The Effect of Cantharellus Cibarius Addition on Quality Characteristics of Frankfurter during Refrigerated Storage

The antioxidant and antimicrobial properties of Cantharellus cibarius decoction and the effect of mushroom addition on the physicochemical and microbiological properties of frankfurters during refrigerated storage were studied. Mushroom addition significantly reduced (p < 0.05) the formation of total aerobic mesophilic bacteria during storage. Regarding the texture, there was no negative effect in frankfurters with the mushroom added, compared to the control group of sausages. Generally, C. cibarius can be used as a natural ingredient in order to prevent the growth of microorganisms in cooked pork sausages, causing an extension in shelf life during chilled storage.


Introduction
Emulsification technology for frankfurter-type sausage production has been used over several hundred years. Frankfurters, as a type of emulsified meat product, do not have a permanent recipe, so their ratio of ingredients varies during production, and therefore these sausages are subjected to various undesirable changes [1]. Lipid oxidation is possibly the biggest problem in the storage of meat products and can cause negative changes that directly influence the product's quality and its shelf life, as well as causing a change in color, taste, and deterioration [2]. In the food industry, traditional (synthetic) antioxidants are used to slow down the degradation process of the final product. However, more and more data are pointing out that these compounds can cause toxicity problems and negatively affect consumers' health. A new way of preventing these effects is to use herbal antioxidants as the substitute for conventionally used antioxidants [3].
Edible mushrooms, whether dry or fresh, contain valuable compounds from a technological and nutritional aspect (proteins, phenolic compounds, and taste enhancers) [4]. Thus, they could be a potential source of natural antioxidants if incorporated into food products. In the previous studies, Lentinus edodes was added to frankfurters [5] and to fermented sausages [6], as well as Agaricus bisporus in cooked ground beef, but no reference has been found for the use of Cantharellus cibarius in cooked sausages.
Thus, the goal of this paper was to inspect the antioxidative and antimicrobial properties of C. cibarius decoction in vitro and in frankfurters, in order to examine the influence on the technological parameters, microbiological profile, and sensory quality during storage.

Sample Preparation
The powdered mushroom was bought as a commercial product from B.M.B. (Arilje, Serbia). In order to obtain a decoction, a mixture of dry powdered mushroom and Milli-Q water (Merck, Darmstadt, Germany) (1: 10), was heated at 80 • C for 1 h. The resulting decoction was subsequently used for the evaluation of the antioxidant and antimicrobial capacity.

Antioxidant Activity of C. cibarius Decoction
The antioxidant activity was tested according to the Petrović et al. [7] method adapted for a 96-well microplate reader. The percentage of neutralization of the ABTS radical was calculated according to the formula: L-ascorbic acid was used as the standard. All measurements were performed in triplicates.

Antimicrobial Activity Assay of C. cibarius Decoction
Three Gram positive bacterial strains-Staphylococcus aureus 25923, Bacillus cereus 11778, and Listeria monocytogenes 19111, three Gram negative bacterial strains-Salmonella Typhimurium 14028, Escherichia coli (O157:H7) 35150, and Yersinia enterocolitica 27729, as well as two yeasts-Candida albicans 10231 and Pichia fermentans 28789, were used to check the antimicrobial potential of C. cibarius decoction. All the selected bacteria and yeasts belonged to ATCC (American Type Culture Collection, Rockville, MD, USA).
The broth micro-dilution method was used to designate the minimum inhibitory (MIC) and minimum bactericidal/fungicidal (MBC/MFC) concentrations, as already explained earlier [8]. C. cibarius decoction concentrations in the range of 0.0097 to 20.0 mg/mL were used for the experiment.

Manufacturing of Frankfurters
The decoctions of C. cibarius were prepared as follows-60 g of powder was added to 2 L of distilled water and preheated at 80 • C. The decoctions obtained in this way were poured into a plastic container, cooled, and frozen in order to obtain ice. This was repeated in triplicate (T1). The same procedure was applied for the following three batches (T2), only with the exception that this time 120 g of powder was used in an individual batch. The control formulation (C) was prepared with ice obtained from distilled water (without mushrooms). All the treatments were formulated to obtain 8 kg batter (Table 1). Fresh pork hams and pork back fat were bought from a local abattoir. All connective tissue and visible fat were removed from the ham muscles. Spice mixtures, primarily garlic and onions, were not used. Also, during the thermal treatment, smoke was not used for the same reason-to obtain the real antioxidant and antimicrobial effect of the decoction added to the sausages. After production, the frankfurters were kept in the refrigerated chamber (1-4 • C) for 60 days.

Proximate Composition Analyses
The protein (nitrogen × 6.25; ISO 937) [9], moisture (ISO 1442) [10], and total fat (ISO 1443) [11] contents of the frankfurters were calculated according to the recommended methods from the International Organization for Standardization. All the analyses were done in triplicate and using two technical replicates.

Color
The method was performed according to Tomasevic et al. [12] and the equipment used was calibrated according to the procedure explained in Tomasevic et al. [13]. The total color difference (∆E) of each sample (S) was calculated with respect to the control sausage (C) using the following equation:

pH Measurement
The pH value was determined using the portable pH meter (Consort C931, Turnhout, Belgium) equipped with a piercing glass reinforced electrode (Mettler Toledo, Greifensee, Switzerland) for direct measuring of pH in meat products. The instrument was calibrated with standard phosphate buffers (pH was 7.02 and 4.00 at 20 • C). All the analyses were done in triplicate and using two technical replicates.

Texture Profile Analyses (TPA)
The textural traits were carried out using the TA.XT Plus Texture Analyzer (Stable Micro Systems Ltd., Surrey, UK) with 5 kg load cell, at room temperature. The textural profile analyses (TPA) were performed using frankfurter cores from the middle (20 mm high and 12 mm diameter), positioned upright on a platform and compressed twice to 25% of their original height with a cylindrical aluminum probe (P/25). The pre-test speed and post-test speed were 180 mm min −1 , while the test speed was 60 mm min −1 . The attributes determined were-hardness, springiness, cohesiveness, and chewiness, according to Pons and Fiszman [14].

Microbological Analyses
In order to assess the microbiological safety of the frankfurter during storage in the refrigerator, determination of the total mesophillic, aerobic organisms-TPC, Salmonella spp., Escherichia coli, and Listeria monocytogenes was applied, according to the procedures described by the standards ISO 4833-2 [15], ISO 6579 [16], ISO 7251 [17], and ISO 11290-1 [18], respectively. The results were expressed as a log CFU/g. The microbiological analyses were performed in triplicate.

Sensory Evaluation
In total, 50 consumers from Belgrade, capital of Serbia, scored their liking for odor, taste, and overall quality of each type of sausage. A 9-point hedonic scale was used (1 = dislike extremely; 5 = neither like nor dislike; and 9 = like extremely) according to Peryam and Pilgrim [19].

Statistical Analyses
An analysis of variance (ANOVA) at p < 0.05 was done for statistical significance. The results of proximate composition, antioxidant properties in vitro, and MIC/MBC were subjected to the one-way ANOVA, Tukey's HSD test. The other analyses were subjected to two-way ANOVA. The C. cibarius decoct concentration and storage time were set as independent variables and the readings of the results were set as the dependent variable. A Bonferroni test was conducted for the mean comparison at different levels of C. cibarius concentrations and storage days. All the data was analyzed using SPSS 17.0 (Chicago, IL, USA).

ABTS Neutralization Activity of C. cibarius Decoction
It was found that the decoct of C. cibarius exhibits neutralization of the ABTS radical in a dose-dependent manner ( Figure 1). However, 50% of the radical neutralization had not been reached in the range of tested concentrations. Moreover, mushroom decoction was a weaker radical scavenger in comparison with ascorbic acid. Other authors found C. cibarius as a very potent source of antioxidative compounds, like polyphenols, ascorbic acid, and β-carotene [20,21]. The ability of the C. cibarius ethanol extract to scavenge ABTS radicals was tested by Dimitrijevic et al. [20], who reported an EC 50 of about 14 mg/mL. In our study, the EC 50 was a bit lower, around 12 mg/mL.

Microbological Analyses
In order to assess the microbiological safety of the frankfurter during storage in the refrigerator, determination of the total mesophillic, aerobic organisms-TPC, Salmonella spp., Escherichia coli, and Listeria monocytogenes was applied, according to the procedures described by the standards ISO 4833-2 [15], ISO 6579 [16], ISO 7251 [17], and ISO 11290-1 [18], respectively. The results were expressed as a log CFU/g. The microbiological analyses were performed in triplicate.

Sensory Evaluation
In total, 50 consumers from Belgrade, capital of Serbia, scored their liking for odor, taste, and overall quality of each type of sausage. A 9-point hedonic scale was used (1 = dislike extremely; 5 = neither like nor dislike; and 9 = like extremely) according to Peryam and Pilgrim [19].

Statistical Analyses
An analysis of variance (ANOVA) at p < 0.05 was done for statistical significance. The results of proximate composition, antioxidant properties in vitro, and MIC/MBC were subjected to the one-way ANOVA, Tukey's HSD test. The other analyses were subjected to two-way ANOVA. The C. cibarius decoct concentration and storage time were set as independent variables and the readings of the results were set as the dependent variable. A Bonferroni test was conducted for the mean comparison at different levels of C. cibarius concentrations and storage days. All the data was analyzed using SPSS 17.0 (Chicago, IL, USA).

ABTS Neutralization Activity of C. Cibarius Decoction
It was found that the decoct of C. cibarius exhibits neutralization of the ABTS radical in a dosedependent manner ( Figure 1). However, 50% of the radical neutralization had not been reached in the range of tested concentrations. Moreover, mushroom decoction was a weaker radical scavenger in comparison with ascorbic acid. Other authors found C. cibarius as a very potent source of antioxidative compounds, like polyphenols, ascorbic acid, and β-carotene [20,21]. The ability of the C. cibarius ethanol extract to scavenge ABTS radicals was tested by Dimitrijevic et al. [20], who reported an EC50 of about 14 mg/mL. In our study, the EC50 was a bit lower, around 12 mg/mL.  Having in mind that our sample was processed (dried) prior to the decoct preparation, and that other authors usually tested fresh samples, the results obtained in our study show a high antioxidant potency. Another restricting factor might be the material extraction method, which is simple cooking in water (decoction), instead of extraction. Water decoction or cooking in water is the simplest and the cheapest method for the extraction. In this study, the authors would like to test exactly this method due to its convenience (easy, fast, simple, and cheap) that would later easily find its way to industrial application. Thus, less amounts of non-polar compounds, like ergosterole and β-carotene, can be extracted and react with radicals.

Broth Microdilution Method
The antimicrobial property of C. cibarius decoction was studied against selected bacteria and yeasts, which are well known as foodborne pathogens ( Table 2). The decoction has displayed significant antibacterial activity against Yersinia enterocolitica (MIC-10 mg/mL) and slight activity against Listeria monocytogenes at the highest concentration (MIC-20 mg/mL) tested. Regarding bactericidal activity, the decoction exhibited a desirable effect only at the highest concentration (20 mg/mL) towards Listeria monocytogenes and Yersinia enterocolitica. The fungistatic and fungicidal activity of C. cibarius decoction, against Candida albicans, was significantly better, expressed through the same value of MIC/MFC (10 mg/mL). Kosanic et al. [22] reported that acetonic and methanolic extracts of C. cibarius were active against S. aureus (MIC-5 and 10 mg/mL, respectively), E. coli (MIC-10 mg/mL for both extracts), and C. albicans (MIC-5 and 10 mg/mL, respectively). Also, Kozarski et al. [23] found that the antimicrobial effect of C. cibarius methanolic extracts, obtained with the same broth microdilution method, were active against B. cereus (MIC-0.3125 mg/mL), L. monocytogenes (MIC-0.625 mg/mL), S. aureus (MIC-1.25 mg/mL), E. coli O157:H7 (MIC-10 mg/mL), and Y. enterocolitica (MIC-20 mg/mL). Mushrooms are known as a valuable source of natural antibiotics [24]. More intense antimicrobial effects of mushrooms may occur due to the specific antimicrobial mechanisms of phenol, i.e., hydroxylation of the phenolic ring [25]. As several authors have reported, antimicrobial activity of the same species may vary depending on the origin of the mushroom, the solvent, and the bacterial strain [26]. As there was no difference between the measurements (n = 3), standard deviations were not shown. Within the same column, means followed by different uppercase letters were significantly different (p < 0.05).
Different species of the genus, Candida, accounting for less than 3% of the microbial population, originate from pastures, spreading via animal fur and gut on carcasses. In general, due to the newly created environmental conditions, ascomycetous yeasts become dominant over basidiomycetic yeasts during the processing of fresh meat and storage. The procedures used for the processing of fresh meat such as salting, drying, and fermentation (acidity and nitrates) favor the growth and development of Candida spp., including C. albicans [27].
Our study showed that C. cibarius decoction was most effective in inhibiting the growth/killing of C. albicans. Considering that this pathogenic yeast may be present not only during the various stages of processing fresh meat and refrigerated storage, but also in finished meat products, it seems that the addition of C. cibarius decoction would be highly desirable in protecting such products, e.g., frankfurter.

Proximate Composition, Color and pH
The results obtained with the addition of C. cibarius were at the normal ranges (Table 3) for these types of products [28,29]. The quality of meat is most commonly judged by consumers through the meat color [29]. Parameters of the color lightness (CIE L* value), redness (CIE a* value), and yellowness (CIE b* value) of examined sausages are shown in Table 4. In the present study, the incorporation of C. cibarius caused a kind of lower, but statistically significant (p < 0.05) the L* (lightness) value of frankfurter compared with the control, on day 1 of examination. T1 and T2 had higher values for yellowness than C. The decline in the lightness and the rise in yellowness presumably results from the suppression of the product's natural color (fallow) owing to the addition of mushrooms. Similar results for lightness and yellowness was reported by Pil-Nam et al. [5], who added shiitake powder in frankfurters. The T2 treatment were generally "redder" than T1 and C. Taking into consideration the total color difference (∆E) between frankfurters with added mushroom with respect to the control samples (at day 1), both values were lower than 3. ∆E is the difference between two colors in L* a* b* color space. Fernández-López et al. [30] stated that only ∆E higher than 3 CIELAB units would be differentiated by an observer. When likened to the addition of 0.5-5% grape seed flour, or sunflower seed oil/makgeolli lees fiber mixture, the result was a decline in lightness, redness, and yellowness of a similar product [31,32]. Therefore, the addition of C. cibarius has a trivial influence on the color characteristics of this product.
During the storage time, all the treatments increased in redness. In parallel, all the frankfurters decreased in lightness after 60 days of storage. Regarding the yellowness, there was decrease during storage, but on the 60th day of storage, it showed a statistically significant increase compared to the 1st day after the production of the frankfurters. Similar results for the yellowness during the storage time were provided by Fernández-López et al. [30]. Throughout the storage, all the color differences (∆E) were increased, all of them being higher than 3.
The pH values are presented in Table 4. The obtained results were inside the usual ranges for this kind of meat product [33]. On the 60th day of storage, there was a decline in pH values compared to the first day of storage, but it was only statistically significant in T1. The decrease in pH value throughout the storage is presumably an aftereffect of the creation of organic acids from carbohydrates, produced by lactic acid bacteria [34]. Related results and the decline of pH values in frankfurters at the end of the storage period were reported by Ranucci et al. [35]. Table 4. CIELAB color coordinates (L*, a* and b*), color differences (∆E*) respect to control sample and evolution of pH during storage of frankfurters.

Texture Analyses
The textural traits of sausages are displayed in Table 5. On the first day after production, there was no difference (p > 0.05) in hardness, springiness, cohesiveness, and chewiness between the control group and C. cibarius frankfurters. Additionally, there was no statistically significant difference (p > 0.05) in the springiness and cohesiveness between T1, T2, and C at all days of examination. Regarding the hardness and chewiness, the sausages with added mushroom had higher values than the control group, at all days of examination during the storage. The storage time did not significantly (p > 0.05) influence the hardness, springiness, and chewiness within all the variants of frankfurters between the 1st to 60th days of storage, except the control group on the 60th day for chewiness. Almost identical trends were reported by Pil-Nam et al. [5], namely, hardness and chewiness were similar between the first and last days of storage, after the addition of L. edodes in frankfurters. On the other hand, the cohesiveness all of the three treatments showed a lower, but statistically significant (p < 0.05) increase during the storage. These findings indicate that although C. cibarius was added to sausages in different ratios, the textural traits changed only slightly.

Microbiological Analyses during Storage
The storage stability of the food, as well as meat products in general, is usually reflected by the number of aerobic mesophilic bacteria [6]. The TPC in frankfurters produced with C. cibarius decoction was significantly lower (p < 0.05) than that found in the control on the first day of storage (Table 6). It was probably the consequence of mushroom decoction addition. Namely, it is well known that the antimicrobial effect of mushrooms can be accomplished due to the presence of certain antimicrobial compounds, above all catechin, gallic acid, and rutin [36]. All treatments of the studied sausages increased the number of TPC. Similar results were reported by Tahmouzi et al. [37]. A higher increase in TPC was noticed for the control, whereas lower for the treatments with mushroom decoction added, except on the 40th day. On the 60th day of storage, the TPC (T1, T2) in the present study were lower, compared to the levels in the same product type enriched with L. edodes on the 30th day of storage as reported by Pil-Nam et al. [5]. In all three groups of sausages, Salmonella spp., E. coli, and L. monocytogenes were not detected.

Sensory Analyses
The sensory attributes of the frankfurters during chilled storage are presented in Table 7. The highest odor scores were obtained for T2, except for the 40th day of examination, where C obtained better estimates. However, no statistically significant difference (p > 0.05) between C and T1, T2 were found in the odor. Concerning the taste, the highest scores were given to the treatments with mushroom added on the 1st, 10th, 20th, and 30th day of examination. On the 40th, 50th, and 60th day of the examination, higher marks were given to the control group of frankfurters. There were no differences between C and T1, T2 in the taste (p > 0.05). The panelists marked the taste and odor of C. cibarius frankfurters with higher scores due to the fact that this mushroom contains particular compounds characterized as flavor enhancers (lenthionine and monosodium glutamate). The overall quality analyses showed a similar trend to that of taste. There was also no statistically significant difference between C and T1, T2 for the overall quality (p > 0.05). Regarding the storage effect, there was no statistically significant difference (p > 0.05) within the control group and C. cibarius sausages from the 1st to 60th day of storage, except for T2 for the overall quality.
Özvural and Vural [32] showed that the addition of grape flour powder resulted in lower estimates of taste. As well, Deda et al. [38] displayed that the tomato paste added in frankfurters caused off-taste alterations. In comparison to previously mentioned studies, the utilization of C. cibarius did not cause negative effects on the sensory traits, inversely, it improved the odor, taste, and overall acceptability during the first month of cold storage.

Conclusions
Sausages with mushroom added increased the shelf life by reducing the number of TPC during chilled storage. Frankfurters produced with C. cibarius had a partial improvement in the estimates for odor, taste, and overall quality, compared to the control group. Considering all the results of this research, hot water extract, or C. cibarius decoction, possesses certain antioxidative and antimicrobial potential and could be used in the production of frankfurters, to potentially prevent lipid oxidation and microorganisms growth in the final product.