**Towards More Sustainable Meat Products: Extenders as a Way of Reducing Meat Content**

## **Tatiana Pintado <sup>1</sup> and Gonzalo Delgado-Pando 2,\***


Received: 3 July 2020; Accepted: 31 July 2020; Published: 3 August 2020

**Abstract:** The low efficiency of animal protein (meat products) production is one of the main concerns for sustainable food production. However, meat provides high-quality protein among other compounds such as minerals or vitamins. The use of meat extenders, non-meat substances with high protein content, to partially replace meat, offers interesting opportunities towards the reformulation of healthier and more sustainable meat products. The objective of this review is to give a general point of view on what type of compounds are used as meat extenders and how they affect the physicochemical and sensory properties of reformulated products. Plant-based ingredients (pulses, cereals, tubers and fruits) have been widely used to replace up to 50% of meat. Mushrooms allow for higher proportions of meat substitution, with adequate results in reduced-sodium reformulated products. Insects and by-products from the food industry are novel approaches that present an opportunity to develop more sustainable meat products. In general, the use of meat extenders improves the yield of the products, with slight sensory modifications. These multiple possibilities make meat extenders' use the most viable and interesting approach towards the production of healthier meat products with less environmental impact.

**Keywords:** meat extenders; meat products; meat substitutes; sustainability; plant-based proteins; insects; by-products; pulses; mushrooms

#### **1. Introduction**

In 2015, all United Nation Member States adopted "The 2030 Agenda for Sustainable Development" [1]. In this agenda, the countries agreed to 17 Sustainable Development Goals (SDG) to be achieved by the end of 2030. Sustainable food production is one of the main pillars of the document, where foods needs to be sufficient, safe, affordable and nutritious, as well as part of a sustainable production system. The world population growth and industrial development are causing an expansion of food production and an increased demand for animal protein [2]. One of the main concerns is the low efficiency of animal protein production. It is estimated that 7 kg of food from plant origin (animal feed) yields 1 kg of milk or meat for human consumption [3]. In addition, animal production is believed to use around 30% of the global land surface, contributing to deforestation and the loss of biodiversity [4]. However, the environmental impact of livestock production goes further than biodiversity loss: important greenhouse gas emissions, vast use of fertilisers and the deterioration of water quality due to effluents [4,5]. Westhoek et al. [6] estimated that "halving the consumption of meat, dairy products and eggs in the European Union would achieve a 40% reduction in nitrogen emissions, 25–40% reduction in greenhouse gas emissions and 23% per capita less use of cropland for food production". However, meat represents an important source of energy, high-quality protein and micronutrients such as iron, zinc, selenium, vitamin B12 and vitamin D [5,7]. Meat and

meat products currently provide one-sixth of the total energy intake of a European adult and widely contribute to total protein, vitamin D and iron consumptions up to 40%, 30% and 23%, respectively [7]. Hence, meat and meat products should not be disregarded in the diet, as they contribute to the avoidance of essential nutrient deficiencies and can also protect against malnutrition in countries where access to other types of highly nutritious products is limited [5,8]. Deficiencies of iron and vitamin D are of high prevalence around the world [9,10]. A suboptimal vitamin B12 status occurs in 30–60% of the population, mainly in less-developed countries [11]. A recent study by Vatanparast et al. [12] found that decreasing by 50% the red and processed meat consumption and increasing by 100% the consumption of plant-based alternatives in Canadian individuals improved the overall nutritional diet value but adversely affected the intake of protein, zinc and vitamin B12. However, not only undeveloped or developing countries are affected. Rippin et al. [8] detected deficiencies in these micronutrients for certain segments of the European population. There is no unique food alternative to meat or meat products with similar nutritional profiles, and even a combination of several foods does not assure the same nutritional intake. Vitamin B12 is only present in foods of animal origin, which makes people following vegan and vegetarian diets in need of supplementations to achieve the dietary reference intake (DRI) for this micronutrient [13]. Furthermore, non-meat foods contain only 20–60% the protein density of that of the meat, and the digestibility and bioavailability of some micronutrients from these sources are known to be lower [14]. Even though highly desirable, a vast improvement of meat production efficiency and sustainability in the near future is not likely. Current strategies should focus on limiting the environmental impact of our diets without risking nutrition deficiencies.

Meat products are inherent to food culture and are widely consumed all around the world. Imamura et al. [15] estimated a global consumption of processed meat from 3.9 g/day (first quintile) to 34 g/day (fifth quintile). Even though their consumption has been linked with the burden of chronic diseases like coronary heart disease, type 2 diabetes and certain types of cancer [16,17], a number of gaps still exist (such as the underlying mechanisms of cancer development and the role of cooking) that could offer room for mitigation during their processing [16,18]. Versatility, more attractive products, waste reduction opportunities and higher shelf lives are some core characteristics that differentiate meat products from fresh meat. Therefore, the reformulation of meat products to produce healthier and more sustainable versions seems like a robust strategy in-line with the SDG. The research on the development of healthier meat products started in the 1990s but still comprises a big proportion of the current research in this field. Two strategies are primarily followed: the reduction of harmful components to appropriate amounts and the incorporation of potentially health-enhancing ingredients [19]. The former is focused on the reduction of harmful saturated fatty acids [20], salt [21], cholesterol [22] and additives such as nitrite [23] or phosphates [24], whereas the latter studies the incorporation of the so-called "functional ingredients", mainly from plant origin, that provide healthier characteristics to the product [25–27].

In the last decade, meat substitutes or analogues have received much interest as plant-based similar-in-properties alternatives to conventional meat products [28]. However, most of these analogues are produced under heavy processing manufacture and, thus, limiting the environmental sustainability gain and losing the healthier prerogative that they were originally based on. Meat reduction arises as a more meaningful alternative to a complete elimination of meat from the diet and, sometimes, a more sustainable option than meat substitutes [29,30]. The integration of plant-based ingredients into meat dishes has been proven as a successful and consumer-accepted strategy [29] and has opened the way to a different approach towards the reformulation of healthier and more sustainable meat products: meat substitutions with plant-based ingredients. Although originally devised to reduce costs, the use of meat extenders presents an opportunity to reduce the meat content while incorporating some healthier ingredients to the meat product. Meat extenders are non-meat substances with high protein contents that can also modify some of the product's properties, such as water-holding capacity (WHC), texture, palatability and appearance [31].

In this review, we aim to evaluate the use of extenders as meat substitutes and how they affect the physicochemical and sensory properties of the meat products. The review has been structured in different sections, where meat extenders have been grouped based on their origin. In addition, a section discussing the consumer perspective about the acceptance of novel and more sustainable meat products has also been included.

#### **2. Meat Extenders**

#### *2.1. Pulses as Meat Extenders*

"Pulses are edible dry seeds of plants belonging to the *Leguminosae* family" [32]. Pulses' protein contents range from the 18.4% of the Bambara bean to the 34.1% of the lupin. They not only contain a great amount of protein, but they also present the highest protein digestibility score among the plant-origin proteins. In addition, pulses are also a rich source of micronutrients such as iron, zinc and B-vitamins. Even though iron from plant origin is less absorbed by the human tract, when combined with meat, the absorption increases substantially [32]. Therefore, from a nutritional point of view, pulses are a great candidate as meat substitutes, providing high quantities of proteins and similar micronutrients to the ones in meat. Hence, several studies have analysed their role as meat extenders in the past fifteen years (Table 1). Even though soybean is not a pulse as per the definition, it is a legume, and for this reason, a study with texturised soy granules as a meat extender has also been included in Table 1.


**Table 1.** Use of pulses as meat substitutes (extenders).

\* TBARS: thiobarbituric acid reactive substances.

Pulses in different forms have been used in meat product reformulations as binders or to increase their nutritional and healthier properties [40–43]. The starch, fibre and protein contents make pulses great binders, as they can form complex gel networks with meat proteins. These networks can trap the water and other compounds, forming stronger bonds between them and, thus, helping to achieve a higher retention in the meat matrix during processing [44]. Aslinah et al. [45] used adzuki bean flour as a fat and corn flour replacer in meatballs due to its water-holding capacities. Soy protein has been also widely used when developing reduced fat meat products due to its gelling properties [46,47]. The type and quantity of the pulse utilised, as well as the type of product, will determine the overall effect on the product stability in terms of WHC. In this regard, Nagamallika et al. [38] used two different pulses, Bengal gram flour and pea flour, to replace the meat content in chicken patties at two levels: 5% and 10%, yielding a higher stability at the higher level of substitution. Pea flour proved to yield a significantly lower cooking loss (9.5% vs. 30.3%) and higher emulsion stability (4.2% vs. 2.2%) and WHC (64.3% vs. 30.8%) when used at the 10% level compared to the Bengal gram flour patties. Nonetheless, at the 5% level of substitution, the emulsion stability and cooking loss were significantly lower for the patties with gram flour, whereas the WHC was significantly higher (19.8% vs. 47.8%). Yadav and Yadava [37] observed an increase in the yield and emulsion stability with an increasing level of substitution (3–9%) with gram flour in quail meat rolls. In a comprehensive analysis with 23 different type of pulses as meat substitutes in chicken and beef patties, this variation among the pulse types was also found [36]. The authors observed that cooking losses in beef patties ranged from 8.0% in the yellow split pea patty samples to 15.1% in the patties with pink beans, whereas the control had a 37.9% cooking loss. In the case of the pork patties, the control had a cooking loss of 22.9%, and the substitution improved the yield in all cases, with the cooking loss ranging from 5.6% to 10.7%, the lowest being the patties with small red beans and the highest for the ones with speckled butter beans. In an interesting study by Serdaroglu et al. [ ˇ 39], three different pulse flours (lentil, chickpea and blackeye bean) were used in low-fat meatballs, replacing not the meat but the rusk used in the control samples. Lower cooking losses and an increased WHC were found in the meatballs reformulated with pulses. This gives an idea that not only the starch (on higher quantities in the rusk) but the protein and fibre contents (much higher in the pulses) have big impacts on the water-holding capacities of meat products. The pulses with higher protein contents, blackeye beans and lentils, gave significantly higher yields to the meatballs. The substitution percentage also determined the effect of the extender on the product yield. Argel, Ranalli et al. [33] evaluated four different pulses (chickpea, lentil, green pea and bean) as extenders in pork patties with six different levels of meat contents. At the lower level of substitution (10.1%), the patties manufactured with bean flour had the highest cooking yields, followed by lentil and green pea, significantly different from the ones with chickpea flour. However, at the highest level of meat substitution (44.6%), the bean flour had the lowest cooking yield among the four pulses; the reformulated patties had higher cooking yields at all substitution levels than a commercial one. The different compositions of these flours might explain this, as the chickpea flour had the lowest protein and fibre contents but the highest fat levels. On the other hand, no significant yield changes were observed in dehydrated chicken ring meat using soy as a meat extender at a 5% level of substitution [34].

Another property closely related to the water-holding properties of meat products is the texture [48]. Texture is usually evaluated using a texturometer by means of a texture profile analysis (TPA) or a measurement of the hardness with the shear force value. A TPA analysis of pork patties substituted with pulse flour showed that the hardness and chewiness increased when compared to the control and commercial ones, but that this difference disappeared when the substitution level was above 35% and added water was at its highest level [33]. In the same study, the authors found that cohesiveness was lower in all the extended pork patties and that the bean flower had the lowest hardness among the four pulses studied. In addition to the level of substitution, the type of pulse will also affect the textural properties. An increase in hardness was observed when the rusk used in low-fat meatballs was replaced (10%) by the flour of three different pulses, being the meatballs with chickpea flour the ones with significantly higher hardness, followed by black bean and lentil flour [39]. Out of 23 varieties of pulses, only four pulses did not affect the shear force value when substituting 50% of the meat in beef patties and five pulses when pork patties were prepared instead [36]. The overall mean shear force was lower for the majority of the pulses used. As the substitution values for this study ranged between 35% and 50%, this agrees with the aforementioned results. The type of meat product will also affect how the substitution alters the textural parameters, as the networks formed in the matrix will be different depending on the degree of comminution and the quantity of fat, water and proteins. A great example can be observed in the 23-varieties study where the beef patties with green northern beans as the extender had the lowest hardness value, whereas in pork sausage patties, the hardness was one of the highest for this same pulse.

Colour is perhaps the attribute most difficult to mask when substituting meat with pulses, as not many of them have similar colour to meat. In addition, cooking of the meat product can also affect the colour changes generated by the use of pulses as extenders. Any colour comparison should be mainly addressed on the product at the state it is going to be purchased, although extra analyses can also be taken into consideration. Lightness was not affected by pulses as extenders in pork and beef patties with varying levels of substitution [33,36]. However, the use of some pulses as extenders in a variety of meat products have significantly affected the redness and yellowness values [33,34,36,39].

The product appearance is the first attribute the consumer observes before purchasing the product, and even though the instrumental colour measurements are correlated with the appearance, the consumer might not be able to detect the differences as the instrument does, or they could like better the colour change. In the same way, texture results from instrumental measurements and those from sensory panels differ substantially. Serdaroglu et al. [ ˇ 39] found that general appearance scores for meatballs with pulses as extenders did not significantly differ when compared to the rusk, but instrumental colour values for the same products showed significant changes in the yellowness value. In the same study, the meatballs with chickpea flour had the harder texture value, and it was scored lower by the panellists, but the one with the highest score was not the one with the softer texture but the second-hardest. These sensory analyses were done by trained panellists on a nine-point hedonic scale, and even though this practise should be avoided—hedonic analyses should always be carried out by non-trained panellists—it can give an idea of the sensorial properties of the product. When black beans, green grams and cowpeas were used as extenders in chicken seekh kababs, the sensory properties remained unaltered throughout storage, with no significant differences among the pulse varieties [35]. Yadav and Yadava [37] found that gram flour substituting meat in quail meat rolls at levels 3% and 6% did not affect the sensory properties, although, at 9%, they observed a significant decrease in the colour and flavour scores by the panellists. The use of texturised soy granules in dehydrated chicken meat only affected the meat flavour intensity, according to a sensory panel [34]. Argel et al. [33] found that pork patties where the meat was substituted (37%) with chickpea, lentil, green pea and bean flour emulsions had acceptable sensory properties, with no significant differences among the pulse types.

The use of pulses as meat extenders has been researched mainly in patties and similar meat products, but no work on comminuted ones, although some studies with pulses as binders can be found for these types of products [40–42]. In general, pulses seem to be an adequate ingredient to be used as a meat replacer, as they have a very similar nutritional composition and do not affect extremely the physicochemical properties of the finished product. Unfortunately, a limitation from pulses and legumes as extenders can be found on the allergenic potential of some proteins contained in soybean and peanuts that would restrict population access to these products (people with allergies) and would need proper labelling [49].

#### *2.2. Other Meat Extenders of Plant Origin: Cereals, Tubers and Fruits*

Other plants such as cereals, tubers and fruits have also been used in meat product formulations. The main reason of using these food products as ingredients in meat products has been the healthy properties they possess: high fibre contents, vitamins and minerals, important proportions of phytochemicals and antioxidants and void of cholesterol, among others [50,51]. Apart from their nutritional properties, some of these plants also have good functional and technological properties, such as improved water-binding and yield properties, fat emulsifiers, increased flavour, etc. [52]. Even though their main usage has been for the development of functional meat products [53–56], there has been also some research about the use of these ingredients as meat substitutes/extenders. Research about the use of cereals, tubers and fruits as meat extenders in the last thirteen years is summarised in Table 2.


**Table 2.** Use of cereals, tubers and fruits as meat substitutes (extenders).

Fruits and their by-products have been used as ingredients in meat products to improve the shelf lives and provide meat with antioxidants, fibre and other phytochemicals [66]. However, their role as meat extenders is yet to be explored, with only a few studies in the scientific literature. Melon flour, from defatted melon kernels, was used to substitute meat in beef sausages at levels 10–40% [63]. The authors found an increased yield, WHC and better sensory properties with the increasing levels of substitution. No significant differences with control on the overall acceptability and appearance were found at the 20% substitution level. The same authors found that, after two and four weeks of storage, the thiobarbituric acid reactive substance (TBARS) values were significantly lower for the sausages with substitution levels above 20% [64]. Low-fat beef patties where the meat was substituted with plum puree (5–15%) showed an increase in the cooking yield and redness of the patties but a decrease in WHC, lightness and yellowness [52]. The TBARS values of the extended patties with plum were lower at the end of the storage period, irrespective of the substitution level. In addition, the sensory properties were improved at the 10% and 15% levels of substitution, being the former the one with the best scores in overall acceptability, flavour, texture and juiciness. An increased cooking yield has been also found in beef patties extended with olive cake powder at levels 2.6–7.9% [59]. The olive cake powder also increased the amount of polyphenols and the antioxidant activity of the patties, but the instrumental colour was also affected, with a decrease of the lightness and an increase of the yellowness with increasing levels of substitution. The sensory properties were negatively affected, with significantly lower values at the higher levels of substitution. When using plum puree as an extender in beef patties (2.8–6.9% substitutions), the cooking yield and sensory attributes remained unaltered, but the WHC increased with the increasing levels, the redness dropped and the hardness increased [58]. All of these studies proved that fruits can be used as meat extenders, but further research is needed on different meat products (not only patties) and with different types of fruits and substitution levels.

Cereals are crops of the family *Gramineae*, which comprises nine species: corn, barley, millet, oat, rice, rye, sorghum, triticale and wheat. They are an important source of proteins (ranging from 7–18% dry matter) and vitamins (B group and E) [67]. Chicken patties where the meat was substituted by sorghum (5%), pressed rice (5%) and barley flour (10%) showed a significant decrease on the extract

release volume and lower TBARS values at the end of storage, with no significant impact on the sensory properties [65]. Mishra et al. [34] found that rice flour at a 10% substitution level in dehydrated chicken ring meat did not affect the sensory properties, whereas a 5% meat substitution with barnyard millet flour decreased the sensory perception of the meat flavour intensity while not affecting any of the other sensory attributes. The same authors also observed that the yield was improved by these two extenders without affecting the instrumental colour. Both cereals also significantly reduced the cholesterol content and increased the manganese; the millet chicken meats had also a 10-fold increase of their iron contents, while the meats with rice had lower iron contents when compared to the control. Corn flour used as a meat extender in quail meat rolls increased the yield and emulsion stability with the increasing level of substitutions (3–9%) [37]. However, the sensory perceptions of colour and flavour were impacted on the rolls where meat was substituted at a 9% level but remained unaffected at the lower substitution levels. A screening of a combination of five different cereals and six plants and tubers as meat extenders (10%) in sheep meat cubes was performed using a Plackett-Burman design [62]. The authors found that millets, carrots and cabbages gave the cubes the most desirable sensory characteristics and that further research with these ingredients should be guaranteed. Malav et al. [61] analysed the use of a blend of sorghum with potato, lentil and water chestnut flours as extenders (15%) in restructured chicken. The blend of extenders exerted higher yields and similar texture attributes but lower sensory scores. Another study where cereals were combined with other ingredients as meat extenders in the same type of product was done by Gupta and Sharma [57]. Wheat, oat and barley were blended with potato, whey and texturised soy protein in three different combinations that were compared to a control. The three blends increased the cooking yield and decreased the hardness, but only one of them did not differ in the overall acceptability of the product; the other two had lower scores for flavour. With regards to the instrumental colour, the redness was not affected, but the yellowness increased in all the reformulated samples. However, the sensory appearance was higher for the sample with the highest chroma value. Cereals proved to be important and successful ingredients when used as meat extenders, but their behaviours in meat products different than restructured meat and chicken are still unknown. It is also important to highlight that cereals containing gluten (wheat, rye, barley and oats) have allergenic potentials that must be declared in the labelling.

#### *2.3. By-Products of the Food Industry as Meat Extenders*

The food industry (from vegetables or animal products) produces high amounts of residues and/or by-products that are edible compounds with high percentages of proteins and/or fibres. In today's global scenario, the use of these compounds—in many cases, undervalued—could be an opportunity to replace meat for manufacturing more sustainable meat products [68]. Furthermore, many of these residues are a source of polyphenols, organic acids and fatty acids, among others, which are underutilised, providing added value to the products in which they are included [69]. In this regard, some studies have assayed the use of residues from the agri-food industry as meat extenders (Table 3).




**Table 3.** *Cont.*

Okara is a by-product with low commercial value that is generated in massive volumes (about two to three tons for each ton of soybean processed) during the manufacturing of soymilk and tofu [80]. This component presents solvent-binding properties, making it an ideal low-cost ingredient to increase yields in meat products (Table 3). Moreover, okara contains valuable components such as fibre and high-quality protein (40% on a dry weight basis) due to the presence of a good essential amino acids profile and its digestibility [80]. In that sense, okara has been applied to extend meat contents both in fresh and cooked emulsion-based meat products (Table 3). In beef burgers, lean meat has been replaced by wet okara in different quantities, up to 37.5% (Table 3). In general, it was observed an increase of the moisture content and a decrease of the protein level in the reformulated burgers [70,71,73]. Moreover, Tie Su et al. [73] obtained beef burgers with 60% less calories than commercial products when 12% of okara was added. The use of okara as a meat extender improved the cooking yields of the samples [70]. Tie Su et al. [73] noticed that, as the percentage of okara increases, an increase in hardness occurs, while Strada de Oliveira et al. [71] observed an improvement in tenderness with respect to the control samples. The effect of wet okara on the sensory properties was significant, and higher scores for overall acceptability were recorded for products with approximately 20% added okara [70,73]. In cooked emulsion-based sausages, contrary to those observed in fresh meat products, the moisture content was increased with an okara addition [72]. Water and oil-holding capacities were improved as a consequence of okara additions, and in that sense, the cooking yield was improved [72]. For textural properties in cooked emulsion-based products, the incorporation of okara presented contradictory behaviours. The same authors observed an increase for the texture parameters with up to 40% of okara added to beef sausages, while a decrease of the hardness, chewiness and breaking force occurred when okara was incorporated in pork meat batters [74]. On the other hand, the overall acceptability of the samples decreased with the okara incorporation [72].

The residue obtained from the production of cashew apple juice (skin and the husk) has been used to extend beef meat in the formulation of hamburgers. With increasing the concentrations of the residues, the samples showed lower moisture, protein and lipid levels, while their fibre contents were higher. Hamburgers with improved yields and similar flavours than the control were observed with additions up to 10.70% of the residue [75]. Apple pomace powder was employed (2–8%) to replace buffalo meat in emulsion-based sausages by increasing the fibre contents. Moreover, the cooking yield and emulsion stability got enhanced [76].

Mushroom by-products are described as a good source of protein, dietary fibre and phenolic components, with the potential to be strong antioxidants [77]. In that sense, the use of different amounts (2%, 4% and 6%) of enoki (*Flammulina velutipes*) mushroom stem wastes as meat extenders in nuggets enhanced their composition (Table 3). The inclusion of meat extenders improved the oxidative stability and shelf-lives of treated nuggets without impacting the sensory attributes of reformulated nuggets.

Whey is a by-product of the dairy industry, which is generated in massive quantities during the manufacture of cheeses, yogurts and other dairy products [81]. Its great content of high biological value proteins offers interesting possibilities to be used during the processing and manufacturing of meat products. Hale et al. [78] extruded a dry whey protein concentrate (80% protein) to obtain an ingredient that they used to substitute from 0% to 50% of beef in the elaboration of patties. Samples containing up to 40% of whey extrudes were as acceptable to a consumer panel as all-beef patties. Moreover, the cooking yield was improved, and these patties suffered less diameter reductions and less water and fat losses by the cooking process.

The meat industry also generates compounds that hold strong potential for higher-value techno-functional applications due to their high-quality protein contents (Table 3). However, their use as meat extenders in meat products is very limited. For example, Álvarez et al. [79] extracted protein concentrates from different residues of the meat industry to be used as meat replacers in the elaboration of an Irish breakfast-type sausage: blood plasma, exudates generated from ham elaboration, brine solutions and water produced during edible fat processing. Two levels were assayed: 15% and 30% (Table 3). Regarding the composition, raw products showed lower fat contents and higher protein levels than the control ones. However, the technological properties were conditioned by the type of protein used and the level of meat substitution. In general, for all types of protein, the 15% meat replacement offered products with a better overall final product quality. Regarding the type of protein, plasma proteins at both replacement levels had the most positive effect on the technological properties, whereas the use of protein concentrates from brine solutions to substitute meat resulted in sausages with lower fat and water-binding properties and redness values (Table 3).

Based on the foregoing, it seems that the use of residues or by-products from the food industry as meat replacers endows products with compounds that offer positive effects on health without being a detriment to their technological properties. In addition, this strategy offers multiple advantages to maintaining a more sustainable world by both using industrial residues and reducing meat productions.

#### *2.4. Mushrooms as Meat Extenders*

Fungi have been used in human foods for a long time. Of more than 14.000 species of mushrooms, at least 2000 of them have various degrees of edibility [82]. Mycoprotein is fungal in origin, and it is utilised as a high-protein, low-fat, health-promoting food ingredient [49]. Mycoproteins could be obtained by the continuous-flow fermentation of *F. venenatum* on a glucose substrate, and it is used to elaborate meat analogues. However, in the development of more sustainable meat products, some studies were carried out adding mushrooms directly to meat products (Table 4), replacing different proportions of meat proteins by mycoproteins.


**Table 4.** Use of mushrooms as meat substitutes (extenders).


Mushrooms are a good source of dietary fibre, where approximately one-third is chitin and two-thirds β-1,3 glucan and 1,6 glucan. Chitin is a modified polysaccharide with an analogous structure to cellulose and considered an insoluble fibre with potential prebiotic properties in gut microbiota [89]. In addition, mushrooms are also a source of proteins; essential amino acids; vitamins (such as thiamine, riboflavin and niacin) and essential minerals (such as Ca, P, Mg, Cu, Se and Zn). Moreover, these products are low in calories, fat and sodium [90]. In that sense, the application of mushrooms as meat extenders could also be an opportunity to improve the presence of health-promoting bioactive components in meat products.

White mushrooms (*Agaricus bisporus*), the most cultivated edible mushroom, poses a dual opportunity as a meat extender by reducing the meat content while also imparting flavours that can complemented and enhance the saltiness perception [82]. Wong et al. [83] compared two meat extenders, a traditional one (textured soy) and *Agaricus bisporus*, to replace 10% to 50% of meat in the development of beef patties (Table 4). Increasing textured soy improved the cooking yield of patties but did not affect their colour or textural properties. However, increasing the level of mushroom extenders performed statistically similar to an all-meat control in yield, lightness and redness, while decreasing the mechanical properties. Additionally, meat extension using mushrooms yielded sensory liking scores more similar to the all-meat formulations than textured soy in reduced sodium samples. In the same way, white mushrooms were used to replace meat in two meat-based dishes, carne asada and beef taco blends, whose sodium contents were reduced [84]. In carne asada, the beef substitution (50%) with mushrooms did not alter the overall flavour strength of the dish, but the replacement of 50% or 80% of meat in the beef taco blend enhanced its overall flavour. The ability of mushrooms to mitigate sodium reductions in terms of the overall flavour has been attributed to the fact that mushrooms contain umami tastants [82]. White jelly mushroom (*Tremella fuciformis*) is another type of edible mushroom that has been used as a meat extender in pork meat patties (Table 4) [85]. In this case, higher mushroom quantities (30%) decreased the sensory acceptance of patties because of the mushroom flavour. However, patties containing 10% of mushrooms improved significantly the sensory affections due to their oil-holding capacities. Furthermore, this ability, along with its capacity to bind water, allowed improving the cooking yield of patties formulated with white jelly mushroom [85]. In pork sausages, *Lentinula edodes* has been used as meat extender to replace 25%, 50% and 100% of the meat (Table 4). Regarding sensory acceptability, all samples were satisfactory. Although those with 25% of substitutions showed the highest scores for sensory attributes. From a technological point of view, the presence of mushrooms improves the oxidation stability and the cooking yield of sausages [86].

The use of *Pleurotus sajor-caju* as a meat extender (25% and 50% of meat substitutions) in beef patties and in lower proportions (2% to 6%) to replace chicken meat in the formulation of frankfurters produced an increase of their fibre contents. It should be noted that this fibre was insoluble mainly based on β-glucans (0.78 g/100 g in the case of patties and 1.43 g/100 g in frankfurters) [87,88]. As with other mushrooms, the use of *Pleurotus sajor-caju* as a meat replacer improved the cooking yield of the products. The hardness values of the reformulated products were lower. However, the sensory analysis scores indicated that the products were accepted by the panellists [87,88].

Mushrooms seem to be an adequate ingredient to be utilised as a meat replacer. The use of mushrooms allows for the development of healthier meat products with higher fibre and less salt contents (as they have the potential to increase saltiness perceptions) without affecting much the physicochemical properties.

#### *2.5. Insects as Meat Extenders*

Entomophagy, or the practise of eating insects, is a long-time practise and an important nutritional source (high-quality protein, lipids, carbohydrates, mineral elements and certain vitamins) for many cultures, mainly located in Africa, Asia and Latin America [91]. More than 40 years ago, Meyer-Rochow [92] already suggested that insects could supplement traditional animal protein sources. Currently, there is a growing interest in edible insects as a novel source of protein due to their high contents, as well as their functionalities, which have been described similar to conventional proteins (included meat proteins) [91]. However, probably due to insect food neophobia in Western countries, there are only a few studies using insects as meat extenders, and the majority are from Eastern Asian countries (Table 5). With the aim to decrease this well-known food neophobia related to insects, Caparros Megido et al. [93] decided to test the level of sensory-liking of patties in which beef was replaced (53%) by mealworms, allowing them to hide insects and to present them in a familiar way. The authors concluded that insect integration into Western food culture could be feasible, as the taste and appearance of burgers were rated higher than neutral scores, positioning them between a fully meat burger and a fully vegetable burger.



The incorporation of mealworms as meat replacers was also studied to evaluate their effects in the composition and technological properties of new products. Ju-Hye et al. [94] studied the effects of different replacement ratios (10% to 60%) of pork meat in the development of patties (Table 5). The addition of mealworms conditioned significantly the composition of the samples, decreased protein contents and increased fat levels. The cooking yield was improved with the presence of insects. There were no significant differences in the sensory characteristics of burgers, although the shear force was reduced and the lightness was increased with the replacement of meat by insects.

In emulsion-based meat products, mealworms (*Tenebrio molitor* L.) have been used to replace 10–60% of pork meat (Table 5). Reformulated samples had increased protein and fat contents when the meat was replaced at the 10% level [95,96]. However, Choi, Kim, Choi, Park, Sung, Jeon, Paik and Kim [95], who assayed higher levels of extended meat (up to 60%), observed that frankfurters with a higher meat replacement by mealworms increased the protein content but decreased the fat content approximately to 30% in respect to all-pork meat samples. Moreover, the incorporation of edible insects increased the mineral contents of emulsion sausages [96]. The cooking yield was improved with a substitution of meat of 10%; extended higher meat decreased the cooking yield [95,96]. Additionally, replacing pork meat with up to 10% mealworms successfully maintained the sensory quality of frankfurters.

Silkworm pupae (*Bombyx mori*) and the House cricket (*Acheta domesticus*) are two other types of edible insects used as meat extenders (Table 5). Kim, Setyabrata, Lee, Jones and Kim [96] added freeze-dried Silkworm pupae (*Bombyx mori*) to replace 10% of the pork meat in an emulsion-based meat product. They assayed three strategies to incorporate the insects: ground, defatted and acid-hydrolysed. The inclusion of insects had no impact on the protein solubility of emulsion sausages. The protein contents of sausages were increased for all the treatments; however, the fat contents only were increased when insects were ground. Additionally, the mineral content was improved when ground and defatted Silkworm pupae was incorporated [96]. The replacement of pork meat with house cricket flour within a 10% level could fortify the product with proteins and some micronutrients (phosphorus, potassium and magnesium) without a negative impact on the cooking yield and textural behaviours [97].

Edible insects possess the necessary physical properties to be used as an alternative nonmeat ingredient for incorporation within fresh or emulsified meat products, which could be further promoting to improve the image that the consumers have of them. Moreover, the addition of invisible insects in food preparations helps to reduce insect food neophobia [93].

#### **3. Meat Products**0 **Sustainability from the Consumer Perspective**

As stated before, protein production has a large impact on the climate change, with proteins from meat being much less sustainable than plant-based proteins [98]. It seems logical to think that the daily choice of food has a high impact on the environment, and therefore, acting to change consumer preferences seems an appropriate strategy to reduce the negative impact that food production may have [99].

Some alternatives for meat products made entirely of vegetable components (e.g., tofu) can be already found in the supermarkets, although the market shares of these products are still very low compared to meat and meat products. The lower penetration of these products in households could be partially explained by the lack of texture and taste reported for some of them [100]. In addition, the heavy processing conditions to obtain these products and, in consequence, the multiple additives that they contain are sometimes neglected; besides, they can have a really high carbon footprint [28].

Complex external cues (perceived healthfulness, animal welfare, environmental impact and sustainability) are increasingly taken into account in our preference for meat [101]. However, despite a seemingly close match between the consumers' image of a sustainable, healthy and a plant-based diet [102], there is actually low consumer awareness of the environmental impact of meat production, as well as a low willingness to change meat consumption behaviours in terms of reducing or substituting meat in Europe. It is therefore relevant to determine the opportunities and barriers for consumers to adopt such alternative meat protein sources in their diets [100]. Preconceptions towards vegetarian diets, habits and prices and a lack of familiarity with meat substitutes, among others, are barriers to changing meat consumption behaviours [103]. Despite all of the above, it must be taken into account that the complete elimination of meat from our diet is impractical and might even have negative societal consequences [104].

The challenge of developing healthier foods with high consumer appeal underscores the need for integrated culinary, sensory and consumer research in this area [105]. Although Hoek et al. [99] concluded that, for the development of new foods, more emphasis is needed on consumer evaluation instead of on the sensory properties of the individual product. In that regard, studies that also take consumer behaviours into consideration could be an alternative to standard consumer sensory analyses. A recent alternative method called Mind Genomics has been applied on meat analogues, with promising results [106]. In addition, in order to increase the acceptance of novel products, it is necessary to obtain knowledge about the demographics, the consumption patterns and the sensory drivers of consumers [107]. In Western countries, vegetable proteins have a high level of acceptance and are consumed regularly. However, the same does not occur with the inclusion of nonconventional meats, insects or food by-products in our diet.

An alternative to conventional meat production is the use of more sustainable species like rats or other pest rodents [108,109]. Although rats are a regular staple in some Asian regions, the mere suggestion of its consumption in Western countries generates a big consumer rejection. Caparros Megido et al. [93] concluded that insect-tasting sessions are important to decrease their neophobia, because they observed that people with previous entomophagy experience gave globally higher ratings to meat products that contained insects-based proteins. In addition, Meyer-Rochow and Hakko [110] concluded that the acceptability of insect consumption would be higher if they were presented in flours or pastes. The inclusion of food by-products or residues from the meat industry can also present a challenge to consumer acceptance. Even though this practise presents a double opportunity towards healthier and more sustainable meat products, their acceptance is quite limited. Some of the reasons are related with consumer perceptions of these by-products as actual waste and, thus, unhealthy, but even if healthiness would be proven, consumers would also reject some of these reformulated products due to "ideational" reasons [111]. This concept is linked to the sensation of disgust some products produce in consumers just because of their origin (e.g., insects, by-products, etc.) and bad taste.

Meat eating is a habitual behaviour that is difficult to change; there is an unwillingness to reduce or substitute meat among the vast majority of consumers in various European countries [100]. In search of new alternatives, it is necessary to know how different food-related attitudes and behaviours (food choice motives, food fussiness, etc.) and socio-demographics (gender, age, education, etc.) influence the consumption of such protein sources [103]. In that sense, although some studies concluded that there is an urgent need for meat moderation campaigns that provide a broad spectrum of measures and habit-breaking interventions—including the promotion of vegetarian options [112]—the use of extenders to reduce animal proteins in the development of meat products could help to minimise their environmental impact without having to give up entirely the meat products in our diet.

#### **4. Conclusions**

A global demand for high-protein foods is on the rise. Meat and meat products are an important protein source in our diets but also great contributors to environment degradation through the far-from-sustainable production and increased carbon footprint of the finished products. Alternatives to more sustainable protein productions fall into two categories: mitigation of the negative impact and the use of more sustainable protein sources. With the use of meat extenders in meat products, we would be mitigating their negative impact by reducing the meat content, but we would also be maintaining the nutritional properties (i.e., protein and minerals) by using more sustainable sources. Even though pulses are the main extenders we should be looking at—similar nutritional profiles to meat—there are other extenders worth exploring. Apart from mushrooms, cereals, tubers and fruits that can be a great choice for some types of meat products, novel approaches such as insects and by-products from the food industry present an opportunity to develop healthier and more sustainable meat products. However, there is a need to devise strategies to increase consumer awareness and acceptance of these types of products. The plethora of sources and possibilities make the use of meat extenders the most viable and interesting approach towards the production of more sustainable meat products.

**Author Contributions:** Conceptualisation: G.D.-P., occurrence: T.P., writing—original draft preparation: G.D.-P. and T.P., writing—editing: T.P. and review: G.D.-P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We would like to thank the reviewers for their time, effort, insightful comments and suggestions that helped improve this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **A Novel Approach for Tuning the Physicochemical, Textural, and Sensory Characteristics of Plant-Based Meat Analogs with Different Levels of Methylcellulose Concentration**

**Allah Bakhsh <sup>1</sup> , Se-Jin Lee <sup>1</sup> , Eun-Yeong Lee <sup>1</sup> , Nahar Sabikun <sup>1</sup> , Young-Hwa Hwang <sup>2</sup> and Seon-Tea Joo 1,2,\***


**Abstract:** This study assessed the effects of Methylcellulose (MC) at different concentrations on plant-based meat analog (PBMA) patties, comprised of commercial texture vegetable protein (C-TVP) and textured isolate soy protein (T-ISP) as key ingredients, and compared to beef patty control. A significantly higher difference was observed in moisture content in control with increasing MC concentration than the C-TVP and T-ISP patties. However, protein varied significantly among three different protein sources, with control had higher protein content than PBMA patties. Crude fiber content recorded higher values in C-TVP as compared to control. Significantly lower pH values were recorded in control than C-TVP and T-ISP respectively. Regardless, with the addition of MC or ingredient PBMA and control patties tend to reduce lightness (L\*) and redness (a\*) value after cooking. Although control sample before cooking exhibits lighter and redder than PBMA patties (C-TVP and T-ISP). Likewise, water holding capacity (WHC) decreases as the concentration of MC increases (1.5–4%) in control and PBMA patties. Warner-Bratzler shear force (WBSF) and texture profile analysis (TPA), including hardness, chewiness, and gumminess of control, were significantly higher than C-TVP and T-ISP. Consequently, panelists' in the sensory analysis presented that C-TVP patties containing 3% of MC had better sensory properties than T-ISP. Hence, PBMA patties with C-TVP and incorporation of 3% MC are considered ideal for manufacturing of meat analog as related to control (beef).

**Keywords:** plant-based meat analog; commercial texture vegetable protein; texture soy isolate protein; methylcellulose

#### **1. Introduction**

The term "meat analog" denotes food products that are not made from red meat exclusively, commonly known as meat alternatives, meat substitutes, fake, mock, and imitation meat [1]. However, it possesses texture, mouth-feel, taste, and nutritional qualities that resemble meat [2]. Meat contributes to the food industry by supplying specific functionalities and has its attraction on consumers for its organoleptic features. Meat proteins are responsible for their characteristic appearance, textural and functional properties [3]. However, mimicking these meat protein characteristics by any other source of protein is difficult. Moreover, recently the International Agency for Research on Cancer, the cancer agency of WHO (World Health Organization), has classified the consumption of red meat (particularly processed meat) as carcinogenic to humans [4]. Furthermore, Food and Agricultural Organization (FAO) reports have been critical of the ecological impact of high levels of meat consumption and potentially transmissible diseases [5,6]. To mask these disadvantages of red meat, meat analogs are just one example of a variety of

**Citation:** Bakhsh, A.; Lee, S.-J.; Lee, E.-Y.; Sabikun, N.; Hwang, Y.-H.; Joo, S.-T. A Novel Approach for Tuning the Physicochemical, Textural, and Sensory Characteristics of Plant-Based Meat Analogs with Different Levels of Methylcellulose Concentration. *Foods* **2021**, *10*, 560. https://doi.org/10.3390/foods 10030560

Academic Editors: Gonzalo Delgado-Pando and Tatiana Pintado

Received: 18 January 2021 Accepted: 4 March 2021 Published: 8 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

products recently demanded by a substantial portion of the population, especially those concerned about red meat's potential health effects [7]. Additionally, the projections for the increasing demand for animal protein in the coming decades are distressing, while extensive livestock production is also causing severe environmental and ecological imbalance [8]. Consequently, the research community is targeting refining the current production systems, searching for efficient novel technologies, while at the same time focusing on the improvement of consumption habits and food cultures [9].

In the current study, soy-based texture vegetable protein (TVP), and textured isolate soy protein (T-ISP) have been used as a meat replacer with many economic and functional benefits [3]. Soy-based TVP<sup>S</sup> are plant-based protein products with low saturated fat, a high concentration of essential amino acids, and is cholesterol-free [10]. The manufacturing process of TVP involves a high-pressure extrusion process and a final spinning or extraction of the finishing product, which can then be used in meat analogs [7]. The low/intermediate moisture TVP has advantages in handling, storage, and shelf stability but requires time to hydrate before consumption. Upon hydration, it presents a spongy textured, fibrous structure mimicking meat [7]. Furthermore, numerous investigators have reported that by using soy protein and wheat gluten as TVP constituents, the final product could mimic the texture, appearance, taste, smell, and functionality of red meat [5].

In red meat, textural and taste parameters are important to the consumers and represent high economic value as some cuts bring exorbitant prices. In contrast, meat analogs lack these features and are generally regarded as substandard to cheaper meats. Numerous plant proteins, including cereal, oilseed, legume, and soy proteins (textured, flour, concentrate, and isolate), are recommended additions to the meat analogs. These elements have appropriate functional properties (e.g., water and oil absorption capacity, emulsification), which allow them to create numbers of distinctive meat substitutes [5,11].

The binding ability of the different ingredients in plant-based meat is of significant importance as non–adhesive behavior of varying plant ingredients can significantly affect the final analogy. Earlier binding agents such as egg solids, hydrocolloids, starch, and milk protein have been used in various commercial products [12]. In the present study, Methylcellulose (MC) has been used as a binder. Quality characteristics of MC include binding abilities and moisture retention, boil-out control, increase volume, and texture improvement in several types of meat analogs and processed meat [13]. Through synthetic modification, the naturally occurring polymer cellulose is converted to hypromellose or MC and is considered safe for consumption by humans [13]. Moreover, MC is classified as GRAS (generally recognized as safe) by the FDA (21 CFR 182.1480) and is also allowed in USDA regulated meat patties at concentrations up to 0.15% (9 CFR 3 t 8.7). Previously, the use of binding agents in meat analogs has been widely investigated, although no such attempt has been made to study the effects of MC on quality characteristics of Plant-based meat analog (PBMA) patties. Therefore, the objective of the present study was to evaluate the effects of MC on quality characteristics of PBMA with the incorporation of different texturized soy vegetable proteins.

#### **2. Materials and Methods**

#### *2.1. Materials*

Commercial texture vegetable protein (C-TVP) (Anthony's goods, Glendale, CA, USA) and ISP (isolate soy protein) (Shandong, China) were as the base for PBMA and MC (high viscosity, Modernist Pantry, Eliot, ME, USA) was incorporated as a binder. Other ingredients, including molasses, yeast seasoning, umami seasoning, coconut oil, canola oil, garlic powder, and pepper were used in the formulation (Table 1).


**Table 1.** Treatment and formulation of plant-based meat analogs.

C-TVP: Commercial textured vegetable protein. T-ISP: Textured isolate soy protein. MC: Methylcellulose.

#### *2.2. Sample Preparation and Processing*

The flow diagram for processing meat analog is described in Figure 1. For meatless patties, C-TVP and texture isolate soy protein (T-ISP) were used as the base. The texturization of ISP was carried out by mixing ISP powder with water at a ratio of 1:6 (*w*/*v*). The mixture was stirred continuously over a lower flame until it forms a thickened paste. Subsequently, the paste was heated in an oven for two hours at a temperature of 120 ◦C T-ISP was a secondary option for comparing the quality characteristics of the created meatless patties to C-TVP. A total of three hundred g of each C-TVP and T-ISP were mixed with water separately (2 times in volume) and allowed to hydrate for "1 h" at 4 ◦C for a single concentration of MC with three repetitions and two formulations having raw and cooked patties respectively. After that, the hydrated C-TVP and T-ISP were mixed with the ingredients listed in Table 1 using a Kitchen Aid (Classic Plus Stand Mixer, St Joseph, MI, USA). Subsequently from the whole mixture, 50 g of the mixture was then shaped into patties using a patty press maker. The current experiment had three different concentrations of MC (1.5%, 3%, and 4%), from every single concentration of MC three patties (repetition) were prepared with one control and two treatments. In total, for one control, two treatments, and two formulations, 27 raw and 27 cooked patties were prepared. Therefore, in total, 54 patties were shaped. Eighteen patties were allocated for each control and two treatments separately.

A beef patty was used for the control formulated as describe in Table 1. The patties were cooked by dry heat, cooking on a non-stick pan at 150 ◦C for 5 min per side. They were flipped three times or until the internal temperature reached 75 ◦C as measured by a probe thermometer. Patties were allowed to cool at ambient temperature for 30 min before measuring the physicochemical and sensory attributes.

**Figure 1.** Flow diagram for manufacturing the meat analog. C-TVP: Commercial textured vegetable protein. T-ISP: Textured isolate soy protein. PBMA: Plant-based meat analog. **Figure 1.** Flow diagram for manufacturing the meat analog. C-TVP: Commercial textured vegetable protein. T-ISP: Textured isolate soy protein. PBMA: Plant-based meat analog.

#### *2.3. Proximate Analysis 2.3. Proximate Analysis*

Moisture, protein, fat, and ash contents were determined based on the standard AOAC [14]. Moisture content was quantified by the oven (BioFree, BF-150C, Buchen Korea), drying 5 g samples at 105 °C for 16 h. Protein was determined by the established procedure of Kjeldahl assay N analyzer (B-324, 412, 435 and 719 S Titrino, BUCHI, Flawil, Switzerland) (N × 6.25) using 0.1 g of sample. The crude protein was determined by using the following formula. Moisture, protein, fat, and ash contents were determined based on the standard AOAC [14]. Moisture content was quantified by the oven (BioFree, BF-150C, Buchen Korea), drying 5 g samples at 105 ◦C for 16 h. Protein was determined by the established procedure of Kjeldahl assay N analyzer (B-324, 412, 435 and 719 S Titrino, BUCHI, Flawil, Switzerland) (N × 6.25) using 0.1 g of sample. The crude protein was determined by using the following formula.

$$\% \text{N} = \frac{[\text{V}(1) - \text{V}(\text{B1})].\text{F.c.F.M}(\text{N}) \times 100}{\text{M.1000}} \tag{1}$$

$$\% \text{ P} = \% \text{ N} \times \text{PF} \tag{2}$$

V(1): consumption of titrant, sample (mL) V(BI): average consumption of titrant, blank (mL) F: molar reaction factor (1 = HCl, 2 = H2SO4) c: concentration of titrant [mol /L] M(N): molecular weight of N (14,007 (g/mol)) M: sample weight (g) 1000: conversion factor (mL in L) PF: protein factor V(1): consumption of titrant, sample (mL) V(BI): average consumption of titrant, blank (mL) F: molar reaction factor (1 = HCl, 2 = H2SO4) c: concentration of titrant [mol/L] M(N): molecular weight of N (14,007 (g/mol)) M: sample weight (g) 1000: conversion factor (mL in L) PF: protein factor % N: % of weight of N % P: % of weight of protein

Crude fat was measured with 2 g samples by extraction in a Soxhlet apparatus (MS-EAM9203-06, Seoul Korea) by using petroleum ether as a solvent. The crude fat content was calculated by using the following formula.

$$\% \text{Crudefat} = (\text{W2} - \text{W1}) \times \frac{100}{\text{S}} \tag{3}$$

Weight of empty flask (g) = W1

Weight of flask and extracted fat (g) = W2

Weight of sample = S

Ash was determined after incineration of 2 g of sample in a furnace (CFMD2, Changsin, Korea) at 500 ◦C. Crude fiber determination was estimated using an Ankom 200 Fiber Analyzer (Ankom Technology, Macedon, NY, USA) by digesting 0.5 g with H2SO<sup>4</sup> and NaOH. The loss of weight resulting from ashing (2 h at 600 ± 15 ◦C) was collected to calculate the crude fiber content [15].

#### *2.4. Physicochemical Analysis*

The pH values of raw and cooked patties were measured with a digital pH meter (Mettler Toledo, MP230, Schwerzenbach, Switzerland) using 3 g of sample homogenized with 20 mL of distilled water.

The color of raw and cooked patties was measured using a Konica Minolta Colorimeter (Chroma meter, CR-300, Japan). The apparatus was standardized through a white ceramic plate (Y = 93.5, X = 0.3132, y = 0.3198), and lightness (L\*), redness (a\*), and yellowness (b\*) values were recorded.

Release water percentage (RW%) was measured based on a method described by Joo [16]. The cooking loss (CL%) was determined as a percentage method adopted by Biswas et al. [17] using the following formula: Cooking loss (%) = (Weight of the patties after cooking/Weight of the patties before cooking) × 100.

Warner-Bratzler shear force (WBSF) was determined on the cooked sample using the established AMSA procedure [18]. The shrinkage percentage of the patties' diameter was measured at four different locations both before and after cooking. A total of 18 (nine raw and nine cooked) patties were allocated for physiochemical analysis.

#### *2.5. Visible Appearance*

The appearance of the control and PBMA patties were assessed by adding the different concentrations of MC (1.5%, 3%, and 4%) respectively. The external and internal appearance were photographed using a digital camera (EOS 700D, Canon, Tokyo, Japan), and various features were distinguished. In total, 18 (nine raw and nine cooked) patties were used for visible appearance.

#### *2.6. Texture Profile Analysis*

Samples were uniformly cut into 1 × 1 × 1 cm, and they were axially compressed using a Sun Rheometer (Compact-100 II, Sun Scientific Co., LTD., Tokyo, Japan) with a flat pressure adaptor of 25 mm in diameter (No. 1). The samples were compressed at a crosshead speed of 60 mm/min at a final strain of 60% through a 2-cycle sequence with a load cell of 10 kg [19]. The following parameters were determined: hardness, cohesiveness, springiness, gumminess, and chewiness. A total of nine patties were assigned for the determination of texture profile analysis.

#### *2.7. Sensory Evaluation*

A 10-member trained panel from the laboratory of meat science Gyeongsang National University Korea, with 20 members of the untrained panel, includes students and researchers from the Department of Animal Sciences at Gyeongsang National University, Republic of Korea, assessed sensory characteristics of prepared patties. The panelist assortment was approved according to Lawless and Heymann [20], modified by Rahman

et al. [21]. Small pieces of different samples (2 cm × 2 cm × 2 cm) were prepared and marked, random coding was allotted on pre-positioned glass container (Pyrex, Charleroi, PA, USA), and the pieces of samples were permitted to rest for 30 min at room temperature and then disseminated among the panelists. For judging each sample in a triplicates way, fluorescent light was used. For every sensory evaluation procedure, the panelist was provided with drinking water for washing the mouth for every new sample evaluation. Sensory traits that were recorded included appearance, shape, firmness, color, and overall acceptability. The samples were judged using a 9-point hedonic scale ranging from extreme dislike (score = 1) to extreme like (score = 9). A total nine number of PBMA patties were assigned for sensory evaluation.

#### *2.8. Statistical Analysis*

The results of PBMA based on C-TVP and T-ISP content are represented as the mean plus/minus standard error of the mean (SEM). The effect of main ingredients and concentration of MC on the variation of proximate composition, physicochemical properties, and visible appearance was described as mean and standard error of mean (SEM). Analysis of variance (factorial ANOVA) was carried out using SPSS version 23 (IBM Corp., Armonk, NY, USA). For multiple mean comparisons, the Tukey's test was run at the level of 5%.

#### **3. Results and Discussion**

#### *3.1. Proximate Analysis*

The proximate composition of control and PBMA patties are presented in Table 2. Moisture content prepared from a lower MC concentration (1.5%) was not statistically different among treatments. However, control beef patties with a higher concentration of MC (3–4%) expressing a higher moisture content than patties prepared from soy-based C-TVP and T-ISP. The ability of MC in reducing the loss of moisture content was due to the thermal gelation of MC. During heating, MC formed an adhesive layer, which acted as a barrier to prevent moisture loss [22].

**Table 2.** Proximate chemical composition of plant-based meat and control (beef) with different concentration of methylcellulose.


a–f Different superscript letters within the same row mean significantly different between treatments (*p* < 0.05). SEM: standard error of mean; \*: interaction between ingredient and concentration. C-TVP: Commercial texture vegetable protein; T-ISP: Texture isolate soy protein

> The mechanism by which MC gelation is achieved between meat protein and plantbased protein is still unclear. One standard theory is that when in solution, hydrophobic methyl groups along the methylcellulose polymers are surrounded by cage-like structures of water molecules [23]. With increasing temperature, the cage structure is disrupted, and the polymers gradually lose their hydrated water. At the gelation point, polymers' association occurs due to extensive hydrophobic associations between exposed hydrophobic segments [24]. Elevated temperatures highly favor the hydrophobic associations, and strong gels can form [25].

> However, in the current study protein belongs to a heterogeneous mixture of different sources. Therefore, purifying the protein following different sources will result in different protein profiles, quality, and functionality [26]. The protein content of three types of patties' varied significantly between various protein sources, with control (beef) indicated higher

protein than PBMA patties. Although at any concentration of MC, there was no significant difference detected between C-TVP and T-ISP.

Consequently, PBMA patties with different MC concentrations exhibit no major (*p* > 0.05) difference in fat content among control (beef), C-TVP, and T-ISP respectively. It has been reported that the fat content of plant-based meat is rationally varied as compared to traditional patties [27], however, the fat content of the present study was within the range of Bohrer [27]. Generally, meat analogs are considered low in fat and protein content; however, the new generation of meat analogs products contain substantially greater fat and protein content than traditional meat analog products [9]. Therefore, our argument regarding the average level of fat and protein in meat analog was supported by Ahirwar et al. [28] who described that ready-to-eat meat analog has a good percentage of protein and average fat content manufactured from vegetable and cereal sources.

Irrespective with an application of different concentrations of MC or C-TVP and T-ISP ash content showed no difference. As expected, fiber content for PBMA patties was recorded higher than the control sample, with C-TVP represents the highest value. Similar results were also reported by Bohrer [27] in modern meat analogs. The higher fiber in PBMA patties was probably due to the plants and polysaccharides incorporated into the plant-based patties recipe. The fibrous nature of meat alternatives gives good textural and sensory sensation. Additionally, dietary fiber has been considered to play an essential role in preventing large bowel disease, ischaemic heart disease, and diabetes mellitus [29].

#### *3.2. Physicochemical Analysis*

The physiochemical indicators, including pH and colorimetric evaluation, are given in Table 3. There was a significant difference in pH between meat analogs and control (beef patties). The lower pH value of control was likely due to the regular glycolytic changes in meat [30]. However, C-TVP and T-ISP showed a pH of more than 6. The higher pH of PBMA could be due to the slight alkalinity of TVP (pH 7.42–7.43) [31]. Consistent with the current study, Bell and Shelef [32] recorded the pH of minced meat containing vegetable protein had higher pH than as compared to control, while Ahmad et al. [33] also determined that integration of soy protein isolate at 25% expressively increase the pH in meat sausage, which is similar to the outcomes of the present study.

**Table 3.** Physiochemical characteristics of plant-based meat and control (beef) with different concentration of methylcellulose.


a–f Different superscript letters within the same row mean significantly different between treatments (*p* < 0.05). SEM: standard error of mean; \*: interaction between ingredient and concentration. C-TVP: Commercial texture vegetable protein; T-ISP: Texture isolate soy protein.

> Likewise, pH and calorimetric measurements are interconnected with each other. The color coordinates are considered to be one of the essential physical properties in determining consumer acceptance of the product. All patties tended to decrease in lightness (L\*) and redness (a\*) after cooking. The results show that the control sample before cooking was lighter and redder than PBMA patties (C-TVP and T-ISP). However, our results were in contrast with the reported results of the literature on L\* and a\* values. Deliza et al. [34] reported an increase in the textured soy protein concentration in beef patties increased the L\* values, but a\* values were not statistically different. Hidayat et al. [35] also found a

similar observation on the beef sausage. The variation of L\* and a\* values in the present study compared to other studies could be due to the substitution of plant-based proteins (100% substitution) in the formulation. The small globules from meat, such as water and fat, can cause more light reflection, which will probably contribute to higher lightness [36].

The a\* values of control before cooking were higher than C-TVP and T-ISP treatments due to the myoglobin pigment in red meat. However, an increase in myoglobin denaturation can be shown by the lower a\* values after applying the heat treatment [37], which in tandem with our result in Table 3. The cooking did not affect the redness values of textured soybean protein incorporated samples [34]. Similar effects were noticed in the raw and cooked samples incorporated with either C-TVP or T-ISP as described in Table 3. The b\* values of C-TVP and T-ISP before cooking were higher than control. The yellowish coloration of PBMA patties can be associated with the yellow color of soy protein ingredients. Subsequently, the yellowish-brown color initially, affecting the final products' [9]. However, MC's concentration at different percentages only plays a minor role in reducing the b\* values of raw and cooked patties.

In the current study, WHC is expressed in two parts, RW and CL, shown in Table 4. The concentration of MC had a significant effect on the RW and CL. An increase in MC concentration from 1.5% to 4.0% lowered the RW and CL in all treatments. These findings were similar to the result reported by Hill and Prusa [38] for beef patties. They described that cellulose hydrocolloids bind moisture in product formulation, and it can gel upon heating. According to Hill and Prusa [38], surface moisture probably would not be affected by gum addition; therefore, evaporative losses were not affected by treatment. Consequently, the present data shows that MC's incorporation did not increase cooked moisture content, but it generally reduced total cook loss. Previously Arora et al. [3] proved that carrageenan and xanthan gum types binding agents had a higher yield than protein-based binding agents. At the same time, they concluded that WHC depends upon protein binding properties, which consequently agreed with our results.


**Table 4.** Water-holding capacity and tenderness related measurement of plant-based meat and control (beef) with different concentration of methylcellulose.

a–f Different superscript letters within the same row mean significantly different between treatments (*p* < 0.05). SEM: standard error of mean; \*: interaction between ingredient and concentration. C-TVP: Commercial texture vegetable protein; T-ISP: Texture isolate soy protein; WBSF: Warner-Bratzler shear force.

> Subsequently, WBSF for control represents the highest value, and there is no significant difference between C-TVP and T-ISP treatments. The softer textural properties of C-TVP and T-ISP affect their shear force values. Ruiz de Huidobro et al. [39] reported that shear force value was significantly correlated to hardness, springiness, and chewiness. The shear force in meat is a good measure of initial bite tenderness, which can cause changes during the cooking process are related to heat-induced alteration of myofibrillar proteins and connective tissue, as solubilizes the connective tissue leading to meat tenderization. In contrast, the denaturation of myofibrillar proteins causes meat toughening [40]. The finding of the current study aligns with the Danowska-Oziewicz [41], who detected lower values shear force for the samples containing soy isolate protein as likened to control (pork patties).

> Diameter before and after cooking of control and PBMA patties are presented in Table 4. The degree of shrinkage (diameter after) was ranged from about 17.46–22.68% for control,

4.23–12.28% for C-TVP, and 3.64–8.98% for T-ISP. Control represents higher shrinkage due to the connective tissue denaturation and fluid (moisture and fat) loss Table 4. The substitution with plant-based protein reduces the shrinkage markedly in T-ISP, although no difference to C-TVP. According to Gujral et al. [42] the addition of fibers and non-meat protein ingredients may reduce diameter shrinkage and weight loss. Similarly, in the current study, the increase in MC concentration (4%) decreases shrinkage of all patties (control: 17.46%, TVP: 4.23%, and T-ISP: 3.64%). control, 4.23–12.28% for C-TVP, and 3.64–8.98% for T-ISP. Control represents higher shrinkage due to the connective tissue denaturation and fluid (moisture and fat) loss Table 4. The substitution with plant-based protein reduces the shrinkage markedly in T-ISP, although no difference to C-TVP. According to Gujral et al. [42] the addition of fibers and non-meat protein ingredients may reduce diameter shrinkage and weight loss. Similarly, in the current study, the increase in MC concentration (4%) decreases shrinkage of all patties (control: 17.46%, TVP: 4.23%, and T-ISP: 3.64%).

Diameter before and after cooking of control and PBMA patties are presented in Table 4. The degree of shrinkage (diameter after) was ranged from about 17.46–22.68% for

TVP and T-ISP affect their shear force values. Ruiz de Huidobro et al. [39] reported that shear force value was significantly correlated to hardness, springiness, and chewiness. The shear force in meat is a good measure of initial bite tenderness, which can cause changes during the cooking process are related to heat-induced alteration of myofibrillar proteins and connective tissue, as solubilizes the connective tissue leading to meat tenderization. In contrast, the denaturation of myofibrillar proteins causes meat toughening [40]. The finding of the current study aligns with the Danowska-Oziewicz [41], who detected lower values shear force for the samples containing soy isolate protein as likened to con-

*Foods* **2021**, *10*, x FOR PEER REVIEW 9 of 15

#### *3.3. Visible Appearance 3.3. Visible Appearance*

trol (pork patties).

The external and internal appearance of meat analogs before and after cooking has been presented in Figure 2. The external appearance before cooking showed no difference in observation at different concentrations of MC. However, MC's effect can be seen after thermal treatment, in which the higher concentration (4%) can maintain the structure of patties. MC is essentially incorporated in some modern meat analog due to product consistency and binds all ingredients together to be more intact and stable [9]. MC is a useful binder, especially on the meat analog that does not require pre-heat for gel formation due to its unique thermal gelling and right emulsifier properties [43]. The external and internal appearance of meat analogs before and after cooking has been presented in Figure 2. The external appearance before cooking showed no difference in observation at different concentrations of MC. However, MC's effect can be seen after thermal treatment, in which the higher concentration (4%) can maintain the structure of patties. MC is essentially incorporated in some modern meat analog due to product consistency and binds all ingredients together to be more intact and stable [9]. MC is a useful binder, especially on the meat analog that does not require pre-heat for gel formation due to its unique thermal gelling and right emulsifier properties [43].

**Figure 2. Figure 2.** The external-internal appearance of cooked The external-internal appearance of cooked and uncooked plant-based meat patties. and uncooked plant-based meat patties.

The drawback of using TVP and T-ISP is that we can see the patties' surface's granular appearance. The internal appearance of all patties appeared more homogenous and cohesive with a higher concentration of MC. This proved that the addition of MC could bind well all the ingredients. The interior of C-TVP and T-ISP patties show a rough with intact and no crack appearance. Nevertheless, the interior of C-TVP shows more finely structure than T-ISP patties. The probable reason could be due to adequate hydration of C-TVP during the preparation of the dough. Earlier, MC's phenomena as a binder have been reported, which confirmed that MC helps maintain product shape and firm texture in various commercially available products, i.e., impossible burgers and beyond burgers [27].

.

[27].

#### *3.4. Texture Profile Analysis* been reported, which confirmed that MC helps maintain product shape and firm texture

*Foods* **2021**, *10*, x FOR PEER REVIEW 10 of 15

The drawback of using TVP and T-ISP is that we can see the patties' surface's granular appearance. The internal appearance of all patties appeared more homogenous and cohesive with a higher concentration of MC. This proved that the addition of MC could bind well all the ingredients. The interior of C-TVP and T-ISP patties show a rough with intact and no crack appearance. Nevertheless, the interior of C-TVP shows more finely structure than T-ISP patties. The probable reason could be due to adequate hydration of C-TVP during the preparation of the dough. Earlier, MC's phenomena as a binder have

The textural properties are crucial for developing meatless patties because in meat analog's texture is an essential factor in mimicking the organoleptic taste of muscle. Figure 2 illustrates the textural parameters, including hardness, chewiness, gumminess cohesiveness, and meat analogs' springiness with different concentrations of MC%. The hardness, chewiness, and gumminess of control were significantly higher in comparison to C-TVP and T-ISP. The higher hardness in control was expected due to the muscle proteins denaturation phenomenon, which led to hardness in the meat system [19]. It is evident from the shrinkage percentage shown in Table 4, whereby meat protein has a higher degree of shrinkage than plant-based proteins. An increase in MC concentration from 1.5% to 4% increases 'hardness of all patties. The current result was consistent with the results reported by Arora et al. [3], who described that when the binding agent increased, the hardness, chewiness, gumminess, and compression values increased proportionally. Similarly, Ayadi et al. [44] reported that incorporating carrageenan at higher concentration (0.5% to 1.5%) increased hardness of sausage products. The reason for lower hardness values in TVP and T-ISP treatments were due to extensive hydration of textured protein with water at the early stage of the processing phase, ultimately causes the PBMA patties to be softer. According to, Ruiz de Huidobro et al. [39] hardness, chewiness, and springiness are instrumental parameters for assessing meat texture. in various commercially available products, i.e., impossible burgers and beyond burgers *3.4. Texture Profile Analysis*  The textural properties are crucial for developing meatless patties because in meat analog's texture is an essential factor in mimicking the organoleptic taste of muscle. Figure 3 illustrates the textural parameters, including hardness, chewiness, gumminess cohesiveness, and meat analogs' springiness with different concentrations of MC%. The hardness, chewiness, and gumminess of control were significantly higher in comparison to C-TVP and T-ISP. The higher hardness in control was expected due to the muscle proteins denaturation phenomenon, which led to hardness in the meat system [19]. It is evident from the shrinkage percentage shown in Table 4, whereby meat protein has a higher degree of shrinkage than plant-based proteins. An increase in MC concentration from 1.5% to 4% increases 'hardness of all patties. The current result was consistent with the results reported by Arora et al. [3], who described that when the binding agent increased, the hardness, chewiness, gumminess, and compression values increased proportionally. Similarly, Ayadi et al.[44] reported that incorporating carrageenan at higher concentration (0.5% to 1.5%) increased hardness of sausage products. The reason for lower hardness values in

However, in the present study, only springiness values of PBMA patties (TVP and T-ISP) showing marginally or no difference to the control. The hardness and chewiness values were showed a substantial difference between treatments and control. As we mentioned earlier (introduction), to mimic conventional beef patties' textural properties is the most challenging part in the development of meat analogs. TVP and T-ISP treatments were due to extensive hydration of textured protein with water at the early stage of the processing phase, ultimately causes the PBMA patties to be softer. According to, Ruiz de Huidobro et al. [39] hardness, chewiness, and springiness are instrumental parameters for assessing meat texture.

**Figure 2.** *Cont.*

35

**Figure 3.** The texture profile analysis (TPA) parameters of plant-based meat on the type of texture soy isolate protein and different methylcellulose concentrations. (**A**) Hardness (N); (**B**) Chewiness (mJ); (**C**) Gumminess (N); (**D**) Cohesiveness; (**E**) Springiness (mm). Those are just different concen-**Figure 2.** The texture profile analysis (TPA) parameters of plant-based meat on the type of texture soy isolate protein and different methylcellulose concentrations. (**A**) Hardness (N); (**B**) Chewiness (mJ); (**C**) Gumminess (N); (**D**) Cohesiveness; (**E**) Springiness (mm). Those are just different concentrations of MC (Methylcellulose) and the concentration is there on the top right.

trations of MC (Methylcellulose) and the concentration is there on the top right

#### *3.5. Sensory Evaluation*

However, in the present study, only springiness values of PBMA patties (TVP and T-ISP) showing marginally or no difference to the control. The hardness and chewiness val-Sensory parameters are a chief concern for the development of PBMA patties using MC as a binder. The sensory traits for control (beef), C-TVP and T-ISP are presented in Figure 3. Based on the percentage of MC, the control patties expressing higher values in 4% MC for shape, firmness and color, although panelists scored higher, appearance and overall acceptability with 1.5% MC respectively. C-TVP patties obtained the highest score for appearance, shape, firmness, color and overall acceptability with 3% and 4% of MC concentration. Though, T-ISP samples incorporating 3% MC performed well than 1.5% and 4% MC concentration. The subjective evaluation demonstrated a clear preference towards 3% MC in PBMA patties (C-TV and T-ISP). Samples with the integration of 1.5 and 4% of MC were the least preferred on sensory evaluation basis. In contrast to our study, Imkyung et al. [45] described that with hydroxypropyl methylcellulose application as an animal fat replacer for meat patties, there is no significant difference in color, flavor and taste; however, tenderness, juiciness and overall acceptability show a statistically significant difference. 4% MC for shape, firmness and color, although panelists scored higher, appearance and overall acceptability with 1.5% MC respectively. C-TVP patties obtained the highest score for appearance, shape, firmness, color and overall acceptability with 3% and 4% of MC concentration. Though, T-ISP samples incorporating 3% MC performed well than 1.5% and 4% MC concentration. The subjective evaluation demonstrated a clear preference towards 3% MC in PBMA patties (C-TV and T-ISP). Samples with the integration of 1.5 and 4% of MC were the least preferred on sensory evaluation basis. In contrast to our study, Imkyung et al. [45] described that with hydroxypropyl methylcellulose application as an animal fat replacer for meat patties, there is no significant difference in color, flavor and taste; however, tenderness, juiciness and overall acceptability show a statistically significant difference.

ues were showed a substantial difference between treatments and control. As we mentioned earlier (introduction), to mimic conventional beef patties' textural properties is the

Sensory parameters are a chief concern for the development of PBMA patties using MC as a binder. The sensory traits for control (beef), C-TVP and T-ISP are presented in Figure 4. Based on the percentage of MC, the control patties expressing higher values in

*Foods* **2021**, *10*, x FOR PEER REVIEW 13 of 15

most challenging part in the development of meat analogs.

*3.5. Sensory Evaluation*

**Figure 4.** Sensory profile of plant-based meat-based with different soy isolate protein and methylcellulose percentage. **Figure 3.** Sensory profile of plant-based meat-based with different soy isolate protein and methylcellulose percentage.

Based on previous literature, it has been reported that the application of water alone in ground beef patties in control without methylcellulose and hydroxypropyl methylcellulose did not satisfy the sensory panel; they further reported that color and aroma of ground patties were least affected by the application of methylcellulose [46]. The vast variability of PBMA patties as compared to control could be due to plant-derived proteins (soy and wheat protein) in meat analogs expressing more elastic, rubbery and chewy sensation and poor mouth feel due to their agglomeration properties. Based on previous literature, it has been reported that the application of water alone in ground beef patties in control without methylcellulose and hydroxypropyl methylcellulose did not satisfy the sensory panel; they further reported that color and aroma of ground patties were least affected by the application of methylcellulose [46]. The vast variability of PBMA patties as compared to control could be due to plant-derived proteins (soy and wheat protein) in meat analogs expressing more elastic, rubbery and chewy sensation and poor mouth feel due to their agglomeration properties.

Moreover, previous literature confirmed that incorporating a different type of soy family (soy paste, soy protein isolate or texture soy protein) generates a unique beany Moreover, previous literature confirmed that incorporating a different type of soy family (soy paste, soy protein isolate or texture soy protein) generates a unique beany essence in meat products and downgrade sensory scores [41]. Remarkably, in the current study, no beany essence was noticed. The possible reason might be due to various types of plant-based ingredients (Table 1) used to mask the beany flavor in PBMA patties successfully. Furthermore, due to natural differences between muscle and plant materials, i.e., structure and size of protein molecules, amino acid composition, peptide sequence, and the chemical composition of both intracellular and extracellular materials, it is difficult to reproduce the complex and delicate sensory profile of animal meat products.

#### **4. Conclusions**

The present study assessed the physicochemical, textural, and sensory properties of PBMA patties with two types of texturized soy isolate protein (C-TVP and T-ISP) and incorporation of different concentrations of binding agent (MC). The addition of MC significantly affected the quality characteristics of C-TVP and T-ISP-based PBMA patties. C-TVP with 3% MC showed promising results, with adequate physicochemical, textural parameters, and with satisfactory patty visible appearance, thereby improving the comprehensive process yield compared to T-ISP. Although samples with 4% MC also exhibit similar results compared to 3 % MC, they failed to satisfy the sensory panelist in C-TVP and T-ISP. Using beef as a control, it can be concluded that C-TVP with a 3% MC (binding agent) is recommended to prepare acceptable PBMA patties with good physicochemical, textural, and sensory acceptability.

**Author Contributions:** All of the authors contributed significantly to the research. A.B. wrote the manuscript. Contribution to experimental work by A.B., S.-J.L., E.-Y.L. and N.S., S.-T.J. designed experimental work, and the manuscript was reviewed and revised by Y.-H.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1I1A206937911).

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **References**


**Maria Martuscelli \* , Luigi Esposito and Dino Mastrocola**

Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Via R. Balzarini 1, 64100 Teramo, Italy; lesposito2@unite.it (L.E.); dmastrocola@unite.it (D.M.) **\*** Correspondence: mmartuscelli@unite.it; Tel.: +39-0861-266-912

**Abstract:** Coffee Silver Skin (CSS) is the unique by-product discarded after the roasting of coffee beans. This research aimed to evaluate the effect of two levels of CSS (1.5% and 3%) added as a natural ingredient in new formulations of chicken meat burgers. This is one of the first studies proposing a "formulation approach" to control the emergence of off flavours after meat cooking. Physical, chemical, and sensory analyses were carried out, within the CSS content and the evolution of volatile organic compounds in different samples. Newly formulated chicken burgers could limit food waste, while also becoming a source of fibres, minerals, and bioactive molecules. CSS limited weight losses (after cooking process) to 10.50% (1.5% addition) and 11.05% (3% addition), significantly lower (*p* < 0.01) than the control (23.85%). In cooked burgers, the occurrence of hexanal was reduced from 55.1% (CTRL T<sup>0</sup> ) to 11.7% (CSS T<sup>0</sup> 1.5%) to 0 (CSS T<sup>0</sup> 3%). As for the limitation of off-flavours, CSS also showed good activity, contrasting with the emergence of octanal, alcohols and other markers of lipid oxidation. From the sensory test carried out, the volatile profile of CSS does not seem to impair the flavour of burgers, though at higher percentages hydrocarbons and pyrazines are traceable. The thiobarbituric acid reactive substances (TBARS assay confirmed the protective effect of CSS against oxidation.

**Keywords:** coffee by-products; chicken burger; meat formulation; cooking yield; volatile compounds; warmed off-flavours

#### **1. Introduction**

Minimally processed raw by-products are available in large quantities and their reutilization might be enhanced to recover bioactive compounds, on top of their promising technological properties [1]. Many valuable molecules such as phenolic acids, carotenoids, and flavonoids can mitigate oxidation occurrence, so there is an increased demand for new methods and technologies to recover and use these [2]. Many publications attest the positive role of by-products' addition in meat formulations to limit oxidation occurrence [3,4].

Oswell et al. [5] explain how some unprocessed food components can help reducing the list of ingredients of a formulation, supporting trends towards the clean and green label. In fact, many by-products have technological properties, acting as additives and ingredients [6].

Coffee Silver Skin (CSS) is a thin layer tightly adherent to coffee seeds, present in all coffee species and impossible to separate when seeds are unroasted [7]. Among all the by-products of the coffee industry, CSS is unique in being discarded immediately after the roasting step [8].

Common features of CSS are high content in fibres (both soluble and insoluble), in minerals such as Calcium and Potassium, and in capability as an adsorbing material [2].

To our knowledge, there are no studies of the inclusion of untreated coffee silver skin (CSS) in meat-based foods such as chicken products. It is well known that poultry meat is easily oxidised; its content of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) (including phospholipids that are distributed in muscles and cellular

**Citation:** Martuscelli, M.; Esposito, L.; Mastrocola, D. The Role of Coffee Silver Skin against Oxidative Phenomena in Newly Formulated Chicken Meat Burgers after Cooking. *Foods* **2021**, *10*, 1833. https:// doi.org/10.3390/foods10081833

Academic Editors: Gonzalo Delgado-Pando and Tatiana Pintado

Received: 1 July 2021 Accepted: 5 August 2021 Published: 8 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

membranes) make it the elective substrate for lipid oxidation [3]. Feeding of animals is responsible for many qualitative characteristics of meat, progressing to the eventual development of off-flavours (after slaughtering) [9,10]. In chicken products, clove, oregano, thyme, and sage were successfully used as natural substitutes of synthetic antioxidants [11]. Kim et al. [12] used residues of coffee brewing (spent coffee) as antioxidants against meat oxidation in raw and cooked samples. Recently Delgado-Ospina et al. [13] added cocoa pod husk flour, discovering an interesting application for quality improvement of frankfurters. Cooked and refrigerated meat products develop undesired rancidity and a great variety of off flavours known as warmed over flavours (WOF). These defects can also come about by heating ready to eat foods or high-processed meat-based items and can be present in many products where food remains exposed to light, oxygen, and heat for a long time (canteens, fast food outlets, collective restaurants). In the study by Lungu et al. [14], most respondents affirmed exposure to WOF defective foods, especially ready to eat meats; moreover, besides the reduced sensory quality, respondents confirmed a preference for defective foods due to their lower cost. The development of WOFs does not impair food safety, but a high oxidation rate affects the nutritional profile; therefore a huge quantity of safe food is discarded daily due to detrimental sensory defects.

In this scenario, the present research investigated the chemical and physical properties of CSS against oxidative phenomena after the cooking of chicken burgers. This study points to the nutritional advantages of including CSS as a new ingredient for chicken burger recipes, while testing some technological functionalities. Lastly, we were able to evaluate the role of CSS on the shelf life of refrigerated cooked burger, focusing on the spreading of WOF and oxidation markers along with an analysis of the volatile compounds and a sensory test with trained panellists.

#### **2. Materials and Methods**

#### *2.1. Coffee Silver Skin*

Coffee silver skin (CSS) was received from the toasting plant Marcafè Torrefazione Adriatica s.p.a. (Giulianova, Italy). After 10 cycles of roasting (10 × 240 kg of roasted coffee), 3.3 kg of CSS were recovered. CSS used for this experiment was a blend of 5 arabica varieties (*Coffea arabica*) (India Arabica, India Cherry, Vietnam, India Mysore, and India Caracolito) and 5 robusta varieties (*Coffea canephora*) (India Parchment, Santos, Uganda CRV 18, Uganda CRV 17, Togo). CSS was ground at 10,200 rpm for 1 min (Bimby®, mod. TM 31, Wuppertal, Germany) until arriving at a particle size of 125–250 µm. Then, physical, and chemical analyses were carried out. CSS was kept frozen at −20 ◦C until analysis. Qualitative analyses were carried out and results were reported in our previous study [2].

#### *2.2. Preparation of Burgers*

Chicken breast fillets were purchased on the market (antibiotic-free, genetically modified organism-free diet, and high welfare/partial free range system meat).

Chicken burgers were obtained from 1 kg of fresh breast fillets with the addition of 1.4% salt and 5.0% water. These ingredients were cut and mixed for 2 min at 1800 rpm with Bimby® mixer (Wuppertal, Germany), mod. TM 31, to obtain a perfectly homogenised blend.

From the whole mixture (meat, water, and salt) 3 batches were obtained: control, without any addition of coffee silver skin (CTRL), coffee silver skin addition of +1.5% (CSS 1.5%) and coffee silver skin addition of +3.0% (CSS 3.0%). CSS was added by mixing for 30 s at 500 rpm.

8 burgers (45 g each approximately) were prepared for each experimental batch (Figure 1).

30 s at 500 rpm.

30 s at 500 rpm.

(Figure 1).

was replicated on another two independent occasions.

*Foods* **2021**, *10*, x FOR PEER REVIEW 3 of 15

1.5%) and coffee silver skin addition of +3.0% (CSS 3.0%). CSS was added by mixing for

8 burgers (45 g each approximately) were prepared for each experimental batch

Then, burgers were cooked on an electrical griddle Bosch (München, Germany), mod. TFB4431V, potency 2000 W for 4 min until reaching an internal temperature of 90–92 °C. Cooked burgers are shown in Figure 2. After cooking, some burgers were eaten during the panel test, and others were left singularly covered with a plastic oxygen permeable film at +4 °C for 72 and 120 h. The utilization of this covering film was chosen to allow the permeability of O2 and thus the spreading of WOFs and other products of oxidation. Moreover, this condition is the closest to what can happen to consumers at home. The trial

1.5%) and coffee silver skin addition of +3.0% (CSS 3.0%). CSS was added by mixing for

8 burgers (45 g each approximately) were prepared for each experimental batch

**Figure 1.** Uncooked burgers of the three experimental batches: (**a**) control (CTRL) without CSS additions; (**b**) with addition of +1.5% of coffee silver skin (CSS 1.5%); (**c**) with +3.0% of coffee silver skin (CSS 3.0%). **Figure 1.** Uncooked burgers of the three experimental batches: (**a**) control (CTRL) without CSS additions; (**b**) with addition of +1.5% of coffee silver skin (CSS 1.5%); (**c**) with +3.0% of coffee silver skin (CSS 3.0%).

Then, burgers were cooked on an electrical griddle Bosch (München, Germany), mod. TFB4431V, potency 2000 W for 4 min until reaching an internal temperature of 90–92 ◦C. Cooked burgers are shown in Figure 2. After cooking, some burgers were eaten during the panel test, and others were left singularly covered with a plastic oxygen permeable film at +4 ◦C for 72 and 120 h. The utilization of this covering film was chosen to allow the permeability of O<sup>2</sup> and thus the spreading of WOFs and other products of oxidation. Moreover, this condition is the closest to what can happen to consumers at home. The trial was replicated on another two independent occasions. (**a**) (**b**) (**c**) **Figure 1.** Uncooked burgers of the three experimental batches: (**a**) control (CTRL) without CSS additions; (**b**) with addition of +1.5% of coffee silver skin (CSS 1.5%); (**c**) with +3.0% of coffee silver skin (CSS 3.0%).

(**a**) (**b**) (**c**)

Colour was expressed as L\* (lightness, intensity of white colour), a\* (+a, red; −a, green) and b\* (+b, yellow; −b, blue) values. Samples were measured in triplicate and at least **Figure 2.** Cooked burgers of the three experimental batches: (**a**) control (CTRL); (**b**) with addition of +1.5% of coffee silver skin (CSS 1.5%); (**c**) with +3.0% of coffee silver skin (CSS 3.0%). **Figure 2.** Cooked burgers of the three experimental batches: (**a**) control (CTRL); (**b**) with addition of +1.5% of coffee silver skin (CSS 1.5%); (**c**) with +3.0% of coffee silver skin (CSS 3.0%).

fifteen measurements were obtained for each batch. To better define the final color

#### observed, the saturation index (chroma, C\*) was calculated according to Formula (1). *2.3. Physico‐Chemical, Colour, and Compositional Analyses 2.3. Physico-Chemical, Colour, and Compositional Analyses*

C\* = (a\*2 + b\*2)1/2 (1) Proximate analysis on moisture, proteins, and ashes was obtained following the The values of water activity (aw) were obtained with the Aqualab 4 TE kit (Court Pullman, WA, USA). Values of pH were taken with a pH meter (model 3510, Jenway, Stone, UK). All values were measured in triplicate. The values of water activity (aw) were obtained with the Aqualab 4 TE kit (Court Pullman, WA, USA). Values of pH were taken with a pH meter (model 3510, Jenway, Stone, UK). All values were measured in triplicate.

Association of Official Analytical Chemists procedure [15]. Total lipids were measured Colour was determined in different locations of burger samples by a colorimeter CR‐ 5 (Spectrally based, Konica Minolta, Tokyo, Japan) with D65 light source and observer 10°. Colour was expressed as L\* (lightness, intensity of white colour), a\* (+a, red; −a, green) and b\* (+b, yellow; −b, blue) values. Samples were measured in triplicate and at least fifteen measurements were obtained for each batch. To better define the final color observed, the saturation index (chroma, C\*) was calculated according to Formula (1). Colour was determined in different locations of burger samples by a colorimeter CR-5 (Spectrally based, Konica Minolta, Tokyo, Japan) with D<sup>65</sup> light source and observer 10◦ . Colour was expressed as L\* (lightness, intensity of white colour), a\* (+a, red; −a, green) and b\* (+b, yellow; −b, blue) values. Samples were measured in triplicate and at least fifteen measurements were obtained for each batch. To better define the final color observed, the saturation index (chroma, C\*) was calculated according to Formula (1).

$$\mathbf{C}^\* = (\mathbf{a}^{\*2} + \mathbf{b}^{\*2})^{1/2} \tag{1}$$

Proximate analysis on moisture, proteins, and ashes was obtained following the Association of Official Analytical Chemists procedure [15]. Total lipids were measured Proximate analysis on moisture, proteins, and ashes was obtained following the Association of Official Analytical Chemists procedure [15]. Total lipids were measured using a modification of the chloroform to methanol procedure described by Folch et al. [16].

The determination of such micronutrients as calcium, potassium, and total dietary fibers (TDF) was performed by calculating their presence in a 45 g burger at different

formulations; the estimation of Ca, K, and TDF accounts of the values found in our previous work on the characterization of CSS [2]. Quantities found refer to the presence of a defined element and not on bioavailability. We used easy proportions to find percentages.

Here, as example, we propose the calculation used for the calcium determination (2), (3) in 1.5% CSS formulation:

$$546.5\text{ mg}; 100\text{ g} = 3.3\text{ g}; \tag{2}$$

$$\mathbf{x} = \frac{(546.5 \text{ mg} \ast 100 \text{ g})}{3.3 \text{ g}} = 16.54 \text{ mg} \tag{3}$$

where 546.5 mg is the Ca content in 100 g of CSS, 3.3 g is the amount of CSS in a 45 g burger, and 16.54 mg is the intake of Ca in a 45 g burger formulated with 1.5% of CSS.

#### *2.4. Cooking Yield*

The cooking yield parameter is a useful and practical tool to easily calculate the quantity of meat available for consumption after the cooking process. Uncooked samples were prepared and weighted singularly, then underwent the established cooking process and weighed again. This formula was used to arrive at the result (4)

$$100 - \left(\frac{(\text{raw larger weight} - \text{cooled larger weight})}{(\text{raw larger weight})} \times 100\right) \tag{4}$$

#### *2.5. Thiobarbituric Acid Reacting Substances (TBARS) Assay*

A thiobarbituric reactant species test was carried out following the methods of Soyer et al. [17] with some modifications. Raw meat (25 g) was ground in 125 mL of pure water for 2 min to homogenise the mixture. From this, 5 mL were filtered and transferred in falcon tubes (15 mL) with 3 mL of a solution containing trichloroacetic acid (15%, *w*/*v*) and thiobarbituric acid (80 mM) in HCl 0.25 N. Samples underwent a centrifugation step (2000 rpm for 5 min) to precipitate proteins. After centrifugation, 3 mL were transferred in tapped glass tubes and kept at 40 ◦C for 90 min.

Samples obtained were read at 532 nm with a spectrophotometer UV-VIS (Jenway, Stone, UK) after a further filtration with filters 0.45 µm. All samples were read in double, and data were expressed as mean ± standard deviation.

The calibration curve was obtained by using a 1,1,3,3-tetraetoxypropane (Sigma-Aldrich, St. Louis, MO, USA, ≥96%) in methanol, at a concentration range of 0.625–20 µM.

#### *2.6. Volatile Compounds (VOCs)*

The experimental plan was designed to have triplicate samples of each formulation of T<sup>0</sup> cooked samples, T<sup>72</sup> and T<sup>120</sup> samples. Cooked samples T<sup>0</sup> were immediately chopped and put in glassy vials of 20 mL capacity (Perkin Elmer, Waltham, MA, USA) with approximately 3 g of meat each, tightly closed and stocked at −40 ◦C, assuring the highest headspace, until gas chromatograph mass spectrometer (GC-MS) analysis. GC-MS analysis was performed with a gas chromatograph (Clarus 580, Perkin Elmer, Waltham, MA, USA) coupled with a mass spectrometer (SQ8S, Perkin Elmer Waltham, MA, USA). Other samples were left in refrigerated conditions for the time required to obtain T<sup>72</sup> and T120, then carefully chopped and stocked in 20 mL vials at −40 ◦C, until GC-MS analysis.

The GC-MS analysis followed the method proposed by Qi et al. [18] with some modifications. Vials were left for 1 h at room temperature, then put in a water bath at 50 ◦C for 20 min. Volatiles from meat were extracted with a headspace solid phase microextraction fibre (SPME 65 µm Polydimethylsiloxane/Divinylbenzene (PDMS/DVB); Supelco, Bellofonte, PA, USA) and collected for 30 min at 40 ◦C, then inserted into the GC injector and desorbed for 3 min at 250 ◦C. Volatile compounds were separated on a Capillary GC column ZB- Semi Volatiles (30 m length, 0.25 mm internal diameter, 0.25 µm film thickness: Phenomenex, Torrance, CA, USA). The oven temperature was maintained

for 3 min at 40 ◦C, increased at 3 ◦C/min to 70 ◦C, then at 5 ◦C/min to 180 ◦C, then at 10 ◦C/min to 260 ◦C, and maintained for 5 min at 260 ◦C. Helium was the carrier gas with a constant flow of 1 mL/min. The mass-selective detector was operated in the electron impact mode (70 eV) and full scan mode (35–500 *m*/*z* range). The identification was performed using the National Institute of Standards and Technology mass spectral library (NIST Mass Spectral library, Search Program version 2.0, National Institute of Standards and Technology, U.S. Department of Commerce, Gaithersburg, MD, USA).

#### *2.7. Descriptive Sensory Analysis of WOF Assessment, Rancidity and Extraneous Flavours*

Chicken burger samples were evaluated for four classes of descriptors grouped as: odour, flavour, taste, and aftertaste [19]. The vocabulary used for descriptors comes from the review of recent literature about rancidity and warmed off-flavours (WOF) assessment in meat products [20–24].

A panel group of six women and two men from twenty to fifty years old was trained for evaluation of quality assessment of meat burgers. After their recruitment, panellists were screened for their ability to distinguish odours and tastes, then were trained for vocabulary development through a series of triangular tests (ISO), 8586:2012 [25]. Training duration was 80 h, including familiarization with relevant descriptive terms and ways of perceiving the selection and quantification of the sensory characteristics of cooked meat, as well as the use of intensity scales (ISO) 4121:2003 [26].

Panellists were asked to taste cooked burgers (T0) and cooked burgers refrigerated at +4 ◦C for 72 h. Samples of 120 h at +4 ◦C were not tasted to avoid any microbial contamination. A hot bath at 72 ◦C was used to heat up to the core temperature of 70 ◦C. Panellists were provided with individual templates, where descriptors were grouped per section. They used a scale from 1 to 5 where 1 meant the absence of the attribute while 5 the maximum rate. Pie shaped pieces of warm burgers were quickly served to panellists trying to maintain the temperature between 70 and 60 ◦C as recommended by [27]. Samples were codified with random numbers to avoid external influences on liking rating of panellists. Meat pieces were served on white plates. All sensory tests and training sessions were carried out in the sensory laboratory of the University of Teramo that fulfils the required standards for these analyses according to (ISO) 8589:2007 [28].

#### *2.8. Statistical Analysis*

All determinations were done in triplicate. Means and relative standard deviations were calculated. Analysis of variance (ANOVA) was performed to test the significance of the effects of the factor variables (formulation, time of storage); differences among means were separated by the least significant differences (LSD) test.

Statistical analysis of data was performed using XLSTAT software version 2019.1 for Microsoft Excel (Addinsoft, New York, NY, USA). All results were considered statistically significant at *p* < 0.05.

#### **3. Results and Discussion**

Results of qualitative characteristics (proximate composition, colour, pH, aw) of uncooked and immediately cooked burgers are shown and discussed in the first section, highlighting significant differences among treatments. Data shown and discussed in the second section (TBARS, VOCs, sensory analysis) evidence the significant differences between sampling times with respect to the oxidative phenomena occurred in the refrigerated cooked chicken burger samples.

#### *3.1. Effect of CSS on the Qualitative Characteristics of Burger Samples*

#### 3.1.1. Compositive Characteristics

Results from proximate compositional analyses of meat are shown in Table 1 (uncooked burgers); data agree with the literature [29,30]. Generally, macro-elements checked do not change so much. Moisture, proteins, and lipids remained at around their normal

values; ashes had a small significant increase reaching the highest point of 2.69% for 3% CSS sample. TDF for samples (calculated as reported in Section 2.3) are 1.70% and 3.40%, for 1.5% and 3% CSS, respectively.

**Table 1.** Proximate composition (mean ± standard deviation, SD) of raw burger samples (before cooking): control (CTRL), with addition of +1.5% of coffee silver skin (CSS 1.5%) and +3.0% of coffee silver skin (CSS 3.0%).


Legend: data followed by different superscript letters, in the same column, are significantly different (least significant difference (LSD) test, *p* < 0.05); asterisks indicate significance at \*\* *p* < 0.01, n.s. not significant.

pH of breast fillets used for the burger production was 5.93 ± 0.04, not differing with reported values which indicate values around 5.89–6.00 [31]. Formulated burgers (uncooked and cooked) registered pH values, shown in Table 2, in line with other sources [32]. Cooked burgers had similar pH and a<sup>w</sup> values.

**Table 2.** Results (mean ± SD) of pH, water activity (aw) values and cooking yield (%) in burger samples (CTRL, control; CSS 1.5% and CSS 3.0%, with +1.5% and +3.0% of coffee silver skin as ingredient, respectively).


Legend: data followed by different superscript letters, in the same column, are significantly different (LSD test, *p* < 0.05); asterisks indicate significance at \*\* *p* < 0.01; n.s. not significant.

In eating a newly formulated burger (45 g) with a CSS inclusion of 1.5%, 16.54 mg of calcium and 65.7 mg of potassium can be assumed. Harvard Health Publishing in 2019 reviewed the daily intake of calcium for women between 50 and 71 years old fixing this at 1200 mg [33]. The same value was established by the National Institute of Health (NIH) which also defines limits [34] for men at 1000 mg. Potassium was fixed at 3400 mg and 2600 mg for males and females from 19 to 50 years old, respectively.

For marketing within the European Union (EU), it is very important to define whether by-products, such as CSS, need to obtain an approval as ingredient for novel foods [34], with special reference to legal status within the EU and potential options for producers to obtain approval according to Novel Food [35–37].

#### 3.1.2. Cooking Yield

The cooking yield allows calculation of how much water and fats a food item loses after a cooking process. The US Department of Agriculture (USDA) in 2014 [38] released a table of cooking yield and retention factors for many meat products. These factors can be used to calculate nutritional values where analytical data for cooked foods are unavailable. Obviously, meat represents an important class of cooked foods and in this way the cooking yield covers an important aspect. Beside this, the cooking yield parameter tells us how much in terms of weight a formulation has lost, and this is also an index of profitability.

Table 2 shows that the addition of coffee by-product allowed an increase in cooking yield (%) in respect to the control. While the control lost 23.85% of its initial weight, CSS addition limited this loss to just 10.50% (+1.5% CSS addition) and 11.06% (+3% CSS addition). This trait of CSS comes from good water holding capacity (WHC) and oil holding

capacity (OHC).For CSS, values of WHC 5.11 ± 0.20 and 5.5 ± 0.2 were found; for OHC, these were 4.72 ± 0.10 and 4.8 ± 0.2 [39–43]. The increased cooking yield also marked a significant difference (*p* < 0.01) between CTRL and CSS added burgers, while burgers with CSS, at both percentages, were similar as regards cooking yield.

Losses are limited even with little addition, registering a high capacity and a possible increase in economical revenue.

#### 3.1.3. Colour

Breast fillets used to formulate burgers had a value L\* of 45.91, on average according to Ziober et al. [43], L\* > 53 denoted pale soft exudative (PSE), L\* < 44 is analogous to dark firm dry (DFD), and 44 ≤ L\* ≤ 53 is normal meat. Colour values of burgers are in line with what Longato et al. [4] have found in their study on chicken burgers with the addition of hazelnut skin; their results on uncooked (control) samples are L\* a\* b\* values of 53.83 ± 4.48, 0.30 ± 0.69, 9.12 ± 1.8, and 64.98 ± 2.55, 1.64 ± 0.55, 15.48 ± 1.15 for cooked burgers. Data on the colour determination (uncooked and cooked samples) are reported in Table 3.

**Table 3.** Colour values (mean ± SD) of L\* (lightness), a\* (redness), b\* (yellowness), and C\* (chroma) for uncooked burgers and cooked burgers, in control (CTRL) and in samples with +1.5% (CSS 1.5%) and +3.0% (CSS 3.0%) of coffee silver skin as ingredient.


Legend: data followed by different superscript letters, in the same column, are significantly different (LSD test, *p* < 0.05); asterisks indicate significance at \* *p* <0.05; \*\* *p* < 0.01; n.s. not significant.

> CSS is easily reducible to a fine crumb and this determined a uniform distribution. Burgers with CSS are in fact darker, more yellow, and redder with respect to the control. Unfortunately, no studies are available to compare these data. Chroma (C\*) values for uncooked and cooked chicken burgers were searched by de Oliveira et al. [44], who added chia seeds to a blend of breast and thigh chicken skinless meat and pork backfat. They reported levels of 14.0 ± 4.8 (raw samples), and 16.8 ± 2.4 (grilled samples). As in our case, burgers were darker than control. Conversely, their data do not show higher saturation. C\* values of burgers here analysed are shown in Table 3. Generally, the addition of CSS increased the C\* value meaning a higher saturation and, thus, a more vivid colour after cooking.

## *3.2. Effect of CSS on the Shelf Life of Cooked Chicken Burger Samples*

#### 3.2.1. Thiobarbituric Acid Reacting Substances (TBARS) Test

The TBARS values of cooked samples are presented in Figure 3. Generally, over time TBARS values increase in all the cases, but the CTRL set showed significantly higher values immediately after cooking (*p* < 0.05), as well as a statistically significant increase during the refrigerated storage (*p* < 0.05). In all burgers with CSS, TBARS mean values were lower than the acceptance limit of TBARS for rancidity (1.0 mg MDA(Malondialdehyde)/kg) [45] until 72 h; after 120 h CSS3% showed a TBARS mean value near to the critical content, while this limit was exceeded in all samples without CSS.

species.

The TBARS values of cooked samples are presented in Figure 3. Generally, over time TBARS values increase in all the cases, but the CTRL set showed significantly higher val‐ ues immediately after cooking (*p* < 0.05), as well as a statistically significant increase dur‐ ing the refrigerated storage (*p* < 0.05). In all burgers with CSS, TBARS mean values were lower than the acceptance limit of TBARS for rancidity (1.0 mg MDA(Malondialde‐ hyde)/kg) [45] until 72 h; after 120 h CSS3% showed a TBARS mean value near to the

The TBARS test is helpful for a first screening of the oxidation rate of a food, but it does not discriminate which kind of oxidation is occurring. We cannot be sure if what is observed with this assay comes from lipid oxidation itself, or if proteins too have taken part in the process and are the principal cause starting the reactions. As is known, chicken meat is poor in lipids that are mainly unsaturated fatty acids which are the elective sub‐ strate for the oxidation. Moreover, [46] have searched for the lipidic profile of CSS and their results show a small content of lipids, which are mainly saturated fatty acids (SFAs). So, one could refer all the defective odours and tastes to the lipidic oxidation complex of reactions. Unfortunately, the oxidation process is very unstable and sometimes unpredict‐ able. Besides lipids, proteins and iron ions boost the process within other factors (rise in temperature, oxygen and light exposure, salt addition, etc.) making TBARS not such an

Anyway, from our results we can imagine that the contribution of CSS to the global oxidation burgers and its lipids increment are almost zero, while its protective effect seems to be promising. This may depend on the high content in phenolic and bioactive

critical content, while this limit was exceeded in all samples without CSS.

affordable method to establish the lipidic oxidative status of a food [47,48].

**Figure 3.** Results of Thiobarbituric acid reacting substances (TBARS) test, expressed as malondial‐ dehyde (mg MDA kg<sup>−</sup>1) in control (CTRL) and burgers formulated with CSS (1.5% and 3%, respec‐ tively), immediately after cooking (T0) and after refrigerated storage (at 4°C, for 72 and 120 h, T72 and T120 respectively). Results are expressed as means ± standard deviations. Different lowercase letters indicate significant differences (*p* < 0.05) among storage time of each batch; different upper‐ **Figure 3.** Results of Thiobarbituric acid reacting substances (TBARS) test, expressed as malondialdehyde (mg MDA kg−<sup>1</sup> ) in control (CTRL) and burgers formulated with CSS (1.5% and 3%, respectively), immediately after cooking (T<sup>0</sup> ) and after refrigerated storage (at 4 ◦C, for 72 and 120 h, T<sup>72</sup> and T<sup>120</sup> respectively). Results are expressed as means ± standard deviations. Different lowercase letters indicate significant differences (*p* < 0.05) among storage time of each batch; different uppercase letters indicate significant differences (*p* < 0.05) among different batches, at same storage time.

case letters indicate significant differences (*p* < 0.05) among different batches, at same storage time. Table 4**.** Qualitative profile of main volatile compounds (area %) in cooked samples (control, addition of coffee silver skin 1.5 and 3%), immediately after cooking process (T0) and after 72 h of storage at +4 °C (T72). **VOCCs from Burgers' Samples Burgers T0 Burgers T72**  CTRL T0 CSS 1.5% T0 CSS 3% T0 CTRL T72 CSS 1.5% T72 CSS 3% T72 Sign. The TBARS test is helpful for a first screening of the oxidation rate of a food, but it does not discriminate which kind of oxidation is occurring. We cannot be sure if what is observed with this assay comes from lipid oxidation itself, or if proteins too have taken part in the process and are the principal cause starting the reactions. As is known, chicken meat is poor in lipids that are mainly unsaturated fatty acids which are the elective substrate for the oxidation. Moreover, [46] have searched for the lipidic profile of CSS and their results show a small content of lipids, which are mainly saturated fatty acids (SFAs). So, one could refer all the defective odours and tastes to the lipidic oxidation complex of reactions. Unfortunately, the oxidation process is very unstable and sometimes unpredictable. Besides lipids, proteins and iron ions boost the process within other factors (rise in temperature, oxygen and light exposure, salt addition, etc.) making TBARS not such an affordable method to establish the lipidic oxidative status of a food [47,48].

> Anyway, from our results we can imagine that the contribution of CSS to the global oxidation burgers and its lipids increment are almost zero, while its protective effect seems to be promising. This may depend on the high content in phenolic and bioactive species.

#### 3.2.2. Volatile Compounds (VOCs) and Warmed Off-Flavours (WOF) in Chicken Burgers

Among analyses used in this work, GC-MS analysis was used to trace markers of oxidation as a more reliable method than TBARS or any other faster, but less accurate, method. Results show a complex profile of compounds emerging from oxidative phenomena, Maillard reaction occurrence, and by-products addition. Table 4 contains all the volatile compounds found in CSS-containing samples. Data depicted refer to T<sup>0</sup> and T<sup>72</sup> samples. Chromatograms are shown as Supplementary Material (Figures S1–S3).

*Foods* **2021**, *10*, 1833



*Foods* **2021**, *10*, 1833


50

According to Chen et al. [49], most of the typical odorants from cooked chicken meat are caused by phospholipids oxidation/degradation that led to the formation of longchain aldehydes such as hexanal, (Z)-2-decenal and (E)-2-decenal. In any case, even if responsible for WOF development, these aldehydes are key aroma compounds of freshly cooked chicken meat. CSS samples showed these classes even if ketones and esters were not found. T<sup>0</sup> 1.5% added samples had hydrocarbons as the first class traced, followed by aldehydes, nitrogen containing compounds and alcohols. T<sup>0</sup> 3% added samples showed aldehydes at first place followed by Nitrogen containing compounds, alcohols, hydrocarbons, and other compounds. T<sup>72</sup> containing 1.5% of CSS showed nitrogen containing compounds, hydrocarbons, other compounds, aldehydes, alcohols, and ketones, while 3% addition showed aldehydes, hydrocarbons, alcohols, other compounds, and Nitrogen containing compounds.

In general, patties tested in this study, especially CTRL, seem to have a small compounds presence if compared with other articles [50–52]. To our knowledge, this is one of the first studies where cooking conditions did not pass 92 ◦C and were not prolonged for more than four minutes. These conditions were selected to best simulate domestic conditions using an electric device set at medium cooking heat. Most studies on chicken meat burgers have tested grilled or oven-cooked patties. Other references on chicken meat evaluated entire boiled or roasted chicken. This is a fundamental step in explaining, for example, the absence of sulphur containing volatiles. In line with findings of other researchers [53], these compounds come from the interaction among Maillard reaction compounds and lipid oxidation products. Thus, quick cooking processes, medium/low heating, or their combination seem not to favour this interaction. These settings did not allow the development of traceable Maillard reaction products (desired and undesired). T<sup>72</sup> (CTRL) samples showed an increase in concentration of hexanal, the emergence of heptanal, some alcohols such as 2-Nonen-1-ol, (E)-, ketones as 2,3-Octanedione and 7,9- Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione, and just one Sulphur containing compound: N-Methyl-taurine. These are all markers of lipid oxidation and muscle damage. The addition of by-products provoked a mitigation of some WOF species, but also gave to patties specific odorants not conducible to meat oxidation and potentially undesirable.

CSS addition reduced the occurrence of aldehydes such as hexanal that reduced from 55.1% (CTRL T0) to 11.7% (CSS T<sup>0</sup> 1.5%) to 0 (CSS T<sup>0</sup> 3%). At T<sup>72</sup> CTRL contained 72% and reduced to 0 in both concentrations. Heptanal was found only in CTRL T72; octanal too, was just found in CTRL samples and not found in samples 1.5 and 3% at both times. Some alcohols such as 2-Nonen-1-ol, (E)- were limited in T<sup>72</sup> samples, but were present in T<sup>0</sup> CSS 3% with other alcohols, probably from the degradation of lignocellulosic precursors. This same pathway is followed by hydrocarbons which are totally absent in CTRL samples while being present in added patties [54]. According to data here shown, Nitrogen containing compounds present in CSS formulations probably came from the degradation of CSS proteins and from the Maillard complex of reactions which takes place during the roasting process. As for CSS, the significant role of phenols' interaction with Maillard reaction products to produce specific compounds can be assumed. Unfortunately, CSS developed p-xylene and o-xylene, involved in the rise of WOF and referred to as "cardboard-like" [55,56]. No references are available to compare results obtained, especially for CSS properties.

Chromatograms, in all cases, showed a great reduction of WOF or general active odorants. As a demonstration of this, CTRL T<sup>0</sup> and T<sup>72</sup> images had a resolution with an order of magnitude of 1010, while CSS had values of 10<sup>8</sup> . From pictures, the peak of hexanal that eluted at around 6.48 min is always visible and it is clear how much it decreases in respect of additions of by-products. In all samples at around minutes 13.44 and 16.49, long chain aldehydes were eluted (i.e., octanal, decenal). At around minutes 19.35–37 2-Nonen-1-ol, (E)- was eluted in almost all samples. After minute 19.40, the main compounds traced were siloxanes and low matched compounds.

Of course, a better characterization of the aromatic profile of these molecules is fundamental to understand if and how volatiles from these substrates can have a negative impact on the final flavour of meat products.

#### 3.2.3. Sensory Analysis

Panellists involved in this analysis were asked to try samples T<sup>0</sup> and T<sup>72</sup> of all formulations on separate days without knowing what they were eating, with the objective of evaluating the presence and the intensity of WOF markers and possible perceptions of extraneous flavours in cooked burgers after refrigerated storage. Descriptors were carefully explained, especially those difficult to associate with food, such as "cardboard-like" or "paint".

In Table 5 are reported all the average values for burger samples tasted immediately after cooking (T0) and after 72 h of refrigerated storage (T72).

**Table 5.** Average scores for cooked CSS containing samples of panelists for each descriptor in all formulations; immediately after cooking process (T<sup>0</sup> ) and after 72 h of refrigerated (+4 ◦C) storage (T72).


Legend: data followed by different superscript letters, in the same row, are significantly different (LSD test, *p* < 0.05).

T<sup>0</sup> samples of all formulations did not show significative differences, and panellists were not able to trace significant variability from control. Statistical analysis did not show significance between samples even if, looking at the average values, some considerations can be made. The score reported for the bitter descriptor rose to 2.25 for the 3% CSS formulation, while CTRL was 1.25 and CSS 1.5% was 1.87. CSS addition did not influence the average score for all descriptors, and, in most cases, they were the same as CTRL or very near to it. CSS seemed to influence the perception of cooked meat odour rising from 1.75 in CTRL, to 2.87 in 1.5% addition, to 2.75 in 3% addition. This is one of the few situations in which 1.5% received higher scores than 3%. Astringency also registered an increase from CTRL to the 1.5% CSS addition of 3%: from 1.62 to 1.75 to 2.37, respectively.

Generally, all the descriptors for all formulations received increased scores, but some significant differences were traced via the statistical analysis or can be noted from the direct comparison among the average scores. Only two descriptors for CSS formulations were significant (*p* < 0.05): cooked meat odour and bitter. Cooked meat odour was mitigated, mainly by CSS addition. For this descriptor, the score decreased from 4 (CTRL) to 2.75 (1.5%) and 2.62 (3%). This limitation can be seen neither as negative nor positive. If the cooked meat odour can be directly linked with positive sensations, we do not know the considerations of each panellist regarding cooked meat. Nevertheless, from the explanation of each descriptor and the training, these lower scores do not directly show a positive thing. For better discrimination, a comparison with the roasted descriptor can be made; although not significant, it received lower scores than cooked meat.

The presence of this descriptor was not casual, because it helped in separating what flavours, odours, and aromas can come from a fresh grilled or oven/pan-cooked burger, instead of an already cooked and reheated burger. Panelists were not aware of the cooking process. The 'roasted' adjective generally includes those positive flavours coming from the Maillard complex of the reaction, while cooked meat is mainly linked with sensations of staling. In these terms, the mitigation obtained from by-products can be positive, while increasing the stability of the product. Bitterness perceived in CSS formulated patties can be a result of the reheating of meat. The data show an increase from CTRL at 1.37, to 1.62 (CSS 1.5%), and to 2.35 (CSS 3%). Heat can make the condensation or splitting of phenolic species easier. During the roasting process, chlorogenic acids degrade to active taste lactones which give desirable sourness and bitterness [57]. When exposed to further heating, these species undergo greater degradation which leads to the splitting of quinic acid which, in successive steps will give metallic, lingering bitter phenyl-indanes which are undesirable for coffee taste. Caffeine and, in general, methylxanthines-alkaloids give a bitter and astringent note. Probably, the double exposure to heating, even if at lower temperatures, can determine higher bitterness.

Refrigerated storage can also favour the condensation of flavonoids to tannins or bigger phenolic species, but no sources are available at this time. A general positive comment is that none of the descriptors directly linked with the development of WOF (paint, cardboard-like, vegetable oil-like and sulphur/rubber) was increased. Even if not significative, average scores of the CSS added sample were lowered in respect of CTRL. Control had 2, while CSS fell to 1.75 for 1.5% addition, and to 1.5 for 3% addition. Acidity and even metallic sensations were not increased. To better investigate the significance of cooked meat flavour and bitterness, different factors were considered. Time, Formulation, and Time x Formulation were selected as factors. For cooked meat flavour, the time factor, i.e., the effect of time, was significant for *p* < 0.01 while time x formulation factor had a *p* < 0.05 (Table 6). Formulation alone did not influence the results. Conversely, bitterness was influenced only by the formulation with a *p* < 0.05. Neither time nor time x formulation factors made an effect. Overall, T<sup>0</sup> samples did not show any significant difference for the cooked meat odour descriptor. After 72 h of refrigerated storage, the time effect greatly influences (*p* < 0.01) this characteristic.


**Table 6.** Anova matrix results for significant descriptors (cooked meat odour and bitterness).

Legend: asterisks indicate significance at \* *p* <0.05; \*\* *p* < 0.01; n.s. not significant.

#### **4. Conclusions**

The present study provides data on technological performance, nutritional aspects and effects on stability of newly formulated meat products.

Coffee silver skin could be considered as a food ingredient that can solve a complex problem in limiting the decay of meat foods (especially of poultry origin) and lowering the food waste caused by coffee production. CSS can become a cheap but valuable integrator of fibres and bioactive molecules; moreover, it is a great source of minerals such as calcium, potassium, and others.

Data obtained are the starting point of a deeper study to comprehend what are the best conditions to use this by-product and how to develop "tailor-made" formulations enjoyable to consumers. Burgers were among the easiest preparations, allowing a direct comparison with reality.

CSS has shown potential for being implemented in meat formulations to limit losses connected with the cooking process.

Considering the principal aim of this study (to understand the role of CSS on WOF occurrence), encouraging results are provided. The sensory analysis conducted gave confirmation that the spreading of WOF, or in general of oxidation markers, was arrested, even if bitterness and astringency can emerge with time. The inclusion of CSS among new ingredients for food production is hoped for, even if further analysis is needed with further consultation procedures regarding current novel food statuses.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/foods10081833/s1, Figure S1: Chromatogram of volatile compounds (VOCs) in chicken burgers immediately after cooking (a, CTRL T<sup>0</sup> ) and after 72 h of refrigerated storage (b, CTRL T72); Figure S2: Chromatogram of volatile compounds (VOCs) in chicken burgers formulated with CSS 1.5%, immediately after cooking (a) and after 72 h of refrigerated storage (b); Figure S3: Chromatogram of volatile compounds (VOCs) in chicken burgers formulated with CSS 3%, immediately after cooking (a) and after 72 h of refrigerated storage (b).

**Author Contributions:** Conceptualization, M.M., D.M. and L.E.; methodology, M.M. and L.E.; software, M.M. and L.E.; validation, M.M.; formal analysis, L.E.; investigation, L.E., M.M.; resources, M.M. and D.M.; data curation, M.M. and L.E.; writing—original draft preparation, L.E. and M.M.; writing—review and editing, M.M., D.M. and L.E.; visualization, M.M. and D.M.; supervision, M.M., D.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** Authors acknowledge to the Torrefazione Adriatica spa, Giulianova (Teramo, Italy) for providing the coffee silver skin sample for this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

