Effect of applying lime essential oil (Citrus latifolia) on the physicochemical and microbiological characteristics of beef meat sausage

Efeito da aplicação do óleo essencial de limão (Citrus latifolia) sobre as características físico-químicas e microbiológicas da linguiça bovina frescal

ABSTRACT

The objective of this study was to investigate the potential of lime essential oil as a substitute for synthetic preservatives in beef sausage, considering consumer demand for healthy meat products produced with natural ingredients. Lime peel essential oil (LEO) was obtained by hydrodistillation and subjected to an evaluation of antibacterial activity by the disc diffusion and microdilution method. Its chemical composition was determined by gas chromatography coupled with mass spectrometry. Three sausage formulations were developed in this study: the first without preservatives (LC), the second with synthetic preservatives (L1), and the third containing 0.5% lime essential oil (L2). Physicochemical and microbiological analyses indicated that all treatments followed current legislation, although the moisture content exceeded the maximum limit. The pH and color varied significantly during refrigerated and frozen storage, reaching stability after 20 days. lime essential oil, with D-limonene as the majority component, proved to be effective in inhibiting microbial growth at a concentration of 0.5%, preserving the physicochemical composition of the sausage. Furthermore, there is a tendency for the color to stabilize during frozen storage. Therefore, 0.5% lime essential oil is a viable and natural alternative for application in meat sausages, such as fresh sausage, and adds a different flavor and aroma to this product.

Keywords
hydrodistillation; citrus fruits; microbiological analysis; meat sausages

RESUMO

O objetivo desse estudo foi investigar o potencial do óleo essencial de limão como substituto para conservantes sintéticos em linguiça de carne bovina, considerando a demanda dos consumidores por produtos cárneos saudáveis e produzidos com ingredientes naturais. O óleo essencial da casca do limão (OEL) foi obtido por hidrodestilação, e submetido a avaliação da atividade antibacteriana pelo método de difusão de discos e microdiluição, assim como, foi determinado a sua composição química por cromatografia gasosa acoplado a espectrometria de massas. Três formulações de linguiça de carne bovina foram desenvolvidas neste estudo: a primeira sem conservantes (LC), a segunda com conservante sintético (L1) e a terceira contendo 0,5% de óleo essencial de limão (L2). As análises físico-químicas e microbiológicas indicaram que todas as amostras estavam em conformidade com a legislação vigente, embora o teor de umidade tenha excedido o limite máximo. Durante o armazenamento refrigerado e congelado, o pH e a cor variaram significativamente, atingindo a estabilidade após 20 dias. O óleo essencial de limão, com d-limoneno como componente majoritário, mostrou-se eficaz na inibição do crescimento microbiano na concentração de 0,5%, preservando a composição físico-química da linguiça. Além disso, evidenciou-se uma tendência de estabilização da cor durante o armazenamento congelado. Portanto, a aplicação de 0,5% do óleo essencial de limão é uma alternativa viável e natural para aplicação em embutidos cárneos, como a linguiça frescal, além de agregar sabor e aroma diferenciados a este produto.

Palavras-chave
hidrodestilação; frutas cítricas; análise microbiológica; produtos cárneos

1. Introduction

Meat products are foods that stimulate great preference among Brazilian consumers, as they are an accessible form of animal-origin protein, low cost, with quick and practical preparation, in addition to presenting a variety of products available on the market, such as sausages, hamburgers, nuggets, seasoned meats, and others (Vessoni et al., 2019).

Sausage is a meat product obtained using ground meat from different animal species, seasoned, containing other ingredients or not, stuffed in a natural or artificial casing, and subjected to a specific technological process. This product may contain 30 to 35% fat in its composition (Brasil, 2000a; Brasil, 2000b; Brasil, 2017).

According to Vessoni et al. (2019), despite the high consumption of these meat products, consumers still have imminent concern regarding the high content of saturated fats and chemical preservatives. The occurrence of the COVID-19 pandemic affected people’s eating habits in two ways, with an increase in binge eating due to anxiety or dietary re-education aimed at healthier habits. In the first case, the consumption of industrialized meat products increased due to the practicality of preparation and greater accessibility. In the second case, the population’s concern with healthier habits, including food, led to a reduction in the consumption of industrialized products (Durães et al., 2021).

According to the National Cancer Institute, the consumption of processed meats, such as ham, sausage, salami, and bacon, can cause stomach and intestinal cancer (Instituto Nacional do Câncer, 2022; Ribeiro et al., 2019). The risks associated with the consumption of these products are related to the presence of nitrates and nitrites used as preservatives, which can undergo endogenous nitrosation and become potentially carcinogenic to humans. Recently, nitrate and nitrite have been linked to the risks of breast and prostate cancer, respectively (Chazelas et al., 2022). Therefore, the replacement of these preservatives with natural products has been the subject of many studies.

Plant essential oils are natural products widely used in meat products, such as hamburgers and sausages, due to their antioxidant and antimicrobial properties. These oils have demonstrated a promising role in controlling the growth of pathogenic microorganisms, such as Escherichia coli and Staphylococcus aureus, in addition to helping to prevent lipid oxidation and preserve the color of foods (Sharafati-Chaleshtori et al., 2015; Gahruie et al., 2017; Sharma et al., 2019; Pateiro et al., 2021).

Lime essential oil (Citrus latifolia) is a bioactive compound rich in polyphenols and acts as an antioxidant, with D-limonene as its main terpene. Another biological property of this essential oil is its antibacterial action, which sparks interest in its application as a natural preservative in foods. Additionally, the Food and Drug Administration considers it safe for intentional use in foods (Teixeira et al., 2013; Ben Hsouna et al., 2017; Food and Drug Administration, 2023).

The presence of antioxidant compounds in Citrus latifolia extracts reveals great potential for maintaining the quality and shelf-life of marinated chicken meat by reducing the levels of mesophilic aerobic bacteria, inhibiting the oxidation of lipids and proteins, stabilizing color, and improving its acceptability (Lotfy et al., 2023; Budiarto et al., 2024).

Considering the antioxidant and antibacterial potential of Citrus latifolia essential oil, this work aimed to apply the essential oil extracted from lime peel (Citrus latifolia) in preparing beef sausage as a substitute for synthetic preservatives, thus proposing healthier meat product forms.

2. Material and methods

2.1. Obtaining lime essential oil

Tahiti limes (Citrus latifolia) were purchased from a supermarket chain in São Luís, Maranhão, Brazil, to obtain the essential oil. The essential oil was extracted from the lime peel by the hydrodestillation process using the Clevenger system in the Chemistry laboratory at the IFMA − Campus Maracanã (Santos et al., 2004). The density was determined using a pycnometer, and the yield was expressed on a dry basis, according to the method described by Santos et al. (2004).

2.2. Antimicrobial activity of essential oils

Antibacterial activity was evaluated by the disc diffusion method as referenced by Clinical and Laboratory Standards Institute (2015) and two strains of standard bacteria were used, E. coli ATCC 25922 and S. aureus ATCC 29213. The bacterial suspension was prepared in saline solution, and the turbidity of this solution was determined using the MacFarland scale (initial concentration of 10⁷ CFU/g). This solution was inoculated on plates with Mueller Hinton Agar (Merck, Darmstadt, Germany) and the essential oil (75 µL) embedded in sterile filter paper discs. Later, the discs were transferred to plates containing Muller Hinton Agar. The plates were incubated at 37°C for 24 hours, and transparent inhibition zones were measured using a millimeter ruler. The broth dilution method evaluated the minimum inhibitory concentration (MIC) according to Lertsatitthanakorn et al. (2014). The Brain Heart Infusion broth (Kasvi, S.A, Spain) was supplemented with an initial concentration of the essential oil and subjected to serial decimal dilutions in tubes, with subsequent inoculation of 0.1 mL aliquots of the standardized bacterial suspension and incubation of the tubes at 37°C for 24 hours. The MIC was considered the lowest concentration that inhibited bacteria growth.

2.3. Chemical composition of essential oil by gas chromatography (GC-MS)

The chromatographic analyses were conducted at the Central Analytical Laboratory of the University of São Paulo (USP). The lime essential oil constituents were identified using the Gas Chromatography technique coupled with Mass Spectrometry (GC-MS). The compounds detected were identified using the mass spectra database of the National Institute of Standards and Technology (NIST105, NIST21) and WILEY139.

2.4. Preparation of beef sausage with lime essential oil

The meat product was processed at the Meat Technology Laboratory at IFMA − Campus São Luís Maracanã, in Brazil. All raw materials used in this study were obtained from a local supermarket in São Luís, Maranhão (Table 1). First, a cut of the Semimembranosus muscle (topside cut) and animal fat (pork) was used to prepare the meat products. The meat was cleaned, and the skin and other tissues were removed. Next, the meat and fat were ground. The essential oil concentration used was 0.5%. Three formulation treatments were evaluated: control beef sausage, without the addition of preservatives and without the addition of lime essential oil (LC); beef sausage with an addition of 0.005% curing salt and 0.5% ascorbic acid (L1); and beef sausage with an addition of 0.5% lime essential oil (L2).

Table 1
Beef sausages formulation (%).

	Treatments
Ingredients	LC	L1	L2
Beef meat	81.4	80.9	80.9
Pork backfat	11.6	11.6	11.6
Onion	4.59	4.59	4.59
Salt	1.7	1.7	1.7
Garlic	0.3	0.3	0.3
Black pepper	0.2	0.2	0.2
Chilli pepper	0.2	0.2	0.2
Nutmeg	0.009	0.009	0.009
Lime essential oil	-	-	0.5
Ascorbic acid	-	0.5	-
Curing salt	-	0.005	-

• LC: Control beef sausage without added preservatives; L1: Beef sausage with the addition of 0.005% curing salt 2 and 0.5% ascorbic acid; and L2: Beef sausage with the addition of 0.5% Lime essential oil.

The mixture of ground meat and fat was divided into polyethylene trays to add condiments and other ingredients (salt, garlic, onion, black pepper, curing salt, nutmeg, antioxidant and chili pepper). The curing salt (Nutri.com, São Paulo, Brazil) used as a synthetic preservative was based on sodium nitrate and nitrite and added at a concentration of 0.005%. In addition, ascorbic acid (ISOFAR, Rio de Janeiro, Brazil) was used as an antioxidant and added to the curing salt at a concentration of 0.5%. The ingredients were manually mixed until a homogeneous mass was obtained. The mixture was embedded in synthetic casings, and then the weight and length of the sausages were standardized. Sausages were refrigerated for a maximum period of 5 days for pH and color analyses, and another part of the sausages were frozen at −18 ºC for 10 days until further physicochemical and microbiological analyses.

2.5. Microbiological analyses

The analyses were conducted according to the parameters established by Normative Instruction 161 (Brasil, 2022b) and methodology recommended by the APHA (2001), which include identification of Salmonella sp., quantification of Escherichia coli and mesophilic aerobic bacteria.

2.6. Physicochemical analyses

The physicochemical analyses of the beef sausage formulations were conducted at the IFMA Chemistry Laboratory – Campus São Luís Maracanã, in triplicate, according to the methodology described by Instituto Adolfo Lutz (2008). Moisture (012/IV), ash (018/IV), proteins (036/IV), and lipids (032/IV) were determined.

2.7. Determination of pH and color

The pH was determined with a skewer pH meter for solids (ASKO, São Leopoldo, Brazil) and color determination using a spectrophotometer (Delta Color, São Leopoldo, Brazil). These analyses were performed while the sausages were refrigerated at a temperature of 5°C (times of 0 and 5 days) and frozen at −18 °C (times of 10 and 20 days). All analyses were performed in triplicate.

2.8. Statistical analysis

The data obtained from the triplicate analyses were subjected to mean and standard deviation calculations using Excel (Microsoft®) software and analysis of variance (ANOVA), with Tukey’s test subsequently applied at a level of 5% significance in the comparison of the means using the Past 4.07b statistical program.

3. Results and Discussion

3.1. Chemical composition of lime essential oil

The essential oil extracted from lime showed an average yield of 1.74% (dry basis) for an average biomass of 416 grams. The essential oil obtained was clear and limpid, with a pleasant aroma characteristic of lime and a density of 0.881 kg/m³.

A total of 30 compounds were found in the lime essential oil belonging to the terpene class (Table 2). D-limonene is the majority compound, representing 45.67% of the compounds found in the oil, corresponding to a concentration of 195.26 µg/mL. The second category of compounds most found were gamma-terpinene and beta-pinene, corresponding to 14.08% and 13.73%, with concentrations of 60.20 and 58.69 µg/mL, respectively.

Table 2
Chemical composition of the lime (Citrus latifolia) essential oil by gas chromatography coupled to mass spectrometry.

Peak	Compound	Retention time (min)	Area (%)	Concentration (µg/mL)
1	Biciclo[3.1.0]hex-2-eno, 2-metil-5-(1-metiletil)-	6.608	0.67	2.84
2	alpha pinene	6.818	2.61	11.15
3	camphene	7.298	0.1	0.41
4	Sabineno	8.190	1.81	7.75
5	beta-pinene	8.295	13.73	58.69
6	beta-myrcene	8.867	2.04	8.70
7	alpha-phellandrene	9.324	0.1	0.43
8	δ-2-careno	9.821	0.31	1.32
9	p-cymene	10.150	5.17	22.11
10	D-limonene	10.408	45.67	195.26
11	beta-ocimene	11.154	0.13	0.55
12	gamma-terpinene	11.574	14.08	60.20
13	δ-2-carene	12.787	0.91	3.89
14	Linalool	13.316	0.77	3.30
15	Carvone epoxide	14.721	0.18	0.75
16	Carvone epoxide	14.927	0.16	0.66
17	Oxirane, 2-(hexin-1-yl)-3-methoxymethylene	15.275	0.11	0.48
18	4-terpineol	16.666	1.06	4.55
19	terpineol	17.278	1.27	5.41
20	decanal	18.029	0.16	0.69
21	Citrol	19.015	0.73	3.14
22	Neral	19.558	1.58	6.77
23	Geraniol	20.208	0.4	1.73
24	Neral	20.889	2	8.55
25	geranyl acetate	25.021	1.24	5.28
26	geranyl acetate	25.841	0.35	1.51
27	beta-elemene	26.118	0.09	0.39
28	Beta-carophyllene	27.199	0.3	1.28
29	Trans-alfa-bergamoteno	27.954	0.87	3.70
30	beta-bisaboleno	30.930	1.41	6.02

Most of the compounds found in lime essential oil are represented by D-limonene, gamma-terpinene, and beta-pinene, which correspond to 73.48%. In comparison, the minority compounds correspond to 25.5%, totaling 99% of the chemical composition of the essential oil. Compounds with less than 0.3% area are trace compounds, corresponding to the remaining 1% of compounds. The minor compounds, which ranged from 5.17 to 0.3% include p-cymene, α-pinene, β-myrcene, neral, sabinene, β-bisabolene, terpineol, geranyl acetate, 4-terpineol, δ- 2-carene, trans-α-bergamotene, linalool, citrol, Bicyclo[3.1.0]hex-2-ene, 2-methyl-5-(1-methylethyl)-, geraniol, geranyl acetate, δ-2-carene, β-carophyllene.

Teixeira et al. (2013) found D-limonene (67.8 to 46.2%), β-pinene (7.98 to 16.55%), and γ-terpinene (9.33 to 13.54%) as major compounds when evaluating 15 genotypes of Citrus limon. According to literature data, the main and characteristic compounds of lime essential oil are limonene, γ-terpinene, and β-pinene, as found in this study. Furthermore, these compounds can be used to identify this essential oil.

3.2. Antibacterial activity of lime essential oil

The lime essential oil did not show antibacterial activity against E. coli by the disc diffusion or broth dilution method. An inhibition zone of 9 mm was found for S. aureus, and none of the oil dilutions inhibited the growth of this bacterium (Table 3). Regarding the antibiotic used as control, it appears that the evaluated strains were sensitive to gentamicin, presenting inhibition halos of 17 and 18 mm for E. coli and S. aureus, respectively.

Table 3
Antibacterial activity and minimum lime essential oil inhibitory concentration.

	Inhibation halo (mm)¹
Microorganism	Lime essential oil	Gentamicin (20 µg/g)	MIC (mg/mL)
E. coliATCC 25922	0	17 (S)	0
S. aureusATCC 29213	9	18 (S)	0

• ^¹
  R - Resistant (≤12 mm);
• I - intermediary (13-14 mm) and S - Sensitive (≥15 mm) (Clinical and Laboratory Standards Institute, 2012).

These results demonstrate that only the pure essential oil applied had a bacteriostatic action on the growth of S. aureus. According to Altun and Yapici (2022), the chemical composition of essential oils is a determining factor for their antibacterial activity. These authors report that essential oils containing aldehydes or phenols, such as citral, carvacrol, eugenol, or thymol, have strong antibacterial activity, such as oregano essential oil. On the other hand, monoterpenes are less efficient. Limonene, γ-terpinene, and β-pinene belong to the class of monoterpenes. This fact may justify the low efficiency in inhibiting bacteria in this study and the need for higher essential oil concentrations (Ben Hsouna et al., 2017).

Another factor that influences the antibacterial activity of essential oils is the cell wall composition of the target bacteria. Most essential oils applied to bacteria are more effective against Gram-positive bacteria (Delaquis et al., 2002). Gram-negative bacteria have an outer membrane that reduces the permeability of antimicrobials, which may have restricted the diffusion of terpenes present in lime essential oil (Nascimento et al., 2010; Araújo et al., 2015). Therefore, only the action of essential oil was observed against S. aureus.

Nieto-Velázquez et al. (2021) also reported that lime essential oil did not affect the growth of E. coli, even at concentrations of 0.872 g/mL, six times higher than that applied in this study. Other studies report that other Citrus species and other plant parts have antibacterial activity. For example, Ben Hsouna et al. (2017) verified the antibacterial activity of essential oil from the Citrus limon flower, both pure and diluted, against Gram-positive and Gram-negative bacteria and fungi. Moreover, Lemes et al. (2018) verified the antibacterial action of the essential oil from peels of Citrus aurantifolia in concentrations of 200 μg/mL against Gram-positive bacteria isolated from the oral cavity of humans.

3.3. Physicochemical composition

The results of the physicochemical analyses of the beef sausages are presented in Table 4. There were no significant differences between the sausage formulations evaluated in this study for the parameters of moisture, proteins, lipids, and ash, demonstrating that adding 0.5% of lime essential oil did not affect its physicochemical composition.

Table 4
Proximate composition of beef sausage formulations.

	Treatments
Parameters	LC	L1	L2
Moisture	73.90 ± 0.79a	73.66 ± 1.94a	71.51 ± 0.99a
Protein	21.55 ± 0.75a	21.68 ± 0.83a	21.30 ± 1.26a
Lipid	22.47 ± 0.91a	22.89 ± 0.46a	22.39 ± 0.59a
Ash	2.63 ± 0.13a	1.25 ± 0.39a	2.40 ± 0.10a

• Means ± Standard Deviation. Values with identical lowercase letters in the same line do not differ in the analysis of variance – ANOVA and Tukey’s test (p > 0.05). Treatments: LC: Control beef sausage without added preservatives; L1: Beef sausage with the addition of 0.005% curing salt 2 and 0.5% ascorbic acid; and L2: Beef sausage with the addition of 0.5% lime essential oil.

Moisture levels ranged from 71.51% to 73.90%, above that for fresh sausages (70%). Meanwhile, the protein and lipid contents found in the treatments follow current legislation, establishing a minimum protein content of 12% for the product and a maximum of 30% for lipid content, respectively (Brasil, 2000b). The high moisture content in the sausage may be related to other condiments present in its formulation, such as onion and fresh garlic. According to the Brazilian Food Composition Table, the moisture content of the Semimembranosus muscle (topside cut) used in sausages is 68.6%. The meat content added to the other condiments used in the formulations may have influenced the final moisture value. This table also provides the carbohydrate, protein, lipid, and ash values of the meat cut used in this study, corresponding to 0%, 21.2%, 8.7%, and 1.0%, respectively (TACO, 2011).

3.4. pH determination and color analysis

The pH results evaluated during 0, 5, 10, and 20 days are expressed in Table 5 for the three beef sausage formulations stored under refrigeration and freezing. There were no significant differences in pH values for the LC treatment during storage. On the other hand, there were significant differences in the L1 and L2 treatments. The L1 and L2 treatments showed decreased pH on the 5th day during the refrigerated storage period. The pH of the L1 and L2 treatments increased on the 10th day and decreased during freezing storage.

The pH value of fresh meat is an important parameter that directly influences the growth of microorganisms and can vary from 5.3 to 6.5. The lower the pH, the slower the growth rate of pathogenic bacteria. Meat with a high pH (equal to or greater than 6.0) is more prone to microbial deterioration than meat with a pH lower than 6.0, as it favors the growth of these microorganisms (Brasil, 2022a). The sausages analyzed in this study had a pH lower than 5.8, indicating they are suitable for consumption. Interestingly, the pH of the L1 sample produced with curing salt and ascorbic acid was lower than the other samples. Furthermore, it is also observed that the pH of the L2 formulation with the addition of lime essential oil was the highest at the end of the 20th day of the storage period.

Table 5
pH values of beef sausage formulations stored under refrigeration and freezing for 0 to 20 days.

	0 days	5 days	10 days	20 days
Treatments	T = 25 ºC	T = 5 ºC	T = −18 ºC	T = −18 ºC
LC	5.39 ± 0.04aA	5.33 ± 0.04aA	5.68 ± 0.02aA	5.66 ± 0.01aA
L1	5.44 ± 0.20aA	5.25 ± 0.05bA	5.64 ± 0.08cA	5.45 ± 0.01aB
L2	5.57 ± 0.10aA	5.39 ± 0.02bA	5.72 ± 0.02cA	5.70 ± 0.01aC

• Means ± Standard Deviation. Values with identical lowercase letters in the same row and values with capital letters in the same column do not differ in the analysis of variance – ANOVA and Tukey’s test (p > 0.05). Treatments: LC: Control beef sausage without added preservatives; L1: Beef sausage with the addition of 0.005% curing salt 2 and 0.5% ascorbic acid; and L2: Beef sausage with the addition of 0.5% lime essential oil.

Animal fat lipolysis and the formation of free fatty acids can cause a reduction in pH in meat products (Tomovic et al., 2020). Monoterpenes, such as D-limonene and β-pinene present in lime peel essential oil, are reported in the literature to have antioxidant activity, acting to block free radicals, which can delay lipolysis and prevent pH reduction (Budiarto et al., 2024).

The use of lemon essential oil in ground beef showed an increase in the pH of the meat with the addition of 0.06 and 0.312% of the essential oil during 10 days of storage at 4°C, from 5.59 and 5.57 to 6.24 and 6.19, respectively (Ben Hsouna et al., 2017). This differs from the study by Xin et al. (2022), who found a decline in pH value when applying 0.150 µL/g of lemon seed essential oil to meat aged at 4°C for 15 days, which reduced it from 6.52 to 5.69.

To date, there is no data in the literature on the use of lime essential oil in beef sausage. When analyzing our results, it appears that adding lime essential oil in the L2 formulation promoted a slight increase in pH compared to the control formulation (LC) at time 0. Furthermore, the increase in pH during the 20 days of storage under freezing may be related to the antioxidant action exerted by this oil, which is rich in monoterpenes and delayed the lipid oxidation of this product.

The color of the sausages was measured by the L*, a*, and b* parameters, which represent the luminosity and the intensity of the red and yellow colors, respectively (American Meat Science Association, 2012). All treatments showed a significant increase in L* and a* values over time, indicating that the sausage became lighter and increased the red color intensity with storage. The L1 and L2 treatments also showed a significant increase in b* values between 0 and 10 days, indicating an increase in the intensity of the yellow color in the sausage over time. According to Xin et al. (2022), lime essential oil is rich in antioxidant bioactive compounds, such as terpenes, that can inhibit the oxidation of myoglobin, as well as the rancidity of fatty acids, resulting in better luminosity and color stability. The L1 and L2 treatments presented L*, a*, and b* values that were generally higher than the LC treatment, demonstrating that the synthetic preservatives and essential oil added changed the color of the sausage more than the absence of preservatives.

When evaluating different smoked salami formulations, Tomovic et al. (2020) observed that the pH remained stable in the presence of 75 and 150 mg/kg of nitrites and that Juniperus communis L. essential oil generated a slight increase in pH. However, no significant differences existed between 0 to 0.10 µg/g concentrations. For the color, the authors found that L* reduced, and the a* and b* parameters remained stable.

Table 6
Color parameters (L*, a*, and b*) of beef sausage formulations stored under refrigeration and freezing for 0 to 20 days.

		Storage period
		0 days	5 days	10 days	20 days
Parameter	Treatments	T = 25 ºC	T = 5 ºC	T = −18 ºC	T = −18 ºC
L*	LC	50.76 ± 2.11aA	64.96 ± 3.44bB	67.97 ± 2.30bB	68.24 ± 1.22bB
	L1	52.29 ± 0.15aA	67.00 ± 3.31 bB	68.12 ± 1.53 bB	69.03 ± 0.52 bB
	L2	50.28 ± 0.02aA	70.49 ± 1.83bB	75.89 ± 0.08cC	71.14 ± 0.31bC
a*	LC	10.36 ± 3.12aA	14.22 ± 4.05bA	15.04 ± 1.49bA	16.51 ± 1.12bA
	L1	11.73 ± 1.54aA	14.59 ± 3.49bA	18.27 ± 1.38bB	18.85 ± 0.51bA
	L2	7.92 ± 1.13aB	13.10 ± 1.04aA	14.01 ± 0.33 bA	14.34 ± 0.59 bA
b*	LC	11.14 ± 4.26aA	21.75 ± 6.07bA	23.85 ± 4.61bA	22.09 ± 1.38bA
	L1	12.02 ± 2.39aA	28.08 ± 4.13bA	31.09 ± 1.61bA	26.74 ± 3.72bA
	L2	11.57 ± 0.40aA	25.28 ± 0.83bA	24.56 ± 1.65bA	30.61 ± 1.66bA

• Means ± Standard Deviation. Values with identical lowercase letters in the same row and values with capital letters in the same column do not differ in the same column do not differ in the analysis of variance – ANOVA and Tukey’s test (p > 0.05). Treatments: LC: Control beef sausage without added preservatives; L1: Beef sausage with the addition of 0.005% curing salt 2 and 0.5% ascorbic acid; and L2: Beef sausage with the addition of 0.5% Lime essential oil.

3.5. Microbiological quality

Table 7 presents the microbiological analysis results for the beef sausage formulations. These results were satisfactory for all microorganisms evaluated, being within the standards required by current legislation and indicating that the addition of lime essential oil did not affect the microbiological quality of this product (Brasil, 2022b). The results obtained for the quantification of mesophilic aerobic bacteria were satisfactory. They ranged from 1.7 x 10⁴ to 4.5 x 10⁴ CFU/g, following the microbiological standards (Brasil, 2022b). A higher count of mesophilic aerobic bacteria was found in the L1 treatment (with the addition of 0.005% curing salt and 0.5% ascorbic acid) compared to the L2 treatment (with the addition of 0.5% of lime essential oil), which showed a reduction in contamination by these bacteria.

Table 7
Microbiological analyses of beef sausages after 10 days of freezing at − 18°C.

	Treatments			Legislation
Microorganisms	LC	L1	L2	m	M
Total coliforms	Abs	Abs	Abs	Abs
Salmonellasp.	Abs	Abs	Abs	Abs
E. coli	Abs	Abs	Abs	Abs
Mesophilic aerobic bacteria	2.1 x 10⁴	4.5 x 10⁴	1.7 x 10⁴	105	106

• m: minimum; M: maximum; Abs: Absent (Brasil, 2022b). Treatments: LC: Control beef sausage without added preservatives; L1: Beef sausage with the addition of 0.005% curing salt 2 and 0.5% ascorbic acid; and L2: Beef sausage with the addition of 0.5% lime essential oil.

Lime essential oil applied in sausages has little been explored in the literature, but it is widely used in meat cuts, especially chicken. A recent meta-analysis study on the quality and safety of additives based on citrus fruits indicates the efficiency of natural extracts in reducing the population of mesophilic aerobic bacteria in chicken meat, corroborating with the data obtained in this study (Budiarto et al., 2024).

Replacing synthetic preservatives with essential oils in fresh sausage is a natural alternative that has demonstrated great potential for controlling spoilage bacteria in this product. In comparing the antibacterial efficiency of four nanoemulsions produced with Mentha piperita, Punica granatum, Thymus vulgaris, Citrus limon, and chitosan in cuts of beef, Lotfy et al. (2023) found that the lemon nanoemulsion had greater efficiency against E. coli.

4. Conclusion

Based on the results obtained in this study, it is concluded that the addition of 0.5% essential oil extracted from lime peel in beef sausage formulations presented beneficial effects for bacteriostatic activity on mesophilic aerobic bacteria compared to the sample with synthetic preservative without affecting the physicochemical composition of the product and maintaining pH and color stability during frozen storage. These data show that lime essential oil is a viable alternative for application in meat sausages, adding a different flavor and aroma to this product.

Acknowledgments

To the Federal Institute of Education, Science and Technology of Maranhão - IFMA - Campus São Luís - Maracanã and the Institutional Scientific Initiation Scholarship Program – PIBIC – FAPEMA.

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Edited by

Editor: José Givanildo da Silva

Publication Dates

• Publication in this collection
  27 Sept 2024
• Date of issue
  2024

History

• Received
  09 Mar 2024
• Accepted
  20 June 2024