Endogenous Enzyme during Meat Processing: Comparison
Please note this is a comparison between Version 1 by Jose Benedito and Version 3 by Conner Chen.

Meat aging is a combination of transformations that originate in the animal’s muscle after slaughter, resulting in changes in colour, tenderness and aroma [1]. The biochemical processes that occur during meat aging are mainly caused by endogenous enzymes, leading to glycolysis, proteolysis and lipolysis.

  • enzymes
  • enzyme reaction
  • meat

1. Introduction

Meat aging is a combination of transformations that originate in the animal’s muscle after slaughter, resulting in changes in colour, tenderness and aroma [1]. The biochemical processes that occur during meat aging are mainly caused by endogenous enzymes, leading to glycolysis, proteolysis and lipolysis. In the glycolysis reactions, glucose is metabolised to produce lactic acid, which lowers muscle pH and depletes the energy reserves (ATP). The energy depletion leads to the degradation of myofibrillar proteins by the action of endopeptidases and the action of exopeptidases. Endogenous proteases (calpains, cathepsins and calpastatin) play a crucial role in the proteolysis of meat; however, exogenous proteases (peptidylpeptidases, aminopeptidases and carboxypeptidases) secreted from microorganisms involved in meat fermentation also contribute to increase the concentration of peptides and amino acids [2]. Another reaction that takes place during the meat aging is the lipolysis in the muscle and the adipose tissue [3].
The products resulting from the degradation of proteins and lipids are precursors of the characteristic flavour and aroma of the meat and meat products [4][5][6][4,5,6]. In addition, the fragmentation of the myofibrils also leads to changes in texture that lead to meat softening [7]. Therefore, post mortem changes in the muscle, which affect the organoleptic properties of meat, are mostly related to the action of enzymes. Moreover, the enzymatic reactions that take place in the transformation of muscle into meat occur at relatively low reaction rates and are affected by numerous intrinsic (animal breed, age or feeding) and extrinsic factors (such as temperature, animal welfare, transport, stress, etc.). On the other hand, exogenous enzymes are also used in the meat industry, mainly to produce restructured meat, obtain bioactive peptides and induce meat tenderization. In certain applications, the enhancement and effective control of the activity of endogenous and exogenous enzymes can be challenging but also of great technological interest.
The application of emerging technologies in meat processing could be used for the intensification of enzymatic reactions involving endogenous and exogenous enzymes. In this sense, during the last few years, there has been a growing interest in non-thermal techniques capable of accelerating enzymatic reactions without affecting the quality of meat and guaranteeing food safety [8]. Currently, some of the emerging technologies that have been used to improve the enzymatic reactions are ultrasound, pulsed electric fields (PEF), moderate electric fields (MEF), high pressure (HPP) or supercritical CO2 (SC-CO2), all of which have shown their ability to preserve the quality and safety of processed food products [9].

2. Endogenous Enzyme during Meat Processing

During the processing of meat, there occurs a series of biochemical reactions, mainly catalysed by enzymes. These reactions involve enzymes responsible for post mortem glycolysis, proteolysis and lipolysis. The pH decline depends on glycogen content, and it is enzymatically controlled by enzymes such as phosphofructokinase [10]. The proteolytic enzymes involved include first muscle endopeptidases: calpains (µ-calpain and m-calpain) and cathepsins (B, H, L and D), the ubiquitin–proteasome system and, subsequently, exopeptidases such as dipeptidases, aminopeptidases and carboxypeptidases [11], which degrade the polypeptides generated by endopeptidases into peptides and free amino acids [12]. In addition to proteolytic changes, the action of lipases brings about hydrolysis reactions of triglycerides and phospholipids, which contribute to the characteristic flavour and aroma of meat [5].

2.1. Conversion of Muscle into Meat

Meat is the result of a series of transformations that the muscle tissue of the animal undergoes after slaughtering. This process entails structural transformations and biochemical reactions, which will produce changes affecting the technological and sensory quality of the meat. The process of converting muscle into meat comprises three stages: pre rigor mortis, rigor mortis and post rigor mortis [13]. The first stage (3–6 h) occurs immediately after the slaughter of the animal due to the interruption of blood circulation caused by bleeding. This process causes the arrival of oxygen and nutrients to be abruptly interrupted [14]. In the second stage (until 24 h), the depletion of energy components takes place, that is, adenosine triphosphate (ATP), phosphocreatinine and glucose. Finally, in the post rigor mortis, endogenous proteolytic systems lead to the disintegration of the muscle structure of the myofibrils, which induces meat tenderization. The duration of the conversion of muscle into meat depends on three aspects: the animal species, the glycogen reserves at the time the animal is slaughtered and the storage temperature. Thus, the three stages of rigor mortis take at least 14 days in cattle, 7 to 10 days in sheep, 5 to 7 days in pigs and approximately 6 h in poultry [15]. The three proteolytic systems involved in the three stages are calpains, cathepsins and ubiquitin–proteasomes [16]. In pre rigor mortis, the meat must be kept at a temperature above 10 °C until reaching the rigor mortis phase (10–10 rule, temperature within meat on a carcass should not be below 10 °C within 10 h after slaughter). Electrical stimulation immediately after the sacrifice of the animal causes a muscular contraction that accelerates the consumption of glycogen, the drop in pH and the establishment of rigor mortis. In this way, it is possible to prevent meat toughening and the apparition of the “cold shortening” problem during rigor mortis [17]. In pre rigor mortis, the metabolism of the animal muscle changes from aerobic to anaerobic and, therefore, undergoes a gradual decrease in energy intake. Muscle needs glycogen and phosphocreatine to synthesise ATP from glucose. Under these circumstances, the enzymes that lead the muscle metabolism begin to act, that is, those responsible for glycolysis. Among the enzymes that participate in anaerobic glycolysis, glucose 6-phosphate and phosphocreatine kinase are particularly relevant. These enzymes act until the glycogen and phosphocreatine reserves are depleted, after which ATP is reduced to form, first, adenosine diphosphate (ADP) and, subsequently, adenosine monophosphate (AMP), which can be deaminated by the enzymes responsible for the degradation of ATP. On the other hand, after the progressive reduction in ATP levels, inorganic phosphate is generated, which stimulates the degradation of glucose to pyruvate, and, subsequently, lactic acid is generated from the enzyme lactate dehydrogenase. Lactic acid causes the muscle pH to drop and the enzymes responsible for anaerobic metabolism (glycolysis) to be inactivated [18]. The decrease in muscle pH is one of the most significant post mortem changes, leading to the start of the rigor mortis. After the depletion of ATP, there is a depolarization of the membranes due to an ionic increase linked to the Ca2+, Na+ and K+ pumps’ standstill, which is dependent on the ATP content. That is why the Ca2+ ions react with troponin, which modifies the configuration of the active sites of actin; subsequently, myosin binds to actin, giving rise to the irreversible formation of actomyosin, which causes a reduction in the water retention capacity (WRC) and, therefore, a hardening of the muscle. The rigor mortis ends with the formation of actomyosin, which is characterised by muscle tension and stiffness. In the post rigor mortis, the enzymatic reactions responsible for the tenderization of meat take place. First, the proteolytic system of calpain plays a central role in post mortem proteolysis and softening [19]. The calpain system is dependent on Ca2+ and its endogenous inhibitors, calpastatins and has been described as the main factor responsible for proteolysis in the early post mortem period (0–24 h) and meat tenderization since the calpain system acts at neutral pH and its activity declines when the pH drops. Caballero et al. [20] postulated that the synergistic action of calpains with cathepsins leads to meat softening. Cathepsins, lysosomal enzymes, are activated at a lower pH than calpains; therefore, they become more important in the later phases of post mortem along with their endogenous inhibitors, cystatins. Finally, the proteasome is responsible for the degradation of most intracellular proteins, with the ubiquitin–proteasome complex responsible for the intracellular turnover of damaged proteins [21]. However, to date, the role of the proteasome in tenderization has not been fully clarified, although it is known that its activation is one of the first cellular responses to oxidative stress. The post-slaughter evolution of pH has a great effect on the technological properties of the meat, affecting the texture (tenderization), colour and aroma due to the generation of the volatile compounds resulting from the proteolytic and lipid degradation of the meat [12]. These are reactions that will be discussed more extensively in SectionSection 2.2 2.2 since they are of greater importance in the maturation and curing of meat. In addition, it should be noted that some factors related to genetics, nutrition and pre mortem and post mortem handling can drastically influence the conversion of muscle into meat [22]. In addition, the role played by the colour of the meat is important as it is indicative of the meat’s freshness and is, therefore, a key factor for consumer acceptance. The colour depends on the concentration and degree of oxidation of the heme compounds, mainly myoglobin (Mb). Mb is a globular protein, which can be found in four chemical forms: deoxymyoglobin (DeoxyMb), carboxymyoglobin (CarboxyMb), metmyoglobin (MetMb) and oxymyoglobin (OxyMb) [23]. Mb is a water-soluble protein, and, among the amino acid residues that it contains, histidine has received the most attention due to its key role in the structure and function of Mb. In addition, there are other heme proteins, such as haemoglobin and cytochrome C, that may also play a role in the colour of beef, lamb, pork and poultry. However, the mechanisms that control colour stability have not been completely elucidated [24]. The bright red colour of fresh meat depends on a triple balance of biochemical factors: the respiratory activities (O2 uptake rate), the auto-oxidation of Mb and the enzymatic reduction of MetMb, which in turn can be affected by time, temperature and muscle pH history [25]. These enzymatic processes affecting texture, colour and aroma occur in all types of meat but are particularly important in the aging of beef cuts [26]. Finally, the treatments carried out, both before and after the slaughter of an animal, determine the final quality of the meat and can trigger two types of meat: dark, firm and dry (DFD) and pale, soft and exudative (PSE). The factors determining this type of meat are those related to the muscle glycogen content, which affects the pH of the meat and the temperature to which the meat is subjected after slaughtering the animal [27].

2.2. Meat Products

There are many different meat products worldwide. The enzymatic reactions leading to meat conversion and initial meat quality influence the yield and final quality of the cooked meat products, as in the case of PSE and DFD meats [28]. However, endogenous enzymatic reactions are of less importance in cooked meat products than in raw meat products because they are inactivated by thermal treatments at temperatures above 40 °C. Accordingly, endogenous enzymes play an important role during the elaboration of dry-cured and fermented meat products.

2.2.1. Dry-Cured Meat Products

The processing of dry-cured meats typically involves the addition of curing salts, such as NaCl, and nitrates and nitrites. Nitrates and nitrites play an important role in dry-cured meat products, particularly in cured ham. The main function of these nitrifying agents is to provide food stability and safety from a microbiological point of view. In addition to ensuring food safety, they are also responsible for the formation and the stability of the characteristic colour of cured meat. Unlike the conversion of muscle into meat, meat curing is a long process, which can be extended for up to 12 months or more in the elaboration of dry-cured ham, with enzymatic reactions being particularly relevant. As previously mentioned in Section 2.1, calpains are the enzymes that act first. These enzymes are very unstable and have an optimal pH and temperature of 5.5–6.5 and 2–6 °C, respectively [10]. Calpains are able to hydrolyse proteins, such as titin, nebulin, troponins T and I, tropomyosin and desmin [19]. On the other hand, cathepsins, along with calpains, also contribute to meat softening during post mortem, as previously mentioned. Cathepsins are mostly active at acidic pH (5.0–6.0). While cathepsins B, H and L are stable and active during the whole meat curing process, cathepsin D disappears throughout the process. To a large degree, the disappearance of cathepsin D is due to the addition of salts (NaCl) [29]. In addition, cathepsins D and L release fragments of proteins from the degradation of myofibrillary proteins, such as titin, troponins T and I and tropomyosin. As in the case of exopeptidases, pyroglutamyl, alanyl, leucyl and arginyl aminopeptidases are enzymes that exert the greatest activity during the processing of cured meat. They present good stability during curing, although NaCl is also considered an inhibitor of these enzymes [30]. The amino acids and peptides generated during this stage by exopeptidases (glutamic acid, alanine, arginine, lysine and leucine) are responsible for the characteristic aroma and flavour of dry-cured products [31]. As in the enzymatic activity of lipases, it consists of the enzymatic hydrolysis of muscle lipids and adipose tissue to generate free fatty acids. These free fatty acids are susceptible to oxidation, which gives rise to some of the aromatic compounds typical of cured products [32]. As in the case of lipases, itwe can be differentiated between lipases (lysosomal and neutral) and muscle phospholipases [33]. Neutral lipases act at the beginning of the curing process, forming free fatty acids. Subsequently, lysosomal acid lipase acts on triglycerides, giving rise to mono- and diglycerides and free fatty acids. Phospholipases act during the first 6 months of curing, forming free fatty acids, especially oleic, stearic, linoleic and palmitic [33]. The proteolytic activity in dry-cured meat products depends on temperature, pH and also on NaCl, which affects the proteolytic activity during the process and the final texture of the cured meat [34]. Arnau et al. [34] and García-Rey et al. [35] studied the texture of Biceps femoris salted at different contents of NaCl and observed that Biceps femoris became pastier when the NaCl content decreased. Ruiz-Ramírez et al. [36] reported that the hardness, cohesiveness and springiness of Semimembranosus and Biceps femoris muscles were affected by the NaCl content. Dry-cured muscles with less NaCl exhibited lower degrees of hardness, cohesiveness and springiness due to the fact that NaCl acts as a strong inhibitor of proteolytic activity [37]. Regarding NaNO2, the reaction of nitric oxide with Mb leads to the formation of nitrosylmyoglobin (NOMb), which is the pigment responsible for the reddish colouration of the dry-cured ham. The NOMb formation requires the presence of nitrites, which generate nitrogen monoxide (NO); under reducing conditions, either directly combined with Mb or indirectly in combination with MetMb, this gives rise to NOMb. This pigment is very stable, maintaining its reddish colour even in very long-lasting hams [38]. Zinc protoporphyrin (ZnPP) is a natural red pigment known for the typical colour that it imparts to the Italian dry-cured Parma ham, which is manufactured without the use of nitrifying agents. In this pigment, the iron ion of the porphyrin ring has been replaced by a zinc ion. There is evidence that, in dry-cured hams, ZnPP is mainly formed endogenously due to the enzyme ferrochelatase (FeCH) [39]. The mechanisms of the formation of ZnPP in Parma hams have recently been reviewed [40]. However, it has been shown that ZnPP can also be formed in different quantities in Iberian and Serrano hams during their processing [41][42][43][41,42,43]. In this regard, the possible relationship between ZnPP and lipolysis or proteolysis has been investigated in hams and other meat models [43][44][45][43,44,45]

2.2.2. Fermented Sausages

There is a wide variety of dry-cured products without anatomical integrity, with or without fermentation. Products that present a fermentation stage during their processing undergo additional enzymatic reactions that will be outlined in this section [46]. The final characteristics and quality of these fermented products depend on the raw material, the microbial population as well as on the processing conditions during fermentation (temperature, 18–26 °C; relative humidity, 90–95% and time, 24–72 h). Microorganisms involved in fermentation include the microbiota of the raw meat and microorganisms added as starter cultures (lactic acid bacteria (Lactobacillus), Gram-positive catalase-positive cocci (Staphylococcus), yeasts and moulds). Lactic acid bacteria, essentially Lactobacillus sakei, play an important role in the technological properties and microbial stability of the final product through the production of lactic and acetic acids and the consequent decrease in pH to approximately 5. At this pH, muscle proteins coagulate and lose their water-holding capacity, leading to an increase in the firmness and cohesiveness of the final product. In addition, the accumulation of lactic and acetic acids inhibits the growth of pathogenic and spoilage microorganisms. On the other hand, Staphylococcus also plays an important role in the fermentation process since it contributes to the development of the characteristic flavour and colour together with the acidic pH promoted by the lactic acid bacteria, which improves the colour stability of the fermented products. The action of these microorganisms (lactic acid bacteria and Staphylococcus) is based on the endopeptidase and exopeptidase enzymes that they generate. Overall, these endopeptidases and exopeptidases contribute to an increase in the concentration of free amino acids that affect flavour development [47]. Finally, yeasts and moulds participate in fermentation through lactate oxidation and the enzymatic reactions of proteolysis and lipolysis [48]. During sausage fermentation, muscle proteins (actin and myosin) begin to degrade the peptides, mainly through cathepsin D, while, at the same time, lipolysis begins. Both the microorganisms (lactic acid bacteria and Staphylococcus) added as starter cultures and the meat endogenous enzymes (lysosomal lipases and phospholipases, explained in Section 2.2) produce lipolysis, which generates free fatty acids; due to successive modifications, these give rise to esters, aldehydes and ketones, among other compounds, and participate in the final aroma of the fermented product [49]. Once the fermentation stage of sausages is complete, the maturation stage begins. This stage implies the maintenance of the sausages during variable periods under controlled relative humidity and temperature conditions. The most common procedures usually consist of 5–10 days at 18–22 °C and a relative humidity of 80–90%; subsequently, they are kept at 12–15 °C and a relative humidity of 65–80%. The maturation stage can range from 20 to 90 days depending on the type of sausage [31]. During maturation, the proteolysis initiated in the fermentation stage continues through the action of exopeptidases, both of endogenous and microbial origins, which release peptides and free amino acids [50]. In addition, the lipolysis initiated in the fermentation continues. Subsequently, oxidative processes involving the release of free fatty acids and the oxidation of unsaturated fatty acids, particularly polyunsaturated acids, along with the production of carbonyl compounds, take place [46].
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