P1: SFK/UKS BLBS102-c18 P2: SFK BLBS102-Simpson March 21, 2012 13:30 336 Trim: 276mm X 219mm Printer Name: Yet to Come Part 3: Meat, Poultry and Seafoods Figure 18.4 Sausage stuffed into a collagen casing, 80-mm diameter, and clipped on both extremes although the operation takes only a short time, just a few minutes, and the ratio of bowl speed to knife speed determines the desired particle size PROCESSING STAGE 2: STUFFING The mixture is stuffed under vacuum into casings—natural, collagen-based, or synthetic—with both extremes clipped The vacuum avoids the presence of bubbles within the sausage and disruptions in the casing The stuffing must be adequate in order to avoid smearing of the batter, and temperature must be kept below 2◦ C to avoid this problem Once stuffed (Fig 18.4), the sausages are in racks and placed in natural or airconditioned drying chambers PROCESSING STAGE 3: FERMENTATION Fermentation Technology Once sausages are stuffed, they are placed in computercontrolled air-conditioned chambers and left to ferment for microbial growth and development A typical chamber is shown in Figure 18.5 Temperature, relative humidity, and air speed must be carefully controlled in order to have correct microbial growth and enzyme action The whole process can be considered as a lactic acid solid-state fermentation in which several simultaneous processes take place: (1) microbial growth and development, (2) biochemical changes, mainly enzymatic breakdown of carbohydrates, proteins, and lipids, and (3) physical changes, mainly acid gelation of meat proteins and drying Meat fermentation technology differs between the United States and Europe High fermentation temperatures (35–40◦ C) are typical in US sausages, followed by a mild heating process, as a kind of pasteurization, instead of drying, to kill any Trichinella Thus, starters such as Lactobacillus plantarum or Pediococcus Figure 18.5 Example of a fermentation/drying chamber with computer control of temperature, relative humidity, and air rate (By courtesy of Embutidos y Conservas Tabanera, Segovia, Spain.) acidilactici, which grow well at those temperatures, are typically used In Europe, different technologies may be found, depending on the location and climate There is a historical trend toward short-processed, smoked sausages in cold and humid countries, as in Northern Europe, and long-processed, dried sausages in warmer and drier countries, as in the Mediterranean area In the case of Northern European countries, sausages are fermented for about days at intermediate temperatures (25–30◦ C), followed by short ripening periods (up to weeks) These sausages are subjected to a rapid pH drop and are usually smoked for a specific flavor (Demeyer and Stahnke 2002) On the other hand, Mediterranean sausages require longer processing times Fermentation takes place at milder temperatures (18–24◦ C) for about days, followed by mild drying conditions for a longer time, usually several weeks or months L sakei or L curvatus are the lactic acid bacteria most often used as starter cultures (Toldr´a et al 2001) The time required for the fermentation stage is a function of the temperature and the type of microorganisms used as starters The technology is quite different in China and other Asian countries Sausages are first dried over charcoal at 48◦ C and 65% relative humidity for 36 hours and then at 20◦ C and 75% relative humidity for days Water activity rapidly drops below 0.80, although pH remains at about 5.9, which is a relatively high value Fermentation is relatively poor, and the sour taste, which is considered undesirable, is reduced Chinese raw sausage is consumed after heating (Leistner 1992) Microbial Metabolism of Carbohydrates The added carbohydrate is converted, during the fermentation, into lactic acid of either the d(−) or l(+) configuration, or a mixture of both, depending on the species of lactic acid bacteria used P1: SFK/UKS BLBS102-c18 P2: SFK BLBS102-Simpson March 21, 2012 13:30 Trim: 276mm X 219mm Printer Name: Yet to Come 337 18 Biochemistry of Fermented Meat as starter The ratio between the l- and d-enantiomers depends on the action of l- and d-lactate dehydrogenase, respectively, and the presence of lactate racemase The rate of generation and the final amount of lactic acid depend on the type of lactic acid bacteria species used as starter, the type and content of carbohydrates, the fermentation temperature, and other processing parameters The accumulation of lactic acid produces a pH drop, more or less, intense depending on its generation rate Some secondary products such as acetic acid, acetoin, and others may be formed through heterofermentative pathways (Demeyer and Stahnke 2002) Acid pH favors protein coagulation, as it approaches its isoelectric point, and thus also favors water release Acid pH also contributes to safety by contributing to the inhibition of undesirable pathogenic or spoilage bacteria The pH drop favors initial proteolysis and lipolysis by stimulating the activity of muscle cathepsin D and lysosomal acid lipase, both active at acid pH, but an excessive pH drop does not favor later enzymatic reactions involved in the generation of flavor compounds (Toldr´a and Verplaetse 1995) Acidulation Protein coagulation Water release Water diffusion Evaporation PROCESSING STAGE 4: RIPENING AND DRYING Figure 18.6 Scheme showing important physical changes during the processing of fermented meats Temperature, relative humidity, and air flow have to be carefully controlled during fermentation and ripening to allow correct microbial growth and enzyme action while maintaining adequate drying progress The air velocity is kept at around 0.1 m/s, which is enough for a good homogenization of the environment Ripening and drying are important for enzymatic reactions related to flavor development and obtaining the required water loss and thus reduction in aw The length of the ripening/drying period takes from to 90 days, depending on many factors, including the kind of product, its diameter, dryness degree, fat content, desired flavor intensity, and so on The reduction in aw is slower in beef-containing sausages The casing must remain attached to the sausage when it shrinks during drying In general, longripened products tend to be drier and more flavorful diffusion rate is much higher than the evaporation rate, water accumulates on the surface of the sausage, causing a wrinkled casing This situation may happen in small-diameter sausages being ripened in a chamber with high relative humidity The progress in drying reduces the water content, up to 20% weight loss in semidry sausages and 30% in dry sausages (Table 18.1) The aw decreases according to the drying rate, reaching values below 0.90 for long-ripened sausages Physical Changes The most important physical changes during fermentation and ripening/drying are summarized in Figure 18.6 The acidulation produced during the fermentation stage induces protein coagulation and thus some water release The acidulation also reduces the solubility of sarcoplasmic and myofibrillar proteins, and the sausage starts to develop consistency The drying process is a delicate operation that must achieve an equilibrium between two different mass transfer processes—diffusion and evaporation (Baldini et al 2000) Water inside the sausage must diffuse to the outer surface and then evaporate to the environment Both rates must be in equilibrium because a very fast reduction in the relative humidity of the chamber would cause excessive evaporation from the sausage surface that would reduce the water content on the outer parts of the sausage, causing hardening This is typical of sausages of large diameter because of the slow water diffusion rate The cross section of these sausages shows a darker, dry, hard outer ring On the other hand, when the water Chemical Changes There are different enzymes, of both muscle and microbial origin, involved in reactions related to color, texture, and flavor generation These reactions, which are summarized in Figure 18.7, are very important for the final sensory quality of the product One of the most important groups of reactions, mainly affecting myofibrillar proteins and yielding small peptides and free amino acids as final products, is known as proteolysis (Toldr´a 1998) An intense proteolysis during fermentation and ripening is mainly carried out by endogenous cathepsin D, an acid muscle proteinase that is very active at acid pH This enzyme hydrolyses myosin and actin, producing an accumulation of polypeptides that are further hydrolyzed to small peptides by muscle and microbial peptidyl peptidases and to free amino acids by muscle and microbial aminopeptidases (Sanz et al 2002) The generation of small peptides and free amino acids increases with the length of processing, although the generation rate is reduced at acid pH values because the conditions are far from optimal for enzyme activity Free amino acids may be further transformed into other products, for example, volatile compounds through Strecker degradations and Maillard reactions; ammonia through P1: SFK/UKS BLBS102-c18 P2: SFK BLBS102-Simpson March 21, 2012 338 13:30 Trim: 276mm X 219mm Printer Name: Yet to Come Part 3: Meat, Poultry and Seafoods Carbohydrates Myofibrillar proteins Triacylglycerols phospholipids Nitrate Catalase Pyruvate Peptides Free fatty acids Nitrite Peroxides destruction Lactic acid Free amino acids Volatile compounds pH Taste Aroma Nitric oxide Color Figure 18.7 Scheme showing the most important reactions by muscle and microbial enzymes involved in chemical and biochemical changes affecting sensory quality of fermented meats deamination and/or deamidation reactions by deaminases and deamidases, respectively, present in yeasts and molds; or amines by microbial decarboxylases Another important group of enzymatic reactions, affecting muscle and adipose tissue lipids, is known as lipolysis (Toldr´a 1998) Thus, a large amount of free fatty acids (between 0.5% and 7%) is generated through the enzymatic hydrolysis of triacylglycerols and phospholipids Most of the observed lipolysis is attributed, after extensive studies on model sterile systems and sausages with added antibiotics, to endogenous lipases present in muscle and adipose tissue (e.g., lysosomal acid lipase, present in the lysosomes and very active at acid pH; Toldr´a 1992, Hierro et al 1997, Molly et al 1997) Catalases are mainly present in microorganisms such as Kocuria and Staphylococcus; they are responsible for peroxide reduction and thus contribute to color and flavor stabilization Nitrate reductase, also present in these microorganisms, is also important for reducing nitrate to nitrite in slow-ripened sausages with an initial addition of nitrate Recently, two strains of Lactobacillus fermentum have proved to be able to generate nitric oxide and give an acceptable color in sausages without nitrate/nitrite This could be used to produce cured meats free of nitrate and nitrite (Moller et al 2003) PROCESSING STAGE 5: SMOKING Smoking is mostly applied in Northern countries with cold and/or humid climates Initially, it was used for preservation purposes, but today its contribution to flavor and color is more important (Ellis 2001) In some cases, smoking can be applied just after fermentation or even at the start of the fermentation Smoking can be accompanied by heating at 60◦ C and has a strong impact on the final sensory properties It has a strong antioxidative effect and gives a characteristic color and flavor to the product, which is the primary role of smoking The antimicrobial effect of some smoking compounds, especially phenols, carboxylic acids, and formaldehyde inhibit the growth of certain bacteria even though some yeasts and molds may be resistant (Sikorski and Kolakowski 2010) SAFETY The stability of the sausage against pathogen and/or spoilage microorganisms is the result of successive hurdles (Leistner 1992) Initially, the added nitrite curing salt is very important for the microbial stability of the mix During mixing under vacuum, oxygen is gradually removed, and redox potential is reduced This effect is enhanced when ascorbic acid or ascorbate is added Low redox potential values inhibit aerobic bacteria and make nitrite more effective as bactericide During the fermentation, lactic acid bacteria can inhibit other bacteria, not only by the generation of lactic acid (and the subsequent pH drop), but also by generation of other metabolic products such as acetic acid and hydrogen peroxide and, especially, bacteriocins (low-molecular mass peptides synthetized in bacteriocin-positive strains; Lăucke 1992) The drying of the sausage continues the reduction in aw to low values (aw below 0.92) that inhibit growth of spoilage and/or pathogenic microorganisms Thus, the correct interaction of all these factors assures the stability of the product Some foodborne pathogens that might be found in fermented meats are briefly described Salmonella is more usual in fresh, spreadable sausages (Lăucke 1985), but can be inhibited by acidification to pH 5.0 and/or drying to aw < 0.95 (Talon et al 2002) Lactic acid bacteria exert an antagonistic effect against Salmonella (Roca and Incze 1990) Staphylococcus aureus may grow under aerobic or anaerobic conditions and requires aw < 0.91 for inhibition, but is sensitive to acid pH So, it is important to control the elapsed time before reaching the pH drop in order to avoid toxin production Furthermore, this toxin is produced only in aerobic conditions (Roca and Incze 1990) Clostridium botulinum and its toxin-producing capability are affected by a rapid pH drop and low aw even more than by the addition of P1: SFK/UKS BLBS102-c18 P2: SFK BLBS102-Simpson March 21, 2012 13:30 Trim: 276mm X 219mm Printer Name: Yet to Come 339 18 Biochemistry of Fermented Meat Table 18.4 Safety Aspects: Generation of Undesirable Compounds in Dry Fermented Meats Compounds Tyramine Tryptamine Phenylethylamine Cadaverine Histamine Putrescine Spermine Spermidine Cholesterol oxides Route of Formation Origin Concentrations (mg/100g) Microbial decarboxylation Microbial decarboxylation Microbial decarboxylation Microbial decarboxylation Microbial decarboxylation Microbial decarboxylation Microbial decarboxylation Microbial decarboxylation Oxidation Tyrosine Trytophan Phenylalanine Lysine histidine Ornithine Methionine Methionine Cholesterol