Preparation and microbiological analysis of Tuscan sausage with added propolis extract

Abstract

The aim of this study was to obtain hydroethanolic extract of propolis by extraction, assisted by focused microwave, and to apply it in Tuscan-style sausage. The extract was used at concentrations of 0.5%, 1.0% and 2.0% (w/v) in the manufacture of the sausage, which was then analyzed in cold storage at 4 °C for 56 days. The following analyses were performed: mesophilic and psychotrophic organisms; coliforms at 35 and 45 °C; positive and negative-coagulase Staphylococcus, sulfite-reducing Clostridium, and Salmonella spp. The results were below the limits established by the Brazilian legislation, with some changes at the end of the study. Consequently, propolis extract prolonged the shelf life of the Tuscan-style sausage for 56 days and it is therefore an ingredient that can be potentially used in the preparation of this product.

Keywords:
sausage; propolis; extraction

1 Introduction

Propolis is a generic term used to describe a complex mixture of resinous, gummy and balsamic substances that are collected by honeybees from shoots, flowers and plant exudates; the bees add salivary secretions, wax and pollen which results in the creation of propolis. The role of propolis in the hive is related to its mechanical properties and it is used to construct, adapt and protect the hive; its antimicrobial activity ensures an aseptic environment (Funari & Ferro, 2006).

The chemical composition of propolis is very complex and it contains more than 180 identified compounds, of which flavonoids are an important feature. Flavonoids, along with phenolic acids and esters, phenolic aldehydes and ketones are considered to be the most important antimicrobial compounds contained in propolis. The other compounds are volatile oils and aromatic acids (5-10%), waxes (30-40%), resins, balms and pollen, which is a rich source of essential elements such as magnesium, nickel, calcium, iron and zinc. The mechanism of antibacterial activity is considered to be complex and it has been attributed to the synergism between flavonoids, hidroxiácidos and terpenes (Fernandes et al., 2006).

The antimicrobial activity of propolis has a wide range of applications in food technology. One particular advantage is that, unlike some conventional preservatives, propolis and its residues generally have a beneficial effect on human health. Given the above, this study aimed to prepare Tuscan-style sausage with propolis extract (PE) and to evaluate the effect of PE on the microbiological stability of the sausages during storage.

2 Materials and methods

2.1 Obtaining the propolis extract

The extractions were carried out using a focused microwave with two cavities and equipped with glass jars with a maximum capacity of 180 mL (Star System 2, 800 W, CEM, Matthews, NC, USA). The ground propolis was initially weighed (6 g) and then transferred to the glass jars. Then 70% grain alcohol solvent (60 mL) (v/v) at a ratio of 1:10 (w/v) was added and submitted to the effect of microwaves for 20 minutes at 70 °C.

After the end of extraction, the extract was filtered on filter paper and centrifuged at 3000 rpm for 20 min. The supernatant was subsequently concentrated in a rotary evaporator (Fisatom 802), packed in amber bottles, and stored in a freezer (–18 °C) until analysis.

2.2 Preparation of the product

The preparation of the Tuscan-style sausages followed the requirements regarding ingredients described by the relevant legislation (Brasil, 2000) as shown in Table 1. The procedures followed were as described by Terra (1998).

Table 1
Formulation of Tuscan-style sausage.

Raw materials and ingredients	Quantity (g/100g)
Pork meat	85
Bacon	15
Water/ice	3
Salt	2.5
Seasoning for Tuscan-style sausage (BREMIL)	0.5*
White pepper powder	0.1
Flavor enhancer	0.05
Garlic Seasoning (BREMIL)	0.2 0.25
Fixative (BREMIL)	0.25

• ^*
  According to the manufacturer's recommendation.

The pork and bacon were initially ground using a grinder (Jamar PJ22, Jamar Ltda, São Paulo, Brazil). Then the raw material was carried to a mixing machine (Jamar MJI 35) where the other ingredients were mixed to obtain a bind. The mixture was subsequently divided into four batches of 5 kg, to which were added the pre-defined concentrations of propolis extract. This provided the following four treatments: Treatment 1 Control (0% PE) - no added propolis extract; Treatment 2 (0.5% PE) – Tuscan-style sausage with 0.5% added propolis extract; Treatment 3 (1.0% PE) - Tuscan-style sausage with 1% added propolis extract; and Treatment 4 (2.0% PE) - Tuscan-style sausage with 2% added propolis extract. After mixing, the mixture was packed into pig intestine and then washed to remove the salt; it was then immersed in 1% lactic acid for 30 minutes to hydrate. For storage, the sausages were packed in polystyrene trays, wrapped with plastic wrap, identified and immediately taken to a D.B.O oven (ELETROLAB, model EL 101) and stored at 4 °C.

2.3 Microbiological analyses

The analyses were performed regarding counts for psychotrophic and mesophilic microorganisms (American Public Health Association, 2001); positive and negative-coagulase Staphylococcus; coliforms at 35 °C and 45 °C; sulfite-reducing Clostridium and Salmonella spp (Brasil, 2003). The analyses were performed on days 0, 7, 14, 21, 28, 35, 42, 49 and 56 of storage at 4 °C.

2.4 Statistical analysis

The data were evaluated by analysis of variance (ANOVA). The means were compared by Tukey's test, with a significance level of 95% (p <0.05) using SPSS 17.0 statistical software.

3 Results and discussion

Counts of mesophilic aerobic microorganisms are commonly used to indicate the sanitary quality of food (Franco & Landgraf, 2005) and to detect the number of aerobic or facultative mesophilic bacteria, which are present both in vegetative form and also as spores in food. In the present study microbiological analyses was performed in relation to the meat, pork intestine and propolis extract used in the preparation of the Tuscan-style sausages. The results (not shown) were within the tolerance limits set by the relevant legislation, i.e. RDC No. 12 (Brasil, 2001) and according to Terra (1998), indicating that the raw materials were properly treated, in optimal hygienic conditions, and that they were well kept, with microbiological quality that was sufficient to be used safely in developing products.

The results obtained for the counts of total mesophilic aerobic bacteria, psychotrophic bacteria, positive and negative-coagulase Staphylococcus, total coliforms at 35 °C, coliforms at 45 °C, sulfite-reducing Clostridium and Salmonella spp for the different formulations of Tuscan-style sausages are shown in Table 2.

Table 2
Microbiological analysis of Tuscan-style sausages during storage at 4 °C.

Mesophilic aerobic bacteria (Log10 CFU.g–1)	0% PE *	0.5% PE	1% PE	2% PE
Day 0	4.72 ± 0.083a	4.78 ± 0.144a	4.79 ± 0.081a	4.73 ± 0.015a
Day 7	4.57 ± 0.063a	4.51 ± 0.133a	4.60 ± 0.048a	4.56 ± 0.058a
Day 14	4.45 ± 0.079b	4.44 ± 0.093b	4.60 ± 0.078a	4.37 ± 0.054b
Day 21	3.97 ± 0.043a	3.98 ± 0.021a	3.97 ± 0.022a	3.85 ± 0.036b
Day 28	3.94 ± 0.038a	3.87 ± 0.048ab	3.84 ± 0.010b	3.59 ± 0.079c
Day 35	3.82 ± 0.042b	3.87 ± 0.035b	3.99 ± 0.020a	3.64 ± 0.082c
Day 42	4.64 ± 0.048b	4.83 ± 0.064a	4.53 ± 0.048c	3.70 ± 0038d
Day 49	5.97 ± 0.022a	5.41 ± 0.091b	3.84 ± 0.130c	3.73 ± 0.043c
Day 56	6.44 ± 0.034a	5.98 ± 0.021c	6.11 ± 0.047b	4.85 ± 0.045d
Psychotrophic bacteria (Log10 CFU.g–1)	C	T1	T2	T3
Day 0	4.26 ± 0.057b	4.49 ± 0.117a	4.06 ± 0.035c	3.94 ± 0.061c
Day 7	4.55 ± 0.050a	4.58 ± 0.068a	4.54 ± 0.061a	4.45 ± 0.104a
Day 14	4.53 ± 0.052a	4.68 ± 0.076a	4.56 ± 0.103a	4.62 ± 0.053a
Day 21	4.32 ± 0.055a	4.34 ± 0.037a	4.13 ± 0.095b	4.14 ± 0.056b
Day 28	4.47 ± 0.048a	4.28 ± 0.075b	4.20 ± 0.142bc	4.04 ± 0.051c
Day 35	4.23 ± 0.263a	4.26 ± 0.041a	4.03 ± 0.038a	3.39 ± 0.030b
Day 42	4.74 ± 0.050a	4.75 ± 0.053a	4.89 ± 0.092a	3.97 ± 0.260b
Day 49	6.00 ± 0.029a	5.88 ± 0.048a	5.28 ± 0.122b	4.75 ± 0.128c
Day 56	6.73 ± 0.044b	6.68 ± 0.119b	6.96 ± 0.036a	5.39 ± 0.067c
Coagulase-negative Staphylococcus (Log10 CFU.g)
Day 0	3.56 ± 0.078a	3.50 ± 0.031a	3.34 ± 0.070b	3.26 ± 0.024b
Day 7	3.56 ± 0.046a	3.33 ± 0.053bc	3.41 ± 0.037b	3.30 ± 0.037c
Day 14	3.79 ± 0.111a	3.71 ± 0.036a	3.59 ± 0.026b	3.43 ± 0.040c
Day 21	3.32 ± 0.145ab	3.41 ± 0.096a	3.47 ± 0.047a	3.21 ± 0.090b
Day 28	3.43 ± 0.133a	3.38 ± 0.059ab	3.26 ± 0.099ab	3.21 ± 0.088c
Day 35	3.29 ± 0.059a	3.27 ± 0.094a	3.48 ± 0.114a	3.11 ± 0.068c
Day 42	3.33 ± 0.085a	3.19 ± 0.073bc	3.31 ± 0.026ab	3.15 ± 0.079c
Day 49	3.57 ± 0.073a	3.22 ± 0.102b	3.06 ± 0.064c	2.81 ± 0.087d
Day 56	3.73 ± 0.029a	3.41 ± 0.087b	3.29 ± 0.070bc	3.14 ± 0.118c
Coagulase-positive Staphylococcus (Log10 CFU.g-1)
Day 0	< 1.00	< 1.00	< 1.00	< 1.00
Day 7	< 1.00	< 1.00	< 1.00	< 1.00
Day 14	< 1.00	< 1.00	< 1.00	< 1.00
Day 21	< 1.00	< 1.00	< 1.00	< 1.00
Day 28	< 1.00	< 1.00	< 1.00	< 1.00
Day 35	< 1.00	< 1.00	< 1.00	< 1.00
Day 42	< 1.00	< 1.00	< 1.00	< 1.00
Day 49	< 1.00	< 1.00	< 1.00	< 1.00
Day 56	< 1.00	< 1.00	< 1.00	< 1.00
Total coliforms at 35 °C (Log10 CFU.g–1)
Day 0	3.28 ± 0.149b	3.50 ± 0.037a	3.16 ± 0.128b	3.21 ± 0.111b
Day 7	3.18 ± 0.107a	3.44 ± 0.452a	3.16 ± 0.054a	2.98 ± 0.059a
Day 14	3.04 ± 0.033b	2.95 ± 0.026b	3.16 ± 0.039a	2.87 ± 0.043c
Day 21	3.92 ± 0.040a	3.21 ± 0.123b	3.25 ± 0.046b	3.04 ± 0.033c
Day 28	3.63 ± 0.145a	2.87 ± 0.066b	2.85 ± 0.101b	2.83 ± 0.047b
Day 35	2.80 ± 0.059ab	2.91 ± 0.030a	2.68 ± 0.070b	2.53 ± 0.116c
Day 42	2.81 ± 0.039a	2.79 ± 0.071a	2.90 ± 0.013a	2.55 ± 0.071b
Day 49	3.71 ± 0.100a	2.66 ± 0.065b	2.50 ± 0.059c	2.33 ± 0.064d
Day 56	2.61 ± 0.040b	2.82 ± 0.022a	2.67 ± 0.063b	2.28 ± 0.078c
Coliforms at 45 °C (Log10 CFU.g–1)
Day 0	< 1.00	< 1.00	< 1.00	< 1.00
Day 7	< 1.00	< 1.00	< 1.00	< 1.00
Day 14	< 1.00	< 1.00	< 1.00	< 1.00
Day 21	< 1.00	< 1.00	< 1.00	< 1.00
Day 28	< 1.00	< 1.00	< 1.00	< 1.00
Day 35	< 1.00	< 1.00	< 1.00	< 1.00
Day 42	< 1.00	< 1.00	< 1.00	< 1.00
Day 49	< 1.00	< 1.00	< 1.00	< 1.00
Day 56	< 1.00	< 1.00	< 1.00	< 1.00
Salmonella spp./25g of sample
Day 0	absent	absent	absent	absent
Sulfite-reducing Clostridium 46 °C/ 25g of sample
Day 0	< 1.00	< 1.00	< 1.00	< 1.00

• ^*
  Values presented as mean ± standard deviation; Different small letters in the same line indicate significant difference (p <0.05) by Tukey’s test; Different capital letters in the same column indicate significant difference (p <0.05) by Tukey’s test; PE: propolis extract.

From the results presented in Table 2, it can be seen that in terms of the means of the total aerobic mesophilic counts at zero storage time there was no significant difference between the treatments; they all showed a value lower than 10^–6 CFU/g, which has been cited by Terra (1998) as an acceptable level of bacterial contamination.

During the final period of storage (days 49-56) the mesophilic aerobic microorganism count rose and the treatments with added propolis extract showed significant difference compared with the standard; these values ​​were lower than 10^–6 CFU/g. Therefore, it was possible to see the influence of the addition of propolis extract in the sausages because the lowest counts ​​for mesophilic aerobic bacteria occurred in the sausages with added propolis extract. These values were 5.41; 3.84 and 3.73 Log₁₀ CFU/g on day 49; and 5.98; 6.11 and 4.85 Log₁₀ CFU/g on day 56 for the treatments with 0.5%, 1% and 2% of added extract, respectively. The standard treatment had average values of 5.97 and 6.44 Log₁₀ CFU/g for the same period. During this period visual changes such as molds, yeasts and fungi were observed in greater quantity in the standard treatment than in the other treatments. Values ​​for the count of total mesophilic aerobic bacteria in the present study were lower than those reported by Pereira (2009) when assessing the use of propolis extract (0.10%) in mechanically separated chicken meat at the end of 10 days of refrigerated storage. The aforementioned study found a value of 6.74 log₁₀ CFU/g, which was even higher than the standard treatment.

Bradford et al. (1993) have argued that the psychotrophic microorganism count is one of the most important criteria for assessing deterioration due to temperature under refrigeration. In the study conducted by the aforementioned authors, the psychotrophic count increased significantly after seven days of storage. The authors attributed these values to the lactic acid-producing bacteria, which were probably responsible for the deterioration of the meat products. The present study also found increased levels of psychotrophic microorganisms between the treatments, but from day 21 of storage. This increase was significantly greater (p<0.05) when compared to the standard treatment (0% PE), with counts higher than the treatment with 2% added PE.

The count for coagulase-negative Staphylococcus and coagulase-positive Staphylococcus (Table 2) was lower than 1.0 Log₁₀ CFU.g^–1 and showed no significant difference between the treatments during the storage period. The RDC No. 12 (Brasil, 2001) approves the Technical Regulation on microbiological standards for food and states that the tolerance in fresh pork sausage for coagulase-positive Staphylococcus is 3×10³ CFU/g; consequently, all the treatments in the present study remained within the legally allowed limit during storage. According to Lee et al. (2007) the ethanol extract of propolis has antimicrobial activity in relation to Staphylococcus aureus.

Table 2 shows that in relation to the average count of total coliforms at 35 °C there was significant difference between the treatments. Once again, the action of propolis extract of 2% can be seen in reducing the number of total coliforms compared to the standard treatment. This reduction was very clear in relation to the treatment with the addition of 2% propolis extract compared to the standard treatment during the period from day 14 to day 56 of storage. Similar results were found by Pereira (2009), who noted a significant reduction in the average total coliform at 35 °C count in mechanically separated meat with propolis extract at the end of the storage period. However, during the course of storage the mechanically separated meat with propolis extract showed no significant difference from the negative treatment (BHT), therefore differing from the present study. Borges et al. (2009) studied the antibacterial and antifungal activity of different concentrations of the hydro-alcoholic extract of propolis in fresh pork sausage and they also found significant differences between the treatments regarding the coliform count at 35 °C. The aforementioned authors found that the use of higher concentrations of propolis extract was more effective in controlling these microorganisms. In the present study, no significant differences were observed between the treatments regarding coliforms at 45 °C; the values ​​were less than <1.00 log₁₀ CFU/g and were in accordance with the limit established by RDC No. 12 (Brasil, 2001), which is 5×10³ CFU/g.

The main pathogenic microorganisms which potentially could have been in the products developed in the present study were Salmonella spp., Staphylococcus aureus, Escherichia coli and Listeria monocytogenes. The latter can enter slaughterhouses through live animals and also humans working in the premises (Birzele et al., 2005). Castagna et al. (2004) investigated the prevalence of Salmonella spp. in a refrigerated pork slaughterhouse and detected the microorganism in 83.33% of the animals. The average prevalence found in the final product (fresh sausage) manufactured using raw materials originating from these animals was 93.94%, with no statistical difference between the prevalence of the carrier animals and that found in the final products. The present study found no presence of Salmonella spp and sulfite-reducing Clostridium in any of the samples throughout the entire storage period. This result was in line with the regulations defined by RDC No. 12 (Brasil, 2001), which establishes a negative limit of Salmonella in 25 g of sample, and a maximum of 3×10³ CFU/g for sulfite-reducing Clostridium.

In recent years, the in vitro antimicrobial activity of propolis has been increasingly reported; this activity is due to flavonoids, aromatic acids and esters, which are present in the natural resin (Gebara et al., 2002). Caffeic acid and ferulic acid also contribute to the bactericidal action of propolis. These factors may explain the positive action of propolis in the present study in relation to certain microorganisms, such as total coliforms, aerobic mesophilic bacteria, psychotrophic bacteria and Staphylococcus aureus, which provided the lowest average scores during the storage period. Nagai et al. (2006) also reported that propolis has a high inhibitory effect in relation to microbial growth during the storage of meat and muscle.

4 Conclusion

It was concluded that the values found in the microbiological analyses were within the tolerance limits established by Brazilian legislation for all the treatments during the storage period, with changes only at the end of that period. Given these results it is suggested that propolis extract can be used as an ingredient in the preparation of Tuscan-style sausage because it extends the shelf life of the product.

Acknowledgements

The authors would like to thank the Coordination of Improvement of Higher Education Personnel (CAPES/FAPERGS) for supporting this study.

• Practical Application: The propolis extract provided microbiological stability to the sausage.

References

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Publication Dates

• Publication in this collection
  29 Apr 2016
• Date of issue
  July 2016

History

• Received
  08 Oct 2015
• Accepted
  16 Feb 2016