex. The isolated fungus was subjected to microscopic analysis for genus identification according to internal protocols for the isolation and purification of fungi. Concentrations of 500, 1000, 1500, and 2000 µL/L were tested for both extracted oils. Tebuconazole (a fungicide) served as the positive control (+), while the negative control (-) consisted solely of the culture medium and mycelium discs. Initially, the essential oil was diluted in Dimethyl Sulfoxide (DMSO) at a 1:1 ratio at the specified concentrations, then introduced into the liquefied culture medium. Following this procedure, three Petri dishes for each dilution were prepared. After the culture medium solidified, 5 mm mycelium discs of an 8-day-old Colletotrichum sp. pathogens were placed at the center of each Petri dish. The dishes were then sealed with cling film and incubated in a BOD incubator (LIMATEC LT 320 TFP) at 28 ºC under a 12-hour photoperiod.

Measurements of mycelial growth (MG) and the mycelial growth index (MGI) or mycelial growth rate (MGR) were calculated according to the modified Nakagava Maguire formula, as adapted by Salgado et al. (2003), and demonstrated through Equation 2.

$$ICM=\frac{C1}{N1}+\frac{C2}{N2}+\mathrm{...}+\frac{Cn}{Nn}$$ (2)

In Equation (2), C1, C2, Cn correspond to the mycelial growth of the colonies at the first, second, and last evaluation, respectively, while N1, N2, Nn relate to the number of days. Evaluations were conducted over 8 days at 24-hour intervals, through measurements of the colony diameters (average of two diametrically opposed measurements). The experiment was concluded when the plates of the negative control fully covered the surface of the culture medium. The results were statistically analyzed using the ANOVA method (p < 0.05).

Particle size distribution of anise flour

The average particle diameter of the flour produced from the residue of anise essential oil (EO) extraction was determined according to the method described by the American Association of Cereal Chemists (AACC International, 1999). Approximately 100 g of the material was measured on an analytical balance and placed in a sieve shaker consisting of five sieves, 30, 40, 60, 80, and 100 mesh, followed by particle separation with agitation for 5 minutes. The results were expressed as a percentage relative to the mass of the original material.

Proximate composition of anise flour

The moisture content (%) was determined by drying the samples (flour and residue) in an oven at 105 °C for 3 hours until constant weight (IAL, 2008). The quantification of ash present in the samples was performed by incineration in a muffle furnace at temperatures up to 550 °C (Instituto Adolfo Lutz, 2008) with an initial mass of 5g.

The lipid content was quantified using the Soxhlet method, initially using 2g of sample, according to Instituto Adolfo Lutz (2008). The protein content was quantified according to The Association of Official Analytical Chemists (AOAC, 1990). A sample of 0.5g of flour and residue was used, undergoing digestion, distillation, and titration steps according to the method for protein content quantification. The carbohydrate content was determined by difference.

Mineral composition of anise flour

The mineral composition of anise flour was determined at the Soil Microbiology Laboratory of Embrapa Tropical Agroindustry. Initially, the flour samples underwent digestion in a digestion block using nitric acid and perchloric acid. Subsequently, the inorganic components were analyzed using inductively coupled plasma atomic emission spectrometry (ICP-AES), following the methodology described by Miyazawa et al. (2009). This method was employed to quantify phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), copper (Cu), iron (Fe), zinc (Zn), and manganese (Mn).

Physicochemical analyses

pH

pH analyses were conducted using a pH meter as described by Instituto Adolfo Lutz (2008). The pH meter was calibrated with a pH 7 buffer solution and subsequently with a pH 4 solution. A 10g sample was weighed and diluted in 100 mL of distilled water. A probe and thermometer were used for readings in a laboratory environment at 20 °C.

Total titratable acidity

The standards of the IAL (2008) were followed, using 0.1N sodium hydroxide (NaOH) for volumetric titration. A 2.0g sample dispersed in 50mL of distilled water with 2 to 4 drops of phenolphthalein solution was used. The titration with 0.1N NaOH continued until a permanent pink coloration was evident.

Hygroscopicity

The hygroscopicity analysis followed the methodology described by Goula and Adamopoulos (2008), using 1.0g of sample in Petri dishes containing hygroscopicity cells at 75% relative humidity for 90 minutes. The hygroscopicity test concluded with the weighing of the plates. Hygroscopicity was calculated using Equation 3, ultimately expressed as a percentage.

$$Hygroscopicity\text{(\%)}=\frac{X}{U\times a}\times 100$$ (3)

In Equation 3, hygroscopicity corresponds to the mass of water absorbed (g) per 100g of sample (solids), with X being the mass of water absorbed (g), U being the moisture of the powder on a dry basis (g/g), and a being the mass of the sample (g).

Bioactive compounds - preparation of extracts

Extracts were obtained from anise residue and anise flour samples, initially using 5g of each sample. Then, the samples were added to 20 mL of 50% ethanol and homogenized in a vortex, remaining in a dark place at room temperature for one hour. The samples were centrifuged at 11000 RPM for 15 minutes. The supernatant was filtered and transferred to a 50 mL amber flask. The remaining residue in the Falcon tubes was added to 20 mL of 70% acetone and homogenized again in a vortex. The tubes were stored in a dark place at room temperature for one hour. Afterwards, the samples were centrifuged under the same conditions, and the supernatant was filtered into the same volumetric flask. The obtained sample was stored in plastic pots without light contact and kept frozen until analysis time.

Total phenolic compounds

For the quantification of total phenolic compounds, 30µL of anise flour extract and 100 µL of anise residue extract were used in test tubes, both made up to 500 µL with distilled water. A 0.5 mL of 1:3 Folin Ciocalteau solution was added, as well as 1.0 mL of 20% sodium carbonate and 1.0 mL of distilled water, vortexed and kept away from light and at room temperature for 30 minutes. The samples were read in a spectrophotometer at 700 nm (Larrauri, Rupérez & Saura-Calixto, 1997).

Total Antioxidant Activity by the ABTS+ Method (2,2´-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid))

The total antioxidant activity by the ABTS+ free radical capture was determined according to Rufino et al. (2007). Volumes of 10, 20, and 30µL of each diluted extract were used, respectively, in 20, 10, and 0 µL of distilled water pipetted into test tubes. Each tube was added to 3.0 mL of ABTS radical solution with absorbance ranging between 0.700 to 0.705 nm. The tubes were vortexed and kept away from light throughout the analysis. Then, the reading was performed in a spectrophotometer at 734 nm after 6 minutes. Ethyl alcohol was used as a blank to calibrate the equipment.

Colorimetric analysis

Colorimetry analysis was determined using a Hunter Labscan colorimeter (Hunter Associates Laboratory, Inc., Reston, VA, USA) with the aid of Easy match QC 4.1 software, expressed in L*, a*, and b*, according to the CIE/Lab color space. The coordinates vary in the spectrum from blue (-a*) to yellow (+a*) and green (-b*) to red (+b), with L corresponding to the sample’s brightness variation, ranging from 0 to 100 (variation from black to white).

Results and Discussion

Yield and composition of anise essential oil

The essential oils exhibited a translucent yellow light with a strong characteristic aroma, in accordance with the characteristics described by Özcan and Chalchat (2006) and Benelli et al. (2018). The yields obtained were 5.83 and 10.37 mL Kg^-1 for the oils extracted at 97 °C and 145 °C, respectively. The yield found for the oil extracted at 97 °C was lower than the values reported in the literature, which expressed variations between 10.0 to 56.0 mL kg^-1 (Orav, Raal, & Arak, 2008; Ullah & Honermeier, 2013). Authors have reported that variations in yield can be caused by various intrinsic and extrinsic factors. Iannarelli et al. (2017) in his study highlighted the influence of temperature and precipitation on the yield of P. anisum essential oil, where conditions of higher temperatures during the vegetative cycle were favorable for a higher yield of essential oil. Abd El-Wahab (2006) in his work of evaluating the influence of water salinity on irrigation, observed a decrease in the yield of anise essential oil and a reduction in the percentage of trans-anethole with the use of saline water. Orav, Raal and Arak, (2008) and Ullah and Honermeier (2013) also reported that variations in yield could be attributed to differences in harvest time, development stage, soil type, pedoclimatic conditions, sowing density, relative humidity on the day of collection, and storage conditions.

Using gas chromatography coupled with mass spectrometry (GC/MS), it was possible to confirm the presence of the compound E-anethole (Figure 1) in the oil extracted at 145 °C. On the other hand, the oil extracted at 97 °C showed levels of 98.71% E-anethole and 1.29% of the secondary compound γ-himachalene (Figure 2). These data are expressed in Figures 2 and 3, respectively. We highlight the presence of the E-anethole compound at 100% and 98.71%, showing higher levels than those found in the literature, which presented a variation between 64.7% (Khalid, 2015) to 93.7% (Orav, Raal, & Arak, 2008). Ullah and Honermeier (2013), studied three cultivars of anise in two different seasons and observed the majority presence of trans-anethole varying between 90.20 to 79.62%, followed by the presence of γ-himachalene between 8.31 to 2.11%. In another study conducted with Pimpinella anisum L. from the island of Lesvos, Greece, by Anastasopoulou et al. (2020), the presence of trans-anethole was identified between 87.00 to 89.99%, and the presence of the compound γ-himachalene varied between 1.31 to 2.90%.

Figure 1:
Chemical structure of the compounds identified in the GC-MS analysis.

Figure 2:
Chromatography of anise essential oil extracted at 97 °C.

Figure 3:
Chromatography of anise essential oil extracted at 145 °C.

In the present study, it was observed that with the increase in temperature, anethole was isolated. This probably occurred because the minor volatile compounds are more prone to volatilization with the temperature rise. There was also a doubling in the amount of essential oil obtained, demonstrating a positive relationship between the increase in temperature, yield, and purity of the compound of greatest interest. Despite being a process under constant pressure, it was observed that the heating temperature of the thermal blanket influences the extraction process. It is believed that the isolation of trans-anethole by the hydrodistillation method opens doors to new possibilities for the purification of this compound. However, studies should be directed towards a better understanding of this procedure. Although there are reports about the influence of time (Anastasopoulou et al., 2020) and pressure (Rodrigues et al., 2003) on the kinetics of the essential oil extraction process from Pimpinella anisum L., information on the effect of extraction temperature on its constituents is scarce.

Evaluation of in vitro antifungal activity

The anise essential oil effectively inhibited the mycelial growth of Colletotrichum sp. The concentrations of 1500 µL/L and 2000 µL/L of the essential oil extracted at 145 °C and the concentration of 2000 µL/L of the essential oil extracted at 97 °C (Table 1) showed no difference from the positive control using commercial fungicide, Tebuconazole (Figure 4). Thus, the concentration of 1500 µL/L is the lowest fungistatic and fungicidal concentration of the oil extracted at 145 °C, and 2000 µL/L is the lowest fungistatic and fungicidal concentration of the oil extracted at 97 °C. Therefore, higher anethole contents are responsible for a better fungicidal action. These results indicate that anethole may be responsible for the antifungal action of anise essential oil and its potential application as a natural food preservative and in the control of anthracnose.

Table 1:
Mycelial growth index of Colletotrichum sp. in different concentrations of EO-A extracted at 97 °C and 145 °C.

Temperature	500 µL/L	1000 µL/L	1500 µL/L	2000 µL/L
97 °C	30.50a	21.53b	17.22c	9.04d
145 °C	26.61b	12.66c	7.12d	6.55d

Figure 4:
Plates with controls and concentrations of EO-A in inhibiting mycelial growth of Colletotrichum sp. a: EO-A extracted at 97 °C - 500 µL/L; b: EO-A extracted at 97 °C - 1000 µL/L; c: EO-A extracted at 97 °C - 1500 µL/L; d: EO-A extracted at 97 °C - 2000 µL/L; e: EO-A extracted at 145 °C - 500 µL/L; f: EO-A extracted at 145 °C - 1000 µL/L; g: EO-A extracted at 145 °C - 1500 µL/L; h: EO-A extracted at 145 °C - 2000 µL/L; i: Negative control; j: Control with DMSO; k: Positive control.

The phenylpropanoid trans-anethole is related to antimicrobial activity and may be the component responsible for this action; however, records in the literature on this hypothesis are scarce (Gülçın et al., 2003; Das et al., 2021). Thus, trans-anethole alone may be responsible for this activity.

Colletotrichum sp. is the phytopathogen responsible for anthracnose, a post-harvest disease of significant impact on the agricultural system, as it generates significant losses in fruit culture. Many studies using essential oils are directed at controlling this pathogen (Rozwalka et al., 2008; Bosquez-Molina et al., 2010; Andrade & Vieira, 2016; Ramos et al., 2016; Correa-Pacheco et al., 2017; Diánez et al., 2018; Peralta-Ruiz et al., 2020). In the present study, the anise essential oil proved effective in controlling this pathogen.

At eight days of measurements, the difference between the plates containing 100% anethole (a, b, c, and d) and 98.17% anethole (e, f, g, and h) is visible (Figure 4). These results suggest that anethole may be responsible for the reduction in activity of Colletotrichum sp.

Andrade and Vieira (2016) reported fungitoxic and fungistatic effects of P. anisum essential oil on C. gloeosporioides in their study with different essential oils in the control of the phytopathogen. The authors assert that P. anisum oil can be used for the control of anthracnose, corroborating the results obtained in the present work.

Das et al. (2021), in their study with encapsulated essential oil of P. anisum, observed the fungitoxic effect and the inhibition of aflatoxin of Aspergillus flavus (AF LHP R14). In the same study, the biochemical action mechanism of the oil against fungal growth inhibition was also verified, and damage to the biosynthesis of ergosterol, leakage of cellular ions, and disturbance of the fungus’s oxidative defense system were found. The use of P. anisum in this work opens perspectives that anethole may be responsible for this action.

Hong et al. (2015) demonstrate that the carvacrol, trans-cinnamaldehyde, citral, p-cymene, and linalool compounds are the substances responsible for inhibiting the germination of C. gloeosporioides in pepper fruits. Another study conducted by Xie et al. (2017) points to the cinnamaldehyde and citronellal compounds as effective against Rhizoctonia solani and Fusarium oxysporum. Although studies are focusing on the evaluation of isolated compounds and their respective mechanisms of action, similar information about anethole is still scarce. Studies with scanning electron microscopy are necessary to evaluate the mechanism of action profile of anethole against Colletotrichum sp.

Centesimal composition of anise residue flour

In Table 2, one can observe the flour composition obtained from the residue of the processing of dried and ground anise essential oil.

Table 2:
Centesimal composition of flour obtained from anise residue flour.

Components	Quantity in g/100g flour
Carbohydrates	50.46±0.00
Proteins	19.81±0.09
Lipids	20.31±0.01
Ashes	4.65±0.04
Moisture	4.77±0.07

Regarding the proximate composition of the obtained flour, the total protein content was around 20.3%. The product exhibited a high protein content when compared to flours from vegetables commonly associated with a protein-focused diet, such as sweet potato. De Araújo et al. (2015) reported a 6.32% protein value in sweet potato flour after drying at 60 °C, while Jaime et al. (2020) achieved a content of 7.38%, and of 9.85% in biofortified sweet potato flour obtained from different states in Brazil. Thus demonstrating that the protein content of the flour obtained in the present study is more than twice than that found in sweet potato flours.

The process of essential oil extraction and subsequent drying of the residue contributes to the concentration of the remaining major compounds present in the anise seed, increasing its nutritional value. De Souza et al. (2016) also found a high protein value in their studies with Brazil nut residue, with values around 37.54%, being considered an excellent protein source and undergoing drying and lipid extraction processes, similar to the raw material of the present study.

As for the lipid content, the flour from the study in question has around 20.31%, a considerable amount when compared with commercially used flours, such as wheat flour and barley flour, having 1.68% and 1.73% respectively (Adhikari, Bajracharya, & Shrestha, 2016). Comparing the result obtained with the Brazil nut residue flour, an oilseed studied by De Souza et al. (2016), the lipid percentage is much lower than that obtained in their work, reaching 35.33% even with the flour being partially defatted. Gouda (2019) reported a lipid value of 14.33% in a powder obtained from dried, ground, and crushed anise seeds for bread supplementation, a value below that obtained in the present study. One of the factors that may have influenced the lipid content is the time versus temperature binomial used for drying the residue, considering that a greater removal of moisture from the sample results in the concentration of other components.

In his work, Gouda (2019) obtained a moisture content in his anise powder of 9.88%, compared to that obtained from the residue, which was of 4.77%, halving the value of free water present in the processed flour. A longer process time during drying allows for greater water removal from the product, being an important factor in both the safety of the product, not offering conditions for the development of microorganisms, and the enzymatic reactions, as well as in the storage of the product, allowing for storage at room temperature.

The ash content showed a value of 4.65% after drying the residue. This value is below what is required by the Brazilian legislation for the maximum ash content allowed in anise, which is a maximum of 12% (Agência Nacional de Vigilância Sanitária - ANVISA, 2019). The ash content is related to the inorganic matter present in the sample, which is associated with the number of minerals present in the sample. The analyzed flour showed a higher ash content when compared with the flour obtained from oilseed residue, which presented 4.08% in 100g of Brazil nut residue (De Souza et al., 2016) and also a higher content than flour obtained from fruit residues, such as bacaba residue flour, which presented 2.97% (Alvares et al., 2021) and acerola residue flour, which had an ash content of 1.76% (Magalhães et al., 2021).

Mineral composition of anise flour

Table 3 presents the values obtained from the inorganic matter analysis of the flour and the daily reference values (DRV) established by the National Health Surveillance Agency in Normative Instruction No. 75, dated October 8, 2020 (Brasil, 2020).

Table 3:
Mineral composition of flour obtained from anise residue flour.

Minerals	Quantity in mg/100g flour	DRV (Brazil, 2020)
Phosphorus (P)	450±0.00	700 mg
Potassium (K)	943,33±0.05	3500 mg
Calcium(Ca)	816.33±0.05	1000 mg
Magnesium (Mg)	250±0.00	420 mg
Sódio (Na)	240±0.00	2000 mg
Cobre (Cu)	1.5±0,00	0,9 mg
Iron (Fe)	22,83±0.75	14 mg
Zinc (Zn)	6.56±0.06	11 mg
Manganese (Mn)	2.63±0.06	3 mg

• All values given are means of three determinations means ± SD deviation.

According to Brasil (2020), foods classified as mineral sources contain a minimum of 15% of the DRV for the respective mineral, while foods with high mineral content must contain at least 30% of the DRV. Consequently, it was observed that anise flour exhibits high content of manganese, calcium, phosphorus, zinc, and magnesium, with 87.66%, 81.63%, 64.28%, 59.64%, and 59.52% of the DRV, respectively. Furthermore, it is a source of potassium with 26.95% of the DRV and has a low sodium content with only 12% of the DRV.

Moreover, we highlight the iron and copper values that exceed the daily reference values set by the Ministry of Health. The intake of iron and copper is essential for the formation of hemoglobin, which constitutes blood and is important for blood transport. The ingestion of these micronutrients is also related to various biological functions such as mitochondrial respiration and DNA synthesis, as well as anti-inflammatory and antioxidant actions, and the proper functioning of the immune system (Skrypnik et al., 2025; Huang et al., 2024). The quantities of minerals found reflect an excellent mineral profile in anise flour. According to the data obtained, it can be used for mineral fortification, especially iron and copper, in food preparations.

Physicochemical characterization of the flour

In Table 4, the results of the physicochemical characterization of the residue flour can be observed, which are important for the stability, storage, and functional quality of the product.

Table 4:
Physicochemical characterization of flour obtained from anise residue.

Componentes	Quantity
Aw	0.25±0.01
pH	5.6±0.02
Titratable acidity (% citric acid)	0.88±0.02
hygroscopicity (%)	48.02±0.13
Polyphenols (mg de EAG/100g)	28.93±0.02
Antioxidant activity (μM trolox/g flour)	5.60±0.89

The water activity (Aw) value found in the anise residue flour was 0.25. A food’s water activity value (a measure of the availability of water to microbes) is inversely correlated with microbial growth and the survival of bacteria in the medium. This is a crucial factor to consider when extending the shelf life of a product (Forsythe, 2020). In studies conducted by De Souza Silva et al. (2023), analyzing flour obtained from plant residues, a water activity value of 0.4 was found, higher than that found in the present work. Gouvea et al. (2020) found a water activity value of 0.4 in their studies with beet stalk flour. Meanwhile, Alvares et al. (2021) demonstrated a water activity of 0.53 in flour obtained from the residue of bacaba oil extraction, both above the value found by the present study. A low water activity indicates that the drying of the food was effective, offering stability, and is considered of utmost importance for flours and dry products.

Regarding the pH value and acidity, a pH value of 5.6 was found, considered to be relatively acidic, and an acidity of 0.88% of citric acid. Magalhães et al. (2021) obtained a pH of 3.23 and an acidity of 3.30% in citric acid, with flour produced from acerola pulp processing residue. Meanwhile, Borges et al. (2021) found a pH value of around 4.30 and an acidity of 1.22% in citric acid with flour produced from açaí residue. The low pH values found in the literature are due to the fact that the flours were obtained from fruit residues, which are naturally more acidic than anis, which has an average pH of 5.38 (Da Silva Neto et al., 2020).

The higher the amount of organic acids present in the food, the lower its pH, a relationship proven in the results obtained and in comparison with the literature. The low pH and, consequently, higher acidity, assist not only in the sensory characteristics of the product but also have an effect on food preservation, acting against the growth of microorganisms. A pH close to neutrality (7.0) favors the growth of microorganisms, while those further away, in both directions, reduce their capacity for multiplication (Forsythe, 2013; Borges et al., 2021).

The hygroscopicity of the residue flour was 48.02%, showing that the product has a tendency to absorb water during storage. Oliveira, Costa and Afonso (2014) obtained a hygroscopicity of 12.93% and 8.51% for powder prepared from whole cajá pulp and powder added with 17% maltodextrin, respectively, showing that maltodextrin can be added to the analyzed product to decrease high hygroscopicity and, consequently, allow a longer storage time for the flour.

Daily consumption of foods containing polyphenols contributes to the health and well-being of the consumer, as their intake in the diet can favor the reduction of various diseases, such as cancer and cardiovascular diseases, due to the antioxidant effect of these bioactives (Medina-Remón et al., 2015). The total amount of phenolics found in the flour sample was 28.93 mg GAE/100g of sample. When compared with fruits and vegetables marketed in Brazil, the value found is below fruits such as pineapple (85.1 mg GAE/100g), banana (215.7 mg GAE/100g), and orange (114.6 mg GAE/100g), but has a higher amount than papaya and tomato, with 15.3 and 13.7 mg GAE/100g, respectively (Faller & Fialho, 2009). Arruda et al. (2024) studied the amount of polyphenols ingested in traditional Brazilian meals and plant-based foods, such as fruits, vegetables, cereals, and derived products. These foods present significant levels of polyphenols, opening an interesting space for the inclusion of these anise residue flour-based products in the population’s diet.

The antioxidant action can delay or inhibit the appearance of free radicals causing oxidative stress, acting through various mechanisms and being important for protection against oxidation and also acting against the increase of this oxidative stress in the body, preventing cellular damage (Fennema, Damodaran, & Parkin, 2010; Teixeira et al., 2016). According to Table 2, the result of the antioxidant activity of the flour was 5.60±0.897 μM trolox/g of flour, not as satisfactory as other flours obtained from residues found in the literature. Zopellaro, Da Silva and Lovato (2019) found an antioxidant activity value of 127.07 μM trolox/g of flour obtained from grape processing residue, superior to that found in the flour of the present study. This observed reduction in the total antioxidant content may be related to the drying conditions used, to the characteristics of the plant itself and even to the degree of ripeness. It is worth highlighting the sensitivity of antioxidant molecules to prolonged exposure to heat, as well as the amount of antioxidants present in the plant. Grapes are rich in anthocyanins and can therefore have high levels of this compound (Lima et al., 2021).

Particle size distribution of the flour

In Figure 5, the particle size distribution of the produced flour is presented. It was observed that approximately 50% of the flour was retained on a 60 mesh sieve, which corresponds to an opening of 250 µm, while the remainder was distributed across five different particle sizes, with a smaller percentage of flour, approximately 1.5%, having a diameter smaller than 150 µm.

Figure 5:
Particle size distribution of anise flour.

Based on the obtained particle diameters, the produced flour is classified as fine flour, according to the legislation for the classification of cassava flour (Brasil, 2011). The particle size of flours is an important parameter for application in food products, as it influences physicochemical parameters, such as water absorption capacity, generating greater dispersibility in various uses, in addition to influencing sensory aspects, such as appearance, flavor, and texture (Storck et al., 2015). In a study conducted by Troilo et al. (2022), negative effects of the addition of grape pomace powder with different particle size fractions were observed as the particle size decreased. Whereas, Cristiano et al. (2019) attributed the lower presence of elastic and viscous modulus to the particle’s size difference between flours, observing different behaviors in the water and flour mixture of different particle sizes.

Different particle sizes are suitable for different products and should be evaluated considering the raw material used. Storck et al. (2015) studied the use of agro-industrial residues and identified that particles smaller than 250 µm are more suitable for incorporation into bakery products.

Colorimetric analysis

The result of the colorimetric analysis of the residue before and after drying is presented in Table 5.

Table 5:
Colorimetric parameters.

Sample	Parameters
	L*	a*	b*	C*	h
Residue	30.41±0.91	6.64±0.09	19.72±1.30	20.81±1.24	71.33±1.12
Flour	35.01±0.38	5.57±0.09	22.97±0,53	22.67±0.52	75.78±0.26

The residue exhibited a* and b* coefficients ranging between 6.64±0.09 and 19.72±1.30, while the flour displayed such coefficients ranging between 5.57±0.09 and 22.97±0.53, respectively. With the formation of a saturation angle (h) at 75.78±0.27 and lightness (L*) 35.01±0.38. The flour sample from the residue after drying is visually presented with a slightly greenish-yellow coloration.

In contrast, the residue showed a lower saturation angle (h) at 71.33±1.12 and lightness (L*) of 30.41±0.91, with lower lightness than that of the flour. The increase in lightness in the anise flour can be explained by the availability of starch which, according to Azevedo et al. (2012), is present in the cotyledon structures that make up the anise seed. Such a difference in lightness was mainly due to the grinding of the seeds and standardization through sieves, uniformizing the color and appearance of the sample.

Conclusions

It is concluded that the extraction conditions of anise essential oil (EO) can influence its in vitro biological action as fungistatic and fungicidal agents in the control of post-harvest diseases such as anthracnose, generating residues with potential for application in the production of anise flour for human consumption as a means of nutritional enrichment. These findings are substantiated by the macro and micronutrient composition and biological properties investigated in the current study.

Acknowledgement

The authors are grateful for the financial support provided by Ceará Foundation to Support Scientific and Technological Development (FUNCAP). This work was funded by FUNCAP (MLC-00191-00087.01.00/22).

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• Editor de seção:
  Renato Paiva

Publication Dates

• Publication in this collection
  13 Dec 2024
• Date of issue
  2024

History

• Received
  16 Sept 2024
• Accepted
  29 Oct 2024