*3.1. Chemical Composition*

The results of chemical composition of di fferent treatments are shown in Table 2. The addition of mushroom flour resulted in a significant (*p* < 0.05) moisture increase (64.34–66.48%) compared with the control (61.05%). The higher concentration of flour resulted in the highest moisture values in LF-Ab and LF-Po. These moisture increments could be attributed to the higher water amount added to compensate the fat reduction in formulation and the presence of mushroom flour, which is rich in dietary fiber, particularly β-glucans [37]. Contents of total dietary fiber of the mushroom flours Ab and Po were 22.82 ± 0.42% and 43.58 ± 1.33% in dry weight, respectively. Several works have pointed out that the porous and hydrophilic capacity of dietary fiber contributes to water holding properties increasing moisture [38,39]. The increased in moisture has been reported in other fat replacers like konjac gel [40], or pineapple dietary fibers [12].

**Table 2.** Proximate composition (%) and content sodium (mg/100 g) of frankfurters type sausage elaborated with flour of edible mushroom (*Agaricus bisporus* and *Pleurotus ostreatus*).


Results are expressed as mean value ± standard deviation. a–f: Different letters in each batch indicate significant differences (*p* < 0.05).

As expected, batches formulated with lower content of fat and salt resulted in significant (*p* < 0.05) lower concentrations for these parameters due to the modification in the formulations. Fat was reduced from 19.16 ± 0.42 to 16.28 ± 0.33, 15.18 ± 0.29 and 14.04 ± 0.47 in medium fat sausages MF-Ab, MF-Po and MF-PoAb, respectively. In LF-Ab (12.99 ± 0.30) and LF-Po (11.79 ± 0.06) was reached the highest reduction of fat. Na contents of flour added samples were approximately half of the control samples. The same behavior was observed in the ash content, quite related with the sodium chloride content reduction in the formulations [36]. The reduction of fat in meat emulsions can provoke changes in emulsion stability parameters, such as fat and water losses during cooking. Therefore, the meat industry has adopted new trends to improve the texture and water holding capacity, substituting animal fat by including the use of non-meat ingredients, such as inulin or β-glucan, considering the fiber´s ability to retain fat and water [15]. However, when Ab and Po performance are compared, Po was less e ffective in fat retention, probably related to di fferent protein content of flours, 28.63 ± 0.10% in Ab flour and 16.04 ± 0.22% in Po flour, expressed in dry basis. When the protein content of frankfurters was analyzed, only sausages with 5% Ab mushroom presented a significant higher protein content (*p* < 0.05) despite the higher protein content of Ab flour in agreemen<sup>t</sup> with the reported by Reis et al. [41] and Cheung [42]. The expected increase in this parameter because of the mushroom addition was overshadowed by the moisture augmentation reached in mushroom flour added products and the reduction of 50% of sodium caseinate in formulations. When the protein content is reported in dry weight (DW) all mushroom formulations presented significant higher contents (over 40% DW) than the 36.94 DW from control samples, being the LF-Ab, LF-Po the samples with the major values 45.77 g/100 g and 43.52 g/100 g DW, respectively.

The addition of these flours as a source of fiber in meat products has to be highlighted, since fiber content is usually absent in these products. Several works have included different vegetables, legumes or fiber sources to increase the fiber content of these products [11,12]. The batches with addition of the Ab and Po flour showed higher fibre content in a range of 0.57 and 2.18 g/100 g of sausage in comparison with the control (0.05 g/100 g sausage). LF-Po presented the higher value of dietary fibre with 6.51 g/100 g sausage (DW) since the Po flour presented the highest content in dietary fiber. The dietary fiber in mushrooms comes from non-digestible carbohydrates mainly chitin, glucans, cellulose and hemicelluloses like mannans, xylans and galactans [18,42]. With the combination of both mushroom flours in MF-PoAb the reduction of fat is accompanied with an interesting fiber content of 1.58 ± 0.17 being a promising alternative for a healthier sausage keeping the protein content with half of the sodium caseinate added.

#### *3.2. Amino Acid Profile*

When the amino acid profile of the treatments is analyzed (Table 3) no significant changes were observed between batches (*p* > 0.05). Some studies have been focused on the addition of non-meat protein sources like legumes or even algae in order to improve the protein profile in meat products [10,33]. In this case the addition of mushroom flour did not modify the amino acid profile keeping the ratio between essential and no essential amino acids in 0.93–0.94. The predominant amino acids in formulations were glutamic acid (ranging 2.58–2.84 g/100 g), aspartic acid (1.44–1.66 g/100 g), lysine (1.38–1.54 g/100 g) and leucine (1.31–1.43 g/100 g). These amino acids with alanine and arginine are also the most important in dried edible mushrooms [18], being glutamic and aspartic acids strongly related to umami taste [17]. However, the concentrations of flours added did not change the amino acid profile although sensorially mushroom taste was noticed. Related to essential amino acids lysine, leucine, arginine and valine were the predominant essential amino acids. The edible mushroom has been reported to contain all nine essential amino acids required for human intake [17,43], even though the protein profile of mushrooms depends not only on the specie but also on the size, composition of the substrate and harvest time [18]. In the case of *A. bisporus* and *P. ostreatus* leucine has been reported to be the limiting amino acid but with a protein quality in terms of digestibility and essential amino acid composition comparable to casein, eggs, and soy [17,37]. In this work, 50% of caseinate was substituted by mushroom flour in the formulation, but considering that non significant differences (*p* > 0.05) were found regarding the essential amino acid profiles, mushroom flours could be even suggested to partially replace meat content or to be considered as meat substitute.


**Table 3.** Amino acid profile (g/100 g sample) of protein from frankfurters with addition of edible mushroom flours.


**Table 3.** *Cont*.

\*: Essential amino acid.

#### *3.3. Lipid Oxidation Analysis*

The oxidative process was evaluated during storage time at 0, 30, 60 and 90 days, and the results are shown in Figure 1. The analysis of variance indicated that TBA values were significantly (*p* < 0.05) affected by the concentration of the edible mushroom flours added and the storage time. Initially, the scores obtained in samples with mushroom flours and less fat were significantly higher (*p* < 0.05) ranging in 0.34–0.72 mg MDA/Kg than the found in control samples with 0.12 mg MDA/Kg. This behaviour remained during cold storage. However, most treatments with Ab and Po flours presented TBA values below the acceptable limit (<1.0 mg MDA/kg) [44]. Mushrooms have been described to possess antioxidant activity, mainly because of the phenolic compounds, although their antioxidant properties depend on the species of mushroom, and the growing, harvest and processing conditions [45,46]. However, the antioxidant effect mushroom flours was not appreciated in this study and the higher concentration of flour (LF-Ab and LF-Po) led to higher initial TBARs, when a reduction of TBARs should be expected, since in these samples a reduction of 50% of fat was applied.

**Figure 1.** Results of TBARS (mg MDA/Kg) of sausages added with Ab and Po flours. (a–d): Mean values (corresponding to the same day) not followed by a common letter differ significantly (*p* < 0.05).

The limited antioxidant activity of mushroom flour and high TBARS found initially in samples with Ab and Po flour could be due to the drying conditions applied to the mushrooms during the obtaining of the flour, which could have promoted browning reaction and protein degradation products, participating in the formation of TBA color complexes and overestimating MDA values, as it has been reported by Papastegriadis et al. [47] in products like dry nuts. Besides, a significant increase was observed during storage for all treatments but after 90 days of cold storage, TBARS values significantly decreased (*p* > 0.05). This behavior was also noticed by Fernandes et al. [48] and attributed to instability or transitory nature of some secondary products from lipid peroxidation like malondialdehyde (MDA). But also, the higher values of TBARs of Ab samples especially from day 60 comparing to Po samples could be attributable to more browning reaction products present in Ab samples reacting with TBA since Ab flour have a darker color.

#### *3.4. pH and Microbial Results*

The inclusion of mushroom flour in the sausage led to a significant increase of pH over 6.0 comparing to 5.94 ± 0.02 Log CFU/g of control samples and the pH values kept during the cold storage. The pH of samples was in the range (5.94–6.11) similar to the pH reported for frankfurters with di fferent levels of shiitake [8], and other cooked sausages with extracts or alternative ingredients added [48]. Microbiologically, the cooking process and post-packing pasteurization eliminated the vegetative microorganisms, so lactic acid bacteria, pseudomonads and psychrotrophic bacteria remained under detection limits. However, counts between 4.52 ± 0.14–6.12 ± 0.08 CFU/g were found in total viable counts for flour mushroom added-frankfurters, much higher than the 1.68 CFU/g reported for control samples, and remained stable during the cold storage (Figure 2). These high levels of microorganisms can be attributed to spore forming bacteria naturally present in agrifoods in contact with soil like vegetables and mushrooms, which survived to the thermal treatment. Ab flour sausages presented significant higher counts (*p* < 0.05) than Po samples, possibly related to the conditioning process, which involved a blanching process, spreading the spore-forming contamination. However, in all cases the high counts are not considered a risk as long as refrigeration temperatures are maintained.

**Figure 2.** Evolution of microbial counts (Log CFU/g) of sausages during storage with Ab and Po flours. (a–d): Mean values (corresponding to the same day) not followed by a common letter differ significantly (*p* < 0.05).

#### *3.5. Color and Texture Profile Analysis*

The color parameters of frankfurters during storage are shown in Figure 3. The addition of mushroom flour significantly reduced (*p* < 0.05) the lightness by the fat reduction procedure, although Po flour decreased the L\* parameter to a lesser extend (LF-Po: 60.63 ± 0.35–MF-Po: 63.90 ± 0.48) than Ab flour (LF-Ab: 57.57 ± 4.31–MF-Ab: 58.57 ± 0.64). Yellowness (b\*) of the frankfurters increased in flour added frankfurters, especially in samples with Po. Ab samples showed significant lower a\* values (*p* < 0.05) comparing with control samples, while addition of Po flour significantly (*p* < 0.05) increased this parameter. So, Ab flour contributed to significantly (*p* < 0.05) reduce the L\* and a\* values

comparing to the control giving a darker color to the frankfurters as can be seen in Figure 4. Visually, the characteristic pink color of the control samples due to nitrosomyoglobine formation was slightly modified into brownish colors. The addition of edible mushroom flours instead of animal fat did not reproduce the color effect of pork backfat on the treated samples, especially in the Ab samples. Fat contributes in emulsions to lighter meat products and usually natural and artificial colorants are added to keep the pink color. The replacement of pork fat from frankfurters or finely comminuted sausages by oleogels [49] have also resulted in lighter and less red meat products. From the brown or greenish color by the incorporation of algae [10,50,51] or chia [11] to the to orange tones by addition of lycopene or carotenoids [52], the change of color will depend not only on the ingredient source, but also on the concentration added [12]. Finally the presence of mushroom flours was not negatively scored in sensory analysis.

**Figure 3.** Evolution of color parameters (**L\***, **a\*** and **b\***) during the storage of cooked sausages with edible mushroom flours (a–e): Mean values (corresponding to the same day) not followed by a common letter differ significantly (*p* < 0.05).

**Figure 4.** Visual appearance of frankfurter with different formulations.

Cold storage marginally affected to color parameters as it has been reported with other fat substitutes in frankfurters like chia or vegetal oils in konjac matrix [11,40]. Although natural pigments use to presenting less color stability [52], color frankfurters with mushroom flour remained mostly stable although a slight increase was observed in lightness probably due to the oxidation of fat. No typical discoloration was observed because of the oxidation of myoglobin pigments during storage [11,12].

The addition of 2.5% and 5% of edible mushroom flours also significantly affected (*p* < 0.05) the texture parameters evaluated in frankfurter with partial decrease of fat (30% and 50%) and salt (Table 4). All mushroom added-samples showed significant (*p* < 0.05) lower hardness, springiness, cohesiveness, gumminess and chewiness values comparing to control samples after elaboration. Samples with Po flour (2.5 and 5%; MF-Po and LF-Po), even in combination with Ab flour (MF-PoAb), gave the lowest values in the textural parameters resulting in softer frankfurters. In the emulsions fat is dispersed in small drops in a continuous phase formed by water, proteins and additives. When the fat is reduced the emulsion can loose stability and affect the texture. In general, when the reduction of fat is compensated by increasing protein, textures tend to be harder. However, when fat content is reduced by increasing the proportion of water, keeping the amount of protein, the structure of low-fat systems becomes softer [40]. In this case the replacement of fat and addition of mushroom flour led to a higher water contents in formulation and protein and fibre from mushrooms could help to bound water reducing hardness, and the rest of textural parameters with concentration of flour added. However, differences found according to the origin of mushroom flour, could be related to the different protein content of mushroom flours, since Ab flour (with a higher protein content) reduced to a lesser extend the textural parameters than Po flour (with around 16% of protein but a higher fiber content, over 40%).

Proteins from different sources have been added to meat products like frankfurters to stabilize and compensate the reduction of meat or fat. Stephan et al. [53] reported suitable properties of mycelia from *Pleurotus sapidus* as meat substitute in vegan boiled sausages comparable to the use of other protein concentrates (soy, pea and sunflower), but lower textural parameters than the original german boiled sausage were observed. In this case, the addition of Ab mushroom flour better helped to stabilize the emulsion, even if other protein source like sodium caseinate was reduced in the formulation.


Results are expressed as mean value ± standard deviation. (X–Z): Means in the same row not followed by a common letter are significantly different (*p* < 0.05). (a–e): Mean values in the same column (for each texture parameter) not followed by a common letter are significantly different (*p* < 0.05).

In general, softer textures have been obtained when different vegetal, cereal or legume, algae or fiber sources have been included in meat formulations with or without replacement of fat or other ingredients. Alvarez et al. [15] found a significant decrease of hardness in frankfurters by addition of rice bran and walnut. Choi et al. [44] reported a significant decrease of hardness in reduced- fat frankfurters with vegetable oils and rice bran fiber, and Cofrades et al. [54] observed that increasing amounts of walnut extracts reduced shear force and elongation values, indicating the formation of softer and less cohesive meat structures. Addition of 1% of pineapple fiber in partial replacement of fat in sausages significantly reduced hardness, chewiness and gumminess but less differences were appreciated in cohesiveness and springiness [12]. The partial replacement of fat and salt by addition of 1% of different seaweeds resulted also in lower hardness and chewiness parameters although the behavior on adhesiveness and springiness was seaweed-species related [50]. The addition of concentration of 0.8–1.2% of shiitake (*Lentinus edodes*) powder in frankfurter also reduced hardness, but increased cohesiveness [8].

In general, texture parameters were also affected (*p* < 0.05) by cold storage increasing all the parameters, but especially hardness in control samples comparing with mushroom added sausages. An increase of hardness during storage has been reported in frankfurters as a consequence to the increase of purge loss [40].

### *3.6. Sensory Evaluation*

In the case of sensory attributes evaluated in samples before cooking (color, decoloration surface), no significant differences (*p* > 0.05) were detected between treatments with color values between good and acceptable (2.17–2.83) and no discoloration was observed (Figure 5). The evaluation of odor indicated that samples with Ab (MF-Ab, LF-Ab, MF-PoAb) and LF-Po presented a stronger mushroom odor, with scores over acceptable level, while MF-Po did not significantly (*p* > 0.05) differ from control. When the samples were cooked the reduction of fat and salt and the inclusion of Ab and Po flours resulted in lower sensorial values for flavor and taste but around acceptability level, without significant differences between flour added samples. In the case of flavor, the samples with 5% of flour (LF-Ab, LF-Po and MF-PoAb) scored over acceptable level (3.0) while control samples scored near good level (1.95 ± 0.76). In the taste parameter, only LF-Ab scored over acceptable level, while de rest of treatments were rated between good and acceptable levels. The characteristic flavor or umami in mushrooms is intense, especially in Ab samples [20], and the acceptability of the frankfurter will depend on how accustomed the consumer to this flavor. When aqueous extract from other edible mushroom, *Cantharella cibarius*, was added to frankfurter, it was not sensorially different from control samples [26]. In general, umami compounds enhance palatability in savory foods which allows to reduce salt content without a negatively perception in the consumer increasing also satiety [55]. But the incorporation of new ingredients into processed meats modifies sensory properties and can limit the consumer acceptability [10]. However, this untypical or unexpected flavor could be moderated by incorporating different seasonings, since no flavor additives were included in formulations here tested, and even control samples were scored as good, but not excellent, because of the plain flavor. Jimenez-Colmenero et al. [51] showed that the addition of konjac-seaweed flour to low fat and low salt frankfurters slightly reduced sensory panel values, due to an intense unfamiliar flavor. However, they did not consider this an obstacle, and suggested the reformulation using less strongly flavored seaweed and considering seasonings.

During cold storage, sensory parameters evaluated in uncooked samples, color, discoloration and odor did not significantly changed confirming the stability of the product during long periods of storage.

**Figure 5.** Sensory results of frankfurters evaluated at day 0.
