TTest results for MIC determination for olive leaves (
This study evaluated the antimicrobial activity of olive leaves in vitro and meat chicken fed with percentages of 5 and 10 g of olive leaves for each kg feed. This is justified by the relevance of obtained safe products, with emphasis on the use of natural additives. The olive leaves presented antibacterial activity in all tested bacteria. For the bacteria Yersinia enterocolitica, Escherichia coli, and Shigella, the minimal inhibitory concentration varied from 0.6 to 1.5 mg/ml. The treatment with an addition of olive leaves showed better microbiological stability of the thighs and drumsticks of chickens than treatment without an addition of olive leaves. The use of 5 g/kg diet inhibited the growth of Staphylococcus aureus and aerobic psychrotrophic and aerobic mesophilic, while the use of 10 g/kg of diet inhibited the growth of Enterococcus spp., lactic bacteria, thermotolerant coliforms, Pseudomonas, Clostridium perfringens, and Escherichia coli.
- antimicrobial activity
- olive leaves
- natural additive
- chicken meat
In order to inhibit microbial growth of raw materials or cuts, it is often more effective than the direct addition of preservatives to add compounds into the diet of the growing animals . The phenolic compounds occurring naturally in the plants have the ability to inhibit the growth of microorganisms, including bacteria, viruses, and fungi, maintaining the quality of meat for a long time.
In a study conducted by Bisignano et al. , the in vitro antimicrobial activity of oleuropein and hydroxytyrosol extracted from olive leaves was evaluated, and the efficiency for the pathogenic bacterium
In a study conducted by ERBAY and ICIER , the main compound found in olive leaves was oleuropein, being 24.54% in dry leaves. In a study realized by Paiva-Martins  with an objective of to assess the influence of OL supplementation at a lower level on feed digestibility and meat quality, the results indicated that olive leaves may be included in pig diets at 25 g/kg in order to improve the tocopherol content of meat without excessively compromising growth performance.
Upon investigating the in vitro activity of a commercial extract of olive leaf (
Botsoglou et al.  evaluated the effect of the use of olive leaves in turkey’s supplement diet in quantities of 5 and 10 g of leaves/kg diet in relation to microbiological quality of breast fillets that were stored at 4°C during 12 days. The turkey fillets that received olive leaves in the diet have had lower numbers of colonies of lactic acid bacteria, psychotropic, mesophilic, and enterobacteriaceae.
This study was designed to evaluate the effects of supplementation of the percentages of 5 and 10 g of olive leaves/kg of feed in the diet of broilers, on microbiological, of the meat the thighs and drumsticks stored at 4°C (±1°C) for 12 days.
2. Materials and methods
2.1 Extraction and chemical composition of olive leaves
Olive leaves (
The determination of total phenolics in olive leaves before followed the methodology described by Swain and Hills , which used as pattern the gallic acid, in concentrations of 50, 100, 150, 200, and 250 mg/l to build the calibration curve. Liquid chromatographic analysis of olive leaves.
We evaluated the oleuropein content present in olive leaves according to the method proposed by Guimarães et al. , and the chromatography conditions were based on Quirantes-pine et al. . The separation of oleuropein was realized by using a HPLC Agilent 1260 Infinity (Agilent Technologies, Germany) liquid chromatography with a diode array detector (DAD).
2.2 Evaluation of different concentrations of olive leaves for antibacterial activity in vitro
The minimum inhibitory concentration (MIC) analysis for the in natura olive leaves (after harvest) and after drying for microorganisms was realized:
The plant extract obtained from the olive leaves was evaluated according to microdilution in all concentrations: 20; 10; 5; 2.5; 1.25; 0.625; 0.312; and 0.156 mg/ml. The extract was put in plaques, and all the plaques were incubated in a greenhouse at 35°C for 24 hours and read with revealing. The read had objective show what concentrations the olive leaves had better effect on microorganisms.
2.3 Animals and diets
The chickens were created in the farm for 42 days and fed with the following diets: T1 (traditional diet without addition of olive leaves), T2 (diet with addition of 5 g of olive leaves for each kg feed), and T3 (diet with addition of 10 g of olive leaves for each kg of feed).
The broilers were slaughtered, and thighs and drumsticks, with skin and bone, were collected, stored in plastic bags of polyethylene without barrier, at 4°C (±1°C), for 12 days to monitor the growth microbiological.
2.4 Microbiological analysis
The poultry meat was analyzed microbiologically on days 0 (zero), 3, 6, 9, and 12 of storage.
The analysis of fecal coliform,
2.5 Statistical analysis
All analyses took place in triplicate runs. Results were statistically analyzed by mean standard deviation, variance, and Tukey test at 95% significance, using the software Statistica 6.1 (Statsoft Inc., USA).
3. Results and discussion
3.1 Extraction and chemical composition of olive leaves
The average for the analysis of olive leaves was 4.65% moisture in dry basis, 4.69% of fixed mineral residue, 1.38% fat, 23.3% crude fiber, and 12.73 g/NT 6.25 × 100 g protein. This result is in accordance with that found by Botsoglou et al. . The low percentage moisture of olive leaves ensures your quality, because it is not favorable to the development of fungi, molds, and yeasts.
The total phenolic content found in olive leaves, before and after the drying, was 12,275 and 9525 mg/g leaves, respectively. Similar values were found by Makris et al.  who reported 40.27 mg of gallic acid equivalents/g of dried olive leaves, and by Botsoglou et al.  who found phenol content of 26 mg of gallic acid equivalents/g of dried leaves.
The oleuropein tenor found in the olive leaves was 15.0 (±0.8) g/kg (CV de 5.1%,
3.2 In vitro antibacterial activity
Table 1 presented the values of inhibitory minimum concentration (MIC) in mg/ml from the olive leaf (
|Minimum inhibitory concentration—MIC (mg/ml)|
|Microorganism||In natural leaf extract||Dry leaf extract|
All the bacteria tested presented sensibility for olive leaves, some with more intensity and others with less. For the bacteria
For the microorganisms
3.3 Effect of olive leaves on microorganisms in meat
The safety and quality of fresh broiler beef can be estimated by counting microorganism indicators aerobic mesophilic and psychrotrophic . Table 2 shows the number of colonies (log10 CFU/g) of aerobic mesophilic, lactic acid bacteria, aerobic psychrotrophic, and
|Aerobic mesophilic bacteria (log CFU/g)||Lactic acid bacteria (log CFU/g)||Psychrotrophic bacteria (log CFU/g)|
|Storage time (days)|
|0||T1||7.30E + 05a||1.00E + 02a||1.10E + 03a||3.20E + 03a|
|T2||3.90E + 03b||4.40E + 01b||7.80E + 01c||7.01E + 02b|
|T3||1.30E + 03c||3.20E + 01c||6.20E + 02b||8.00E + 01c|
|3||T1||1.20E + 06a||5.20E + 03a||2.30E + 04a||1.30E + 04a|
|T2||5.60E + 04b||7.32E + 02b||1.97E + 02c||3.80E + 03b|
|T3||2.90E + 04c||1.80E + 02c||2.10E + 03b||2.10E + 03c|
|6||T1||2.50E + 06a||4.20E + 05a||2.81E + 04a||5.30E + 04a|
|T2||9.70E + 04c||3.50E + 03b||2.10E + 03c||4.70E + 03b|
|T3||6.60E + 05b||3.10E + 03c||1.71E + 04b||3.10E + 03c|
|9||T1||1.91E + 07a||1.70E + 06a||1.30E + 05a||1.80E + 05a|
|T2||2.30E + 05c||2.30E + 04b||6.72E + 03c||2.30E + 04b|
|T3||6.81E + 05b||5.80E + 03c||2.40E + 04b||5.80E + 03c|
|12||T1||4.51E + 07a||3.50E + 06a||3.80E + 05a||4.90E + 06a|
|T2||4.30E + 05b||3.10E + 04b||7.42E + 03c||3.80E + 04b|
|T3||7.51E + 05b||6.00E + 03c||3.60E + 04b||6.20E + 03c|
Analyzing the growth of the microorganism aerobic mesophilic (Table 2), the treatments that received diet supplemented with olive leaves (T2 and T3) remained within the quality standards during the 12 days storage at 4°C reaching a maximum counting of 5.63 and 5.87 log10 CFU/g, respectively, while control treatment showed a counting of 6.07 log10 CFU/g from the third day of storage. The aerobic mesophilic counting of 107 CFU/g or 7 log10 CFU/g is considered an indicator for the end of shelf life of cooled broiler meat . Some studies that are more precise indicate outside the ideal sanitary conditions broilers with a counting of mesophilic 106 CFU/g . According to these parameters, the counting between 106 and 107 CFU/g was considered a limit to end the shelf life, and it can be said that the diet with added olive leaves of broilers provided an increase in the shelf life of meat compared with the control treatment.
The number of colonies of lactic acid bacteria, in the treatments with olive leaves (T2 and T3) in all analyzed days, was lower than that of the control treatment (T1) and differed significantly (P < 0.05) among themselves. These results show that olive leaves present an inhibitory effect on the growth of lactic acid bacteria (Table 2). Between treatments with olive leaves, T3 had throughout the period fewer colonies of lactic acid bacteria than T2, and this result was significantly different (P < 0.05). Botsoglou et al.  added 5 and 10 g olive leaves/kg in the turkey feed and evaluated microbial growth in breast fillets, which were stored at 4°C for 12 days. On the twelfth day of storage, the number of lactic acid bacteria in the control treatment was 6.5 log10 CFU/g, and treatments with 10 and 5 g olive leaves were 4 and 5 log10 CFU/g, respectively. In this study, the number of lactic acid bacteria in meat on day 12 was 3.77 and 4.49 log10 CFU/g for treatments T3 and T2, respectively, and these values were lower than those found by Botsoglou et al.  in the same storage time and the same temperature.
Although the counting of aerobic psychrotrophic microorganisms indicates the degree of deterioration of refrigerated foods, the Brazilian legislation establishes no standard for these microorganisms. However, the International Commission on Microbiological Specifications for Foods  establishes 106–107 CFU/g as a standard. Considering these microbiological standards, the chicken meat in this study was fit for consumption during the 12 days at 4°C (Table 2). Throughout the storage period, T2 had the lowest number of colonies, with a significant difference (P < 0.05) of T3 and T1. The number of colonies of T3 was lower than that found in T1 also with significant difference (P < 0.05), indicating that olive leaves had significant effects on the growth of the counting of aerobic psychrotrophic microorganisms. In breast turkey fillets that received olive leaves in the diet, Botsoglou et al.  found the number of colonies of aerobic psychrotrophic on the twelfth day of storage of 4.6 and 5.7 log10 CFU/g for tests that received 10 and 5 g of olive leaves/kg diet. This counting were largest found that in this study, 4.55 to T3 and 3.87 log10 CFU/g to T2 (Table 2).
The analysis of total aerobes and
Table 3 shows the results of the microbiological analysis of total coliforms,
|Total coliforms (log CFU/g)||Thermotolerant coliforms (log CFU/g)|
|Storage time (days)|
|0||T1||4.40E + 02a||3.31E + 03a||3.80E + 01a||3.20E + 04a||5.00E + 00a||1.20E + 03a|
|T2||1.12E + 02b||1.80E + 02b||1.00E + 00c||2.31E + 02b||3.00E + 00a||6.30E + 02b|
|T3||8.60E + 01c||1.10E + 02b||2.30E + 01b||8.70E + 01b||2.00E + 00a||5.41E + 02b|
|3||T1||6.10E + 03a||4.20E + 04a||4.21E + 02a||5.50E + 05a||1.07E + 01a||3.70E + 04a|
|T2||2.81E + 02b||2.30E + 03b||1.00E + 00c||9.31E + 02b||1.00E + 01a||9.30E + 03b|
|T3||1.52E + 02c||3.41E + 02c||4.83E + 01b||9.51E + 02b||1.00E + 01a||7.30E + 03b|
|6||T1||9.71E + 03a||1.60E + 05a||7.80E + 03a||7.40E + 05a||1.20E + 04a||7.40E + 05a|
|T2||4.80E + 02b||4.10E + 03b||2.10E + 01c||1.40E + 03c||8.40E + 02b||1.60E + 04b|
|T3||1.50E + 02c||1.20E + 03c||2.83E + 02b||3.80E + 03b||2.30E + 02c||1.10E + 04b|
|9||T1||5.50E + 04a||6.30E + 05a||1.90E + 04a||1.50E + 06a||6.21E + 04a||1.30E + 06a|
|T2||3.10E + 03b||4.20E + 04b||5.01E + 02c||7.50E + 04b||3.20E + 03b||1.90E + 05b|
|T3||2.80E + 03c||3.10E + 03c||3.90E + 03b||2.92E + 04c||6.12E + 02c||4.10E + 03c|
|12||T1||7.40E + 04a||8.80E + 05a||5.60E + 04a||5.30E + 06a||8.41E + 04a||5.20E + 06a|
|T2||4.10E + 03b||5.20E + 04b||7.01E + 02c||9.60E + 04b||5.40E + 03b||2.20E + 05b|
|T3||3.20E + 03c||4.20E + 03c||4.40E + 03b||3.12E + 04c||7.22E + 02c||5.10E + 03c|
The Brazilian legislation does not establish microbiological parameters of coliforms. The treatments were subjected to this analysis to know the microbial load and so evaluate the sanitary conditions of the broiler meat of the three treatments, since these parameters reflect the quality of the raw material. The results vary between treatments (Table 3), where T2 and T3 had lower levels of total coliforms than T1 during the 12 days of storage.
The use of olive leaves in the amount of 10 g/kg feed showed a better inhibitory effect than the use of 5 g/kg feed for
According the Resolution no 12/2001, National Agency for Sanitary Surveillance , cuts of broiler cooled or frozen can have a tolerance limit for counting coliforms 45°C/g of 104 or 4 log10 CFU/g. According to this tolerance, T1 would be inappropriate for marketing and consumption because its initial counts were 4.5 log10 CFU/g, while the treatments with olive leaves have had initial counts of 1.93 and 2.36 for T3 and T2, respectively, and had presented counts within the tolerance limit of the legislation until the sixth day of storage at 4°C. The analysis of fecal coliform indicated that T2 and T3 have had significant inhibitory effect (P < 0.05) compared to T1, demonstrating that both concentrations of olive leaves are inhibitory.
The research of
The current legislation in Brazil does not set a standard for
The federal legislation provides the absence of
The samples of broiler meat analysis of the three treatments showed absence of
The research for
The use of 5 g/kg olive leaves reduced the growth of