THE EVALUATION OF TEXTURE AND SAFETY OF FERMENTED BEEF SAUSAGE
PROCESSED WITH HIGH PRESSURE
by
MACC RIGDON
(Under the Direction of Alexander M. Stelzleni)
ABSTRACT
The fermented and (semi-) dried sausage market in the United States has been growing at
both the retail and food service levels over the past decade. Fermented and semi-dry or dry products can have a wide range of water activity, final product pH, and may be produced with variable degrees of thermal processing or none at all. The variability across and within a product category can influence product texture and safety. Traditional products, with lower acidity and lower thermal processing, can be a greater food safety concern due to the reduced intervention parameters. The emergence and implementation of an additional processing technique, high pressure processing (HPP), may have the capability to reduce microbial populations of pathogenic organisms in semi-dry and dried sausages. Therefore, the purpose of the current research was to investigate the effects of HPP on the texture and safety of beef summer sausage and salami with regards to Escherichia coli. Results indicated that the use of HPP in the production of an all beef summer sausage produced at greater pH and lower thermal processing
endpoints, can improve the safety of the sausage, without detrimental effects on color, texture and lipid oxidation. Additionally, the impact of HPP on the texture of non-thermally processed, fermented, and dried salami is negligent. The use of HPP in commercial fermented sausage production can be implemented for improved safety without adverse texture and quality implications.
INDEX WORDS: Salami, Summer sausage, Escherichia coli, Texture, Safety, Beef
THE EVALUATION OF TEXTURE AND SAFETY OF FERMENTED BEEF SAUSAGE
PROCESSED WITH HIGH PRESSURE
by
MACC RIGDON
B.S., California State University, Fresno, 2013
M.S., The University of Georgia, 2015
A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial
Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
ATHENS, GEORGIA
2019
© 2019
Macc Rigdon
All Rights Reserved
THE EVALUATION OF TEXTURE AND SAFETY OF FERMENTED BEEF SAUSAGE
PROCESSED WITH HIGH PRESSURE
by
MACC RIGDON
Major Professor: Alexander M. Stelzleni
Committee: Mark Harrison Ronald Pegg Harshavardhan Thippareddi
Electronic Version Approved:
Susanne Barbour Dean of the Graduate School The University of Georgia August 2019
DEDICATION
I would like to dedicate this dissertation to my Father and Mother who have supported my every decision in life, both financially and emotionally. Without the two of them, I would not be in the position that I am. Secondly, to my wife for her support of me through this process. Without her to come home to every evening, I surely would not have had the perseverance to complete this work. Lastly, yet ironically the most important, my Lord and savior Jesus Christ. I have spent many nights praying that you guide me through the darkness and into the light. I have full faith in my shepherd leading me to your plan for the Jesus is the way, the truth, and the life.
iv
ACKNOWLEDGEMENTS
I would like to thank a whole list of people who have mentored me through this degree.
Each one has had a remarkable impact in one way or another on the man I have become during my 6 years at the University of Georgia. I would like to start with my advisor, and friend, Dr.
Alexander Stelzleni. You have taught me more than just meat science and meat processing, you have encouraged me to be independent and thorough. Your critiques have made me a better writer and conversationalist. I thank you for all the time you have spent having conversations of current events and topics in the field. I also thank you for the many opportunities you have given me to attend conferences, both domestically and abroad, as these are events, places, and times I will never forget. Thank you for understanding me and helping me.
Dr. Harshavardhan Thippareddi, thank you for taking me under your proverbial microbiology wing (insert poultry joke here) and teaching me how to enjoy and be passionate about a new field, with a new species of animal. Thank you for surrounding me with knowledgeable people to learn from and for the opportunity to work with many different projects. Working in your lab, I had the opportunity to learn techniques that I never thought I would use, but would ultimately need for my projects, and for that I am grateful. Your willingness to bring me along with you to conferences and direct questions to me or ask me to speak was an amazing experience that I enjoyed thoroughly, from IPPE, to STEC workshops, to the Cold Pressure Council, I have enjoyed every one. Lastly, I want to thank you for the
v opportunity to submit an abstract to the Center for Food Safety Annual Meeting which awarded me with an opportunity to speak in front of a great deal of industry professionals, as well as a scholarship and my eventual job. I am forever grateful.
I also want to thank Dr. Mark Harrison and Dr. Ronald Pegg for agreeing to serve on my committee. I selected you both because I thoroughly enjoyed the classes that you taught that I was in. I admire both of you professionally, and I hope to one day be as successful as both of you. Thank you for your service on my committee and your mentorship throughout my learning.
Gina McKinney and Ryan Crowe are two of the most wonderful, hardworking lab managers around. I thoroughly believe that you two are the best two unit and lab managers this school has. You are both invaluable in helping students, teaching, and assisting in projects, and for that I thank you.
I would be remiss not to specifically mention my fantastic lab mates who have endured, assisted and concurred this process with me. Robert McKee, you have challenged me and made me a better thinker. I have learned a lot from you, both in the kitchen, and in the field. I am envious of your knowledge of plants and am grateful for the time we have spent working and killing cattle, as well as the endless hours in the meat lab.
Chevise Thomas, my friend, my lab mate, and my role model. There are not words that can express my gratitude for having you in my life. You have taught me so much more about myself than I ever thought I could learn. From long van rides to Eatonton, and Griffin, to plane rides all over the country, and the seemingly endless hours in coolers and cold rooms, as well as the late nights and all-day jobs in the micro labs, I could not have ever asked for a better lab mate. I wish you the best where ever life takes you!
vi
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...... v
CHAPTER 1: INTRODUCTION ...... 1
CHAPTER 2: LITERATURE REVIEW ...... 4
LITERATURE CITED ...... 29
CHAPTER 3: TEXTURE OF FERMENTED SUMMER SAUSAGE WITH DIFFERING PH,
ENDPOINT TEMPERATURE, AND HIGH PRESSURE PROCESSING...... 39
Abstract: ...... 40
INTRODUCTION ...... 41
MATERIALS AND METHODS ...... 42
RESULTS AND DISCUSSION ...... 48
LITERATURE CITED ...... 58
CHAPTER 4: MEETING THE PERFORMANCE STANDARD FOR ESCHERICHIA COLI
O157:H7 AND SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) LETHALITY IN
BEEF SUMMER SAUSAGE USING HIGH PRESSURE PROCESSING ...... 70
Abstract: ...... 71
INTRODUCTION ...... 72
MATERIALS AND METHODS ...... 73
RESULTS AND DISCUSSION ...... 78
Conclusions ...... 84
LITERATURE CITED ...... 85
CHAPTER 5: TEXTURE OF LOW TEMPERATURE FERMENTED SALAMI WITH
DIFFERING WATER ACTIVITIES, AND HIGH PRESSURE PROCESSING TIMES...... 91
Abstract: ...... 92
INTRODUCTION ...... 93
MATERIALS AND METHODS ...... 94
RESULTS AND DISCUSSION ...... 98
Conclusions ...... 104
LITERATURE CITED ...... 105
CHAPTER 6: CONCLUSIONS ...... 114
viii
CHAPTER 1: INTRODUCTION
In 1994, an outbreak of Escherichia coli O157:H7 that was associated with commercially available salami produced in the United States and distributed to several Western states, drastically altered the inspection and production of commercial salami. In Washington and northern California, there were 20 reported and confirmed cases of infection resulting in diarrhea, with 3 patients being hospitalized, one of which resulted in a severe kidney infection
known as hemolytic uremic syndrome (HUS; CDC, 1995). Hemolytic uremic syndrome is a
condition that affects the blood vessels of the kidney, leading to kidney failure, while also
destroying blood platelets and decreasing red blood cell counts (National Kidney Foundation,
2015), and often results in the death of infants and young children. Leaders of the fermented and
dried sausage industry met with the United States Department of Agriculture, Food Safety
Inspection Service in the wake of the outbreak to discuss plans to investigate and improve the
safety of dried and fermented products (Reed, 1995) produced and sold in the U.S. Among the
decisions made were several options for the validation of non-adulterated products, with a
primary focus on cooking or mild heat treatment. However, the final option was the use of total
process controls that are validated to achieve a 5 log reduction of pathogenic E. coli in the final
product (Nickelson II et al., 1996).
The production and replication of traditional Italian style salami products is heavily
dependent on the texture of the finished product, often described by a soft, smooth texture that is
attributed to two factors: 1) lack of cooking and thermal processing; and 2) low acid/low
temperature fermentation of the product (Aquilanti et al., 2012). Traditional methods of
production for these products do not meet the standards outlined by the USDA FSIS for the 5D
inactivation of E. coli O157:H7 (Glass et al., 1992). However, under the final option provided by
the Blue Ribbon Task Force in their meetings with USDA FSIS, a processor using beef could
implement traditional methods of salami production provided it be done in combination with a
novel non-thermal intervention strategy aimed at reducing E. coli O157:H7 and other Shiga toxin
producing E. coli.
High pressure processing is an intervention method that subjects potentially contaminated food substrates to extreme pressures (400-600 MPa) by the injection of water into a confined space resulting in an isostatic and instantaneous distribution of pressure throughout the system
(Cheftel and Culioli, 1997). The use of HPP eliminates pressure gradients, and variability in
process control, that can occur during thermal processing. These pressure vessels also allow the processing of foods at refrigerated temperatures avoiding the negative attributes associated with cooking of traditional salami products. Several studies have evaluated the use of HPP for its effectiveness for pathogen reduction in salami products containing beef (Porto-Fett et al., 2010;
Omer et al., 2010; Gill and Ramaswamy, 2008). However, these studies were often performed on products that were aggressively fermented (low pH) rather than low-acid content, had variable
HPP temperatures applied, and their methods of enumeration were unable to detect injured cells that could potentially recover from the HPP process.
As previously mentioned, the acceptability of traditional salami products is heavily dependent on the texture and over all organoleptic properties of the product (Aquilanti et al.,
2012). While the literature demonstrates there are textural changes present during the fermentation and maturation of dried and fermented products (Barbut, 2015), it is practically
2 devoid of studies investigating the changes in texture of semi-dry and dry products due to HPP.
One such study was found, which showed that during the early stages of maturation sensory panelists were able to detect and did not prefer the texture of pressurized samples (Omer et al.,
2015). The same panelists were unable to detect differences between pressurized and non- pressurized samples later in the maturation stages, suggesting that the use of HPP in finished completely ripened sausages is undetectable. Therefore, the objectives of this research were:
1) Identify and quantify the textural changes present in beef summer sausage products
fermented and cooked to varying endpoint pH and temperature combinations and the
subsequent effects induced by the high pressure processing of those products;
2) Validate the reductions in populations of Escherichia coli O157:H7 and non-O157
Shiga toxin-producing E. coli in summer sausage fermented and cooked to varying
endpoint pH and temperature combinations with the use of high pressure processing
as an intervention;
3) Identify and quantify the textural changes present in a low-temperature fermented
low-acid beef salami dried to varying water activities and the subsequent effects
induced by the high pressure processing of those products.
3
CHAPTER 2: LITERATURE REVIEW
Background
It has been estimated that 31 pathogens cause 36.4 million illnesses each year in the
United States (Elaine et al., 2011). While 9.4 million of these episodes are foodborne, only 5.5
million come from a bacterial source. Elaine et al. (2011) further estimated 55,961 cases caused hospitalization as a result of contaminated food. Shiga toxin producing Escherichia coli (STEC), including both O157 and non-O157 serotypes accounted for approximately 5,283 laboratory confirmed cases of foodborne illness (Elaine et al., 2011). The USDA conducted a study on the prevalence of O157-STEC in raw ground beef and found that 0.39% of bench trimmings produced in the United States were contaminated (USDA, 2011). Shiga toxin-producing E. coli are found to be particularly concerning due to their low infective dose, mortality, and associated morbidity, and therefore, in the production of raw comminuted beef products, are deemed adulterants to the food supply by the USDA FSIS (USDA, 2015).
In the current market, it is noted that consumers are seeking specialty food products as well as traditionally processed foods (Guerrero et al., 2009; Ilbery et al., 1999). However, after an outbreak of E. coli O157:H7 in 1994, that was linked directly to commercial production of dry-cured salami (CDC, 1995), the USDA met with representatives of the nation’s dry sausage makers to discuss processing procedures that would improve the safety of dried sausages, safeguarding them against E. coli O157:H7 (Reed, 1995). Representatives of the meat processing industry agreed to address the issue by validating their processes to ensure the reduction of E.
coli O157:H7 and to make their findings available to the USDA for review (Reed, 1995). Reed
(1995) also stated that the validation process should be a 5D process that addresses at least 5 strains of E. coli O157:H7. These recommendations lead to a conflicting status for the processors that would like to both, provide customers with traditional style products and follow federal guidelines for production of dry fermented sausages containing beef.
The Blue Ribbon Task Force of the National Cattlemen’s Beef Association identified five options to reduce E. coli O157:H7 by 5-log in dry and semi-dry fermented sausages. While some of these options are centered around thermal destruction and ‘test and hold’ programs, the authors also identified the possibility of a validated 5-log reduction process where establishments could use multiple hurdle technology, in which, the collective process achieves a sufficient reduction for E. coli O157:H7 (Nickelson II et al., 1996).
Health Risk and Morphology
Prior to discussion of the elimination of Shiga toxin producing E. coli organisms, it is vital to discuss the organism and its morphology. Doyle et al. (2013) describes E. coli as a facultative anaerobic gram-negative rod that tends to be present in the intestinal tract of humans and animals. Many E. coli serotypes are nonpathogenic and are part of a natural microflora.
However, there are serotypes that are pathogenic and can lead to sickness or even death in humans and animals (Donnenberg and Whittam, 2001). Escherichia coli can be categorized into specific pathotypes based on virulence characteristics (Doyle et al., 2013). These characteristics include adhesion mechanisms, invasion techniques, motility, toxins, surface structures, and genetic characteristics. This genus and species of the organism can be further categorized into five categories of pathogenic E. coli: Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli
5 (ETEC), Enteroinvasive E. coli (EIEC), Enteroaggregative E. coli (EAEC), and
Enterohemorrhagic E. coli (EHEC). Although these groupings can be useful in the categorization
of E. coli, these organisms can be further identified serologically, and differentiated by the
presence or absence of various surface antigens (Doyle et al., 2013). The “O” antigen is
responsible for the identification of the somatic antigen, while the “H” and “K” antigens identify
the flagella and capsules, respectively. When serotyping E. coli diarrheal isolates, it is only required to determine the “O” and the “H” antigen. Although most diarrheal E. coli isolates described under the antigen labeling system fall under the EHEC category, there are several pathogenic E. coli that fall into more than one category, specifically O111 (Doyle et. al., 2013).
The first case of human pathogenesis from EHEC occurred in 1982 from two outbreaks of hemorrhagic colitis from E. coli O157:H7 (Wells et al., 1983). In 1983 the production of shiga toxin was first reported, and later associated with a life-threatening condition called Hemolytic
Uremic Syndrome (HUS; Johnson et al., 1983; Karmali et al., 1985). Since 1983, the identification, and detection of these organisms has increased to now include various non-O157
STEC on USDA’s adulterant list, including O26, O45, O103, O111, O121, and O145, known as
“the big six” (USDA, 2011).
Although the big six STEC are considered adulterants by the USDA FSIS these six serotypes are less likely to cause infections resulting in hospitalizations, bloody diarrhea, and
HUS (Hedican et al., 2009). There does however exist a regional dichotomy in the prevalence and dominance of EHEC isolated outbreaks. It appears that the serotype E. coli O157:H7 is the dominant cause of infection in the countries Japan, the United Kingdom, Canada, and the United
States, while non-O157 STEC are the primary cause of EHEC outbreaks in South Africa,
6 Argentina, Chile, Australia, and Europe (Doyle et al., 2013). This fact justifies the investigation of both O157 and non-O157 STEC in products being produced in the United States.
Reservoirs for EHEC bacterium were initially identified as unpasteurized milk and raw or undercooked ground beef (Doyle et al., 2013). As information became more available other avenues for infection were identified and can include the cattle themselves, fresh produce, contaminated water, and even livestock exhibits and petting zoos. Although it is widely accepted that O157 STEC are colonized in ruminant animals, non-O157 STEC have been traced back to produce, unpasteurized milk, and contaminated drinking water (Mathusa et al., 2009). Davis
(2019) reported that although an on-going outbreak of non-O157 STEC in 2018 and 2019 was traced back to several produce farms in California, the true source of contamination of the leafy greens was likely caused by storm water run-off from a concentrated animal feeding operation contaminating the source of irrigation water, although no empirical evidence exists. This and similar outbreaks indicate that the source of even non-O157 STEC in produce may be originating from ruminant animal vehicles. Davis (2019) continued to describe the best practices for produce farms to include elimination of wildlife intrusion, such as deer, wild birds, and rodents on growing operations, further pointing to ruminant and other fecal contamination as a primary vector.
The mitigation strategies outlined by Davis (2019) are crucial to the safety of the food supply, and it should be noted that the dangers of contaminated food products are particularly alarming when the infectious dose of EHEC is very low (100 cells; Doyle et al., 2013).
Prevalence of E. coli O157:H7 as low as 0.3 colony forming units (CFU) per gram were enumerated from both frozen ground beef patties associated with a 1993 outbreak in the western
United States and retail packaged salami associated with the 1994 outbreak, also in the western
7 United States. Additionally, an outbreak of E. coli O111 in salami was determined to have less than 1 cell per 10 g of product, which caused infections in Australia (Doyle et al., 2013).
The mechanism of pathogenicity is what enables such a low infectious dose associated with E. coli organisms. In general, the mechanism for pathogenicity is described as the attachment of the organism to the host’s cell membrane and colonization of the intestinal lining producing one or more Shiga toxins (Stx). Specifically, E. coli O157:H7 produces a histopathological structure called an attaching-and-effacing (A/E) lesion, which is a formed by the attachment to the host plasma membrane and necrosis of the brush border cells in the microvilli of the large intestine. Subsequently the pathogen constructs an actin structure similar to a pedestal (Garmendia et al., 2005) sometimes referred to as the crown or the A/E lesion. The production of these A/E lesions are encoded by a pathogenicity island identified as LEE gene, or
Locus of Enterocyte Effacement. The LEE encodes for several structural, translocator, and effector proteins associated with the Type Three Secretion System (TTSS; Garmendia et al.,
2005). The TTSS is a secretion system not specific to EHEC or EPEC but an apparatus used by various gram-negative pathogenic organisms; however, this is the system used by EHEC pathogens to inject virulence factors directly into the host cells (Doyle et al., 2013). Another gene associated with the pathogenicity of E. coli serotypes is the eae gene, or the E. coli attaching and effacing gene, which codes for an outer membrane protein called intimin. Intimin functions to create a pore in the outer membrane using a beta-barrel structure that attaches to the plasma membrane of the host cell for the function of translocation of various effector proteins
(Garmendia et al. 2005). These effector proteins make up the structure previously described as the TTSS.
8 The TTSS is responsible for the transfer of virulence factors from the bacteria directly
into the host cell. Shiga toxins (Stx) are one of those virulence factors and EHEC pathogens
produce up to two Stxs. Two distinct types of Stx exist, known as Stx1 and Stx2, of which there
are 3 and 7 known variants, respectively (Feng et al., 2014). Of these 10 Stx, only 6 of them
(Stx1a, Stx1c, Stx2a, Stx2b, Stx2c, Stx2d) cause human disease (Doyle et al., 2013). Melton-
Celsa and O’Brien (1998) outline six key facts that support Stx’s crucial role in the pathogenesis
of STEC: 1). Only Stx producing bacteria cause HUS; 2) In virto, human renal microvascular
epithelial cells are highly effected by Stxs, the same cells that are damaged in human HUS patients; 3) Renal tubular necrosis leading to death in mice fed STEC strains can be inhibited by
Stx antibodies; 4) The inoculation of STEC in rabbits resulted in vascular lesions in the bowel similar to those found in hemorrhagic colitis patients; 5) There is commonality between pigs and humans in the effects Stxs have on targeted small vessel endothelial cells; and, 6) The same vascular lesions found on the glomeruli in the kidney of human patients with HUS are found in greyhounds inoculated with Stx. Significant correlations can be drawn between the pathogenesis for the seven serotypes of E. coli and human consumption of contaminated foods. Sources of these organisms have been outlined and the low infectious dose (100 cells) describe the serious nature of this organism and its need to be eliminated from the food supply.
Comminuted and Fermented Products
The origination of protein for products such as dried and fermented summer sausages and salami is undoubtedly of animal origin. This skeletal muscle tissue is often regarded as sterile prior to harvest (Huffman, 2002), with the exception of imbedded lymph nodes present in intermuscular and subcutaneous fat (Romans, 1985). However, during the slaughter process, and
9 the subsequent removal of the hide and gastrointestinal tract, there are various vectors of contamination as employees process the animal into meat (Lahr, 1996). Even though efforts
employed by slaughter facilities are effective in reducing or eliminating potential pathogens
during the slaughter process, the potential for contamination is assumed for various products
including (bench) trimmings (USDA, 2011). As such, the USDA FSIS recommends subsequent
processing of bench trimmings for ready-to-eat products containing beef should include a documented process that provides a 5D process against Escherichia coli O157:H7 as well as six other non-O157 STECs (Nickelson II et al., 1996; Reed, 1995; USDA, 2017).
After the Washington-California outbreak of foodborne illness due to E. coli O157:H7 and the implementation of the new performance guidelines, Faith et al. (1998) conducted a study that showed a combination of fermentation to pH 4.8 and drying to a water activity (aw) of 0.92 only reduced various E. coli O157:H7 strains by 2.1 log CFU/g. The 5-log performance guideline was not met until one of the following processes was followed: 14 days of aging for a raw batter that had been tempered, frozen, then thawed; 21 days of aging for raw batter that had been frozen then thawed; or 28 days of aging for a raw batter that had been stored at a refrigerated temperature (Faith et al., 1998). Clavero and Beuchat (1996) reported sustained cell death of E. coli O157:H7 serotypes when salami was fermented to pH 4.6 and dried to an aw of
0.95 or 0.90 followed by 32 days of storage at 20°C; however, these reductions did not meet the
5-log performance guidelines. These studies, along with others (Glass et. al, 1992; Nickelson II
et al., 1996), indicate that traditional methods of salami production that include beef are not
sufficient to eliminate STECs.
Balamurugan et al. (2017) conducted a study investigating the differences between the
reductions of the big six non-O157 STEC and O157:H7 during the production of dry fermented
10 sausages. Using highly selective media (Sorbitol MaConkey agar) for the enumeration process,
Balamurugan et al. (2017) reported a 5 log CFU/g reduction for fermentation through drying (aw
= 0.85) for pork and beef salami inoculated with each of the 7 serotypes. In describing the
physiochemical properties of the product Balamurugan et al. (2017) reported that the pH of the
salami reached 5.0 in 24 hours and stabilized at 4.9 after 72 hours of production. However, upon further investigation of the pH decline, the pH of most of the batches tested obtained a pH much
lower than the values reported, some even showing pH values approaching 4.5. Although the information is poorly described, the use of 2.98% dextrose and corn syrup solids at an unknown
ratio could partially explain the pH decline to a much lower value than was reported for the
target pH. The lower pH combined with the use of highly selective media during enumeration
could lead to a misrepresentation of the microbial reductions actually obtained. Although, the
total reductions may be inconclusive or misrepresentative of the actual events occurring, the
conclusions made by Balamurugan et al. (2017) concerning the performance of the non-O157
STEC compared to O157 serotypes is novel. The authors did report that there were differences between the different serogroups at various stages of the production process. For example, E. coli O157:H7 had greater reductions during fermentation than serotypes O145, O26, O103, but had lower reductions than O45. However, ultimately their total process was able to produce a 5- log reduction for all serotypes examined.
Glass et al. (1992) indicated in the conclusion of his study that fermentation and drying alone were not sufficient in the reduction of E. coli O157:H7 without the use of additional intervention strategies. Therefore, additional non-thermal processing steps need to be explored to achieve the 5-log reduction of E. coli O157:H7 in dry and semi-dry fermented sausages that include beef. High pressure processing (HPP) is a non-thermal process that is currently being
11 employed for various ready-to-eat food products targeting microbial reduction of pathogens and
may hold promise for use in non- or low-thermally processed ready-to-eat salami containing
beef.
High Pressure Processing
Mechanism of bacterial inactivation
The complexity of high pressure on food systems begins with the principle of Le
Chatelier, which states that if a dynamic equilibrium is disturbed by changing pressure among
temperature, product concentration or reactant concentration, the equilibrium position of the
reaction changes to counteract that inequality. Therefore, if the pressure of a system is increased
the reaction of the macromolecules in that system will be shifted to a more compact state or
reduced number of total molecules and a lower energy state (Cheftel and Culioli, 1997). The
covalent bonds found in food systems are usually less affected than weak hydrostatic interactions
and may be explained by the low energy state of those covalent bonds (Cheftel and Culioli,
1997). Due to the isostatic and instantaneous distribution of pressure, it has been found that most
food products are not deformed upon the release of pressure, unless a significant volume of gas
is present in the product. Therefore, the traditional inactivation gradient found in thermally
processed products is not apparent in pressure processing, and the time of HPP is independent of
the size and diameter of the sausage (Cheftel and Culioli, 1997). This phenomenon could be
helpful in the inactivation of pathogens present in the center of a sausage without concern of
processing time and uniformity that would be found in a thermal application.
The full mechanism of inactivation of pathogenic bacteria remains elusive; however, the hydrophobic and electrostatic regions of the tertiary and quaternary structures of proteins are
12 extremely sensitive to pressure denaturation (Gross and Jaenicke, 1994; Heremans 1995).
Furthermore, the disruption of cell membranes has been shown to result in the inactivation of key
enzymes resulting in cell death (Erijman and Clegg, 1998; Ritz et al., 2000; Simpson and
Gilmour, 1997). Pressures as little as 100 MPa have been shown to be detrimental to DNA replication, and nuclear membrane functions, while pressures of 400–600 MPa have been shown to further impact the mitochondria and cytoplasm of cells (Shimada et al., 1993). Smelt (1998) describes the protein denaturation and membrane disfunction and a subsequent influx of protons causing major changes in intracellular pH as a major factor in the inactivation of pathogenic organisms during HPP. The addition of more acidic environments, and the subsequent availability of free protons such as those present in salami and summer sausage, could yield an improved, or synergistic role in the inactivation of bacteria.
Early High Pressure Processing Research
Prior to the discussion of HPP on fermented meats, the early research on HPP in various foods will be covered. The use of HPP in food matrices is of great interest, due to its ability to inactivate pathogenic bacteria under low temperature parameters (Cheftel and Culioli, 1997).
Early studies that covered the use of high pressure in meat and meat by-products examined its effects on tenderness and color with pressures up to 500 MPa (Carlez et al., 1995; Spanier et al.,
2000). In 1997, the first successfully high pressure processed food product placed in a U.S. test market was a guacamole product produced by Avomex. High pressure was reported to be used to inactivate enzymes in the avocado, as well as to kill bacteria without the use of heat or chemical preservatives, while having no effect on color, taste, or texture (Mermelstein, 1997). Additional research on buffered milk and raw chicken showed that a 5D inactivation of E. coli O157:H7 was achieved with pressures up to 700 MPa for 15 min (Patterson, 1995). However, the most
13 resistant strain, E. coli O157:H7 NCTC 12079, was able to be recovered after HPP. Bacterial
population reductions reported for various food substrates, with little impact on organoleptic
properties made the use of HPP ideal for inclusion in multiple hurdle technology for fermented
and dried meat products when used in conjunction with fermentation, low temperature cooking,
and/or drying to achieve the desired 5-log inactivation of STECs.
High Pressure Processing - Dried and Fermented Foods
Initial investigations of HPP on salami containing beef inoculated with E. coli began in
2010 when researchers started investigating the reductions of E. coli O103:H25, which was
recovered from a Norwegian outbreak associated with dry fermented sausage (Omer et al.,
2010). The pH of the salami tested in the study was approximately pH 4.77 and was dried to a
water activity of 0.85. After production, the sausage was subjected to HPP at 600 MPa for 10 min. Reductions of E. coli O103:H25 attributed to production (fermentation and drying) were approximately 2.00 log CFU/g, while reductions due to the HPP process were approximately
2.90 log CFU/g. Omer et al. (2010) concluded that the reductions of the HPP process of 600 MPa
for 10 min reduced approximately 3.0 log CFU/g while the production of the product itself
reduced 2.0 log CFU/g making a total process reduction of 5.0 log CFU/g. However, the data
indicate that confidence intervals were as low as 1.92 log CFU/g and 2.71 log CFU/g for sausage production and HPP, respectively. These results indicate that there is a potential for as low as
4.63 log CFU/g, which does not meet the 5D process suggested by USDA FSIS.
Gill and Ramaswamy (2008) conducted a study prior to Omer et al. (2010), where commercially manufactured beef and pork salami were purchased at a delicatessen, inoculated
with E. coli O157:H7 then subjected to HPP at 600 MPa for 3, 6, or 9 min. Upon initial
inoculation of the salami, the pH was reported as 6.30, while the water activity was 0.968. This
14 product reported a moisture to protein ratio (MPR) of approximately 4.5:1, which by the USDA
Standard of Identity, does not qualify as a summer sausage or salami product as the MPR is
required to be below 3.1:1 and 1.9:1, respectively (USDA, 2005). Upon initial observation of the
results the authors claimed a reduction of greater than 4 log CFU/g on cefixime tellurite sorbitol
MacConkey agar (CTSMAC) for samples pressurized at 600 MPa for 3 min. However, after 21
days of storage, the pressurized samples had similar counts to non-pressurized control samples.
These results indicate a sublethal injury occurred during HPP and the organisms were unable to
grow on the CTSMAC, selective media. Interestingly, although Gill and Ramaswamy (2008),
showed sustained cell life during extended storage, the authors concluded that the data indicate
that HPP can be effective in the reduction of E. coli O157:H7 on sliced ready to eat meats. It was
also concluded that the results should be confirmed by the inoculation of the salami batter prior
to production.
Porto-Fett et al. (2010) further investigated the efficacy of HPP on the reduction of
Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Trichinella spiralis in
pork genoa salami. Porto-Fett et al. (2010) used a low temperature fermentation (20-27° C) such
as those found in traditional Italian style salami and fermented using several types of
commercially available starter cultures (Bactoferm HPS, Saga 75, and Saga 444). During
fermentation, the pH decline reached as low as pH 4.56 but was not higher than pH 4.66, much lower than traditional southern Italian style salami. The lower pH reported by Porto-Fett et al.
(2010) was likely due to the inclusion of 1% dextrose in the sausage formulation. This research
identified two different drying regimens for sausages in a 65 mm diameter casing: A) 25 days of
drying, and B) a target water activity of 0.92. These drying regimens resulted in final water
activity of 0.884 and 0.926. Again, a selective agar was used for the enumeration of these
15 organisms after fermentation, drying, HPP, and storage. The data collected from this study
indicated that the fermentation to pH 4.6, drying for 25 days, and storage for 28 days only
reduced E. coli O157:H7 by 3 log CFU/g. However, the use of HPP in the same product for any
hold time up to 5 min reduced >6.9 log CFU/g when plated on a selective media. Porto-Fett et al.
(2010) further enriched samples that were below detectable levels throughout storage. While the
direct plating of samples of HPP genoa salami were below detectable levels, the enrichment of
samples still had viable organisms. Samples subjected to HPP for 5 min and immediately plated
were 100% positive after enrichment. When the same samples were stored for 28 d the
percentage of positives after enrichment declined to 50% positive. This data indicated that E. coli
O157:H7 is reduced or injured during the fermentation and drying stages of salami production,
and the subsequent HPP further kills or injures these cells below detection levels and the
continued storage of these products enables further cell death. While these results are
encouraging, Gill and Ramaswamy (2008) also proved that the use of selective media in the
enumeration of fermented, dried and HPP salami was ineffective in determining the true
reductions of E. coli O157:H7.
Balamurugan (2019), in Food Safety magazine describing unpublished data regarding
beef and pork salami high pressure processed at 600 MPa, stated that when salami was fermented
as low as 4.70 and dried to a aw of 0.85 reductions of 3.25 log CFU/g of E. coli O157:H7 were
observed and further reductions due to HPP were 2.42 log CFU/g. These results are similar to
those reported by Balamurugan et al. (2017) where, as previously mentioned, a 5 log CFU/g
reduction is shown. Interestingly, Balamurugan (2019) also reported that when products were
fermented to 4.72 and dried to an aw of 0.92 a reduction 1.53 log CFU/g was achieved. Further reductions were realized after HPP (MPa and time) for a total process reduction of 6.48 log
16 CFU/g. Although it is unclear if statistical analysis of these data were performed, it appears that all of the products reduced nearly 6 log CFU/g of the target organism, regardless of the product being dried to a lower or higher aw. An important point to note is that as the product got dryer the reductions achieved from HPP were reduced. However, reductions from fermentation and drying processes increase pH and aw decrease. Therefore, it is important to identify the optimal pH, aw,
pressure and time for each targeted product and organism.
Texture of Fermented Meats
Traditional Italian fermented meat products are typically categorized by region which has
a large influence on the organoleptic properties (Aquilanti et al., 2012). Northern Italian sausages
are typically associated with a sweeter taste, while those sausages from southern Italy have more
savory flavor profiles. While there is a vast array of regional categories, the European Union
identifies some of these varieties under their Protected Designation of Origin (PDO), Protected
Geographical Indication (PGI), and their Traditional Specialty Guarantee (STG). Some of these
designations or varieties identified by the EU in their PDO programs include Salame di Varzi,
Salame Sant’Angelo, and Soppressata Lucana. While most of these varieties are tagged with
specific regions of Italy, they share specific rheological properties and descriptors such as a soft
consistency, tender, and soft, respectively (Aquilanti et al., 2012). It is evident that the
rheological properties of these traditional Italian style salami products are important, as is the
measurement of these properties.
Sausage rheological properties are considered equally as important as the safety of the
product and are highly variable between categories of dried and fermented foods. The texture of
fermented and dried meat products are some of the most important attributes to consumers
17 (Claus, 1995). Texture is defined as the mechanical properties of the tissue, and is considered
independent of other organoleptic properties such as juiciness, and flavor (Stanley, 1979).
Stanley (1979) stated that any scientist investigating the texture of meat is tasked with producing
a method for the measurement of a perceived concept of tenderness, which is subjective in their
evaluation. The use of a well-trained sensory panel, whom are instructed on how to measure the
attributes of concern is considered a necessity for the assessment of the human response to
inconsistent meat products. Currently, there is no objective measurement that can replace the
human senses with regards to their organoleptic assessments. It should be stated that the use of
trained sensory panels is considered subjective and correlating trained sensory panels with
objective analysis can lead to erroneous results (Stanley, 1979). A method for assessment of
congruencies between subjective and objective data is examining the agreement between the two
measurements, as opposed to a correlation type evaluation. Sale (1960) also stated that the final
criterion for the determination of tenderness is the mouth, and that the use of objective
measurements is for relating the sensory information to objective findings.
One of the methods commonly employed for the measurement for the texture of sausage
products is texture profile analysis (TPA). Texture profile analysis was developed by the General
Foods Corporation Technical Center and is comprised of a 75% compression of a standardized
food product approximately 1-cm3, two times in a repeating manner (Bourne, 1979). General
Foods created the initial definitions of the texture parameters obtained from the force time curves obtained from their ‘Texturometer’. Hardness was defined as the peak force during the first compression; cohesiveness was defined as the ratio of the positive force area during the first and
second compression (A2/A1); springiness was defined as the recovery distance between the end
of the first compression and the start of the second compression; gumminess was a calculated
18 value of the product hardness and cohesiveness; and, chewiness was an additional calculated
value of the product hardness, cohesiveness, and springiness.
Sausage softness can be considered a product defect in certain dry fermented sausage
categories (Gimeno et al., 1999), while it is a desirable attribute in other product categories
(Melendo et al., 1996). Initial texture development occurs during the mixing of the raw materials
and the subsequent salt soluble protein extraction (Romans et al., 1994). Subsequent texture
development occurs during the fermentation and acidification stages of sausage production and is
primarily a result of protein coagulation and denaturation, often referred to as protein gelling
(Barbut, 2015). Protein gelation properties have been reported to increase the hardness of commercial chorizo de Pamplona by as much as 2 – 4-fold (Barbut, 2015). Fretheim et al. (1985) reported that the decline of pH below 5.5 of a 1% myosin solution at 5°C would spontaneously form a gel, which was attributed to acid-induced denaturation. Acid-induced gelation had a stiffness that was dependent on pH and reached a maximum at pH 4.5. The pH dependant gelation of myosin explains some of the texture differences observed in fermented sausages from different regions. The rheological properties that make Italian style salami soft can be attributed to the low acidity and high pH found in these varieties. This is further substantiated by the observations that at pH > 5.0 the myosin solutions remained dissolved, indicating an incomplete denaturation (Fretheim et al., 1985). Gelation of bovine myosin, the salt soluble protein that is extracted during the blending of salami, was shown to form a spontaneous gel in the presence of pH 4.0 buffers during dialysis (Hermanson et al., 1986). The formation of the gel structure was also demonstrated to be more sensitive to pH than it was to heat, while the orientation of the myosin fibers and the subsequent gel network formed, was dependent on the salt content of the solution (Hermanson et al., 1986). Ngapo et al. (1996) further showed the formation of a gel
19 structure of bovine myosin with a gradual decline in pH using glucono-delta-lactone (GDL). This
gel structure was measured for stiffness using a Youngs modulus, which measures the stress and
strain along an axis of measurement and provides a ratio that is interpreted as stiffness. The
increasing concentration of GDL in the solution cause a more rapid and extensive disruption of
the myofibrillar proteins creating an increase in hardness. Barbut (2005) demonstrated a
difference in a measure of texture called initial storage modulus (G’; defined as shear stress over
shear strain) between raw beef batters (pH 5.6), lactic acid bacteria (LAB) fermented beef (pH
4.60) and liquid lactic acid acidification (pH 4.62). Naturally, the raw beef batter had the lowest
value (13.1 kPa) followed by liquid acidified batters (26.7 kPa) and then LAB fermented beef
which had the greatest values for G’ (41.0 kPa). These data suggest that the slow acidification of
LAB fermentation allows for a more rigid gel structure like the ones observed during the
production of fermented sausages. The use of direct liquid acidification was used for the purpose
of detailing the negative quality effects, and the development of poor or no texture due to the
rapid protein denaturation that could occur (Barbut, 2005). Accelerated acidification methods
may cause protein clumping and loss of smooth textures found in LAB fermented sausage batters
when fermentation is hastened or uncontrolled (Barbut, 2015). Salami makers use these gelation properties for the monitoring of the fermentation process, by its organoleptic properties, specifically the stiffness of the sausage being an indicator of the development of texture during fermentation (Barbut, 2015).
Following the fermentation stages, the ripening or drying stages focus on the release of water from the protein gel matrix (Barbut, 2015). During ripening the systematic drying focuses on the removal of water from the center of the sausage resulting in the release of more tightly bound water. This release of water creates a denser product with a chewier structure due to
20 further denaturation and degradation from endogenous and microbial enzymes (Barbut, 2015).
While these two phenomenon seem counter intuitive, the hardening due to denaturation and the softening due to enzymatic degradation, salami makers often refer to the later stages of drying as the development of bite and or body using both of these properties to increase the firmness, but also not create a product that is hard and unpalatable (Barbut, 2015). Warnants et al. (1998) tracked the penetration force of fermented salami that was dried for 11 weeks, showing a greater than 2-fold increase during the first 3 weeks of drying and a subsequent numeric, but insignificant increase for the remaining 8 weeks. This is likely due to the concentration of products (proteins, fats, and spices) in the sausage and subsequent hardening, but also the enzymatic degradation, and softening that was simultaneously occurring. This can be further substantiated by Benito et al. (2005), where the evaluation of enzymes added to the salami batter were investigated by measuring the changes in myofibrillar proteins. While both control salami and salami with exogeneous enzymes showed a decrease in the amount of myofibrillar proteins over time, the salami with added protease enzymes had a quicker reduction for the same myofibrillar proteins. After the completion of drying, TPA was performed and the control salami was nearly twice as hard as the enzyme supplemented salami. Springiness and cohesiveness of the sausage remained unchanged between the two treatments, but both chewiness and gumminess were reduced by nearly half in the protease supplemented salami (Benito et al.,
2005).
Producers of fermented and (semi) dry sausages in the United States began cooking or heat treating their traditional Italian style products in the 1990s to ensure they were meeting the
Blue Ribbon Task Force’s guidelines for the reduction of E. coli O157:H7. The implications on the U.S. industry caused a shift in the texture of beef containing salami, deviating from the
21 traditional texture of the products prior to the outbreak. The textural changes during cooking are
described by Barbut (2005) where G’ is measured, showing that initial denaturation begins
around 45°C, and protein gelling is observed at 54°C. The subsequent heating beyond 54°C up to
70°C results in an exponential increase in G’. While these are the trends for cooking raw
unacidified batters, the heating of LAB acidified sausages is remarkably different. Barbut (2005)
showed that the heating of LAB fermented sausages from 30°C to 40°C resulted in a decrease in firmness as measured by G’, which is attributed to the softening and rendering of fat. Additional heating from 40°C to approximately 55°C resulted in no additional textural differences; however, heating beyond 55°C resulted in significant protein gelation. Although both products finished with similar G’ values, the texture of the products were different under light micrographs, such that the LAB fermented product showed a less dense structure than the nonacidified control
(Barbut, 2015). The structural differences were attributed to the prior fermentation and gelation
of the proteins prior to cooking, creating a less dense and softer structure than the control.
There are various processing parameters that can impact the texture and eating
characteristics of sausages. Particle reduction of both lean and fat sources, temperature of added
fat, and mixing or blending time are all factors that can have a significant impact on texture
(Barbut, 2015). Van’t Hooft (1999) was able to identify five key components influencing the
texture and bind of semi-dry fermented sausages: 1) chopping time with salt; 2) lean meat
temperature; 3) fat temperature; 4) sharpness of chopping knives; and, 5) application of vacuum
mixing to the batter prior to stuffing. It was determined that increased chopping time with salt,
higher lean and fat temperatures, dull knives, and vacuum mixing all increased the bind of the
sausage and therefore, the hardness (Van’t Hooft, 1999). However, there is an optimization that
should be attained where over mixing with salt, excessively warm lean and fat, combined with
22 dull knives, may smear the product creating an overly hard product with undesirable texture.
Barbut (2015) contradicted Van’t Hooft (1999) indicating that dull chopping blades had a negative impact on the particle definition and therefore the marketability of sausages. Barbut
(2015) further stated that dull knives may induce excessive protein extraction and a subsequent increase in water holding capacity. The increased water holding capacity may impede the release of water molecules during the drying stages, resulting in failure or nonuniform drying. It is demonstrated that although these factors can all influence the texture and drying of dry fermented products, there is an optimization that is required. Not enough protein extraction will result in poor texture, but too much extraction can result in inadequate drying. Barbut (2015) concluded that the manufacture of high-quality fermented products requires an excellent understanding of all processing factors and the interactions of the meat and non-meat ingredients, processing parameters, and the environment parameters after stuffing.
Literature on the rheological properties of high pressure processed fermented products is limited. However, HPP does have an effect on proteins, which is associated with a small change in volume (approximately 1%; Cheftel and Culioli, 1997). The covalent bonds in proteins are uncompressible and therefore are largely unaffected. However, the inter- and intramolecular interactions such as protein bound water or even the hydrogen bonding is often changed.
Breakdowns in the protein that can occur due to the application of high pressure include salt bonds and hydrophobic interactions (Cheftel and Culioli, 1997). This is substantiated by a study reporting pressure induced degradation of proteins in the I-band, M-line, and cleavage of the A- band (Suzuki et al., 1990). MacFarlane et al. (1988) also showed severe damage to the sarcomere of beef pressurized to 150 MPa for up to 24 hours but was unable to improve the tenderness of the cooked meat from the same samples. Breakdown of the sarcomere structure indicates that
23 although there may be some degradation of ultrastructure this has no impact on the tenderness of
the steaks. Cheftel and Culioli (1997) concluded that there is no direct evidence of tenderization
of meat processed using HPP under 30ºC. The authors further explain that the gelation of
proteins subjected to HPP is not fully understood, but myosin heavy chains are likely to be
involved. The evidence of gelation of proteins during HPP is not likely to impact a comminuted product that has already undergone gelation through a natural fermentation process. Although the
effects of HPP on texture of sausage products is quite limited throughout the literature, Mor-mur
and Yuste (2003) investigated the effects of HPP on commercially produced cooked sausage
texture where they reported no difference in the springiness, adhesiveness, hardness and force
cutting in sausage samples when pressurized at 500 MPa for 5 mins. However, the authors did
report differences in cohesiveness, stating that pressurized samples were more cohesive than the
unpressurized controls. The similarities in textures reported between the control and HPP
samples were attributed to the initial cooking of the sausage and the gelation of the proteins prior
to the pressurization. Omer et al. (2015) conducted a study on the effects of high pressure
processing of raw materials prior to comminution and salami production on sensory attributes.
This study conducted a triangle test using HPP and non-HPP to determine whether panelists
could differentiate between the texture. Panelists were able to detect textural differences between
pressurized and non-pressurized samples at two weeks of maturation, however, there were no
differences detected between HPP and non-pressurized samples after six weeks of maturation.
Omer et al. (2015) also stated that the panelists preferred the non-pressurized samples more than
those which had undergone HPP at 600 MPa after two weeks of maturation, and after six weeks of maturation, the inability to distinguish differences made the determination of preference impossible. While early texture differences were attributed to the treatment of raw materials, it is
24 unclear if the use of high pressure itself would have a major impact on texture when employed after the production of the salami. Pressurization of the raw materials prior to grinding and particle reduction as done in Omar et al. (2015) could have a negative impact on the protein structure of the raw material prior to stuffing and fermentation, creating a grainy or undesirable texture that would otherwise not be present in a fermented and coagulated or gelled salami batter that is subjected to HPP.
Meat Color, Lipid Oxidation, and High Pressure Processing
Meat color is determined by the chemical state of myoglobin. Myoglobin is a water- soluble protein constructed of 8 alpha-helices commonly named A-H, with a functional group, the heme ring, with a centrally located iron atom (AMSA, 2012). The iron atom is embedded in the center of the heme group by four bonds to nitrogen residues and has two additional binding sites with one attaching to histidine on the myoglobin molecule and the sixth site is a reversible binding site that depicts the color of the meat by conformational changes that occur when the ligand binding site is vacant (deoxymyoglobin), has elemental oxygen (oxymyoglobin), carbon monoxide (carboxymyoglobin), nitric oxide (nitric oxide metmyoglobin, nitric oxide myoglobin, or nitric oxide hemochrome). The oxidation state of this iron molecule also determines the shape and color of the molecule, such that, an oxidized state (ferric iron) produces a brown color
(AMSA, 2012). For cured meat products, the conversion of myoglobin to nitric oxide hemochrome, or nitrosylhemochrome is an important aspect of color stability. The use of nitrate or nitrite in fermented and dried products, along with a reducing agent such as bacteria or
-2 - erythorbate, reduces NO3 to NO2 which is believed to react with the heme iron before reducing to NO and binding to the heme ring irreversibly (AMSA 2012). After heat or acid is applied to
25 the network, the resulting molecule is in the stable cured pink color indicative of
nitrosylhemochrome.
Bak et al. (2019) summarized in a comprehensive review of the effects of high pressure
on color of animal proteins five phenomenon occurring: 1) severe changes to the secondary
structure of the myoglobin molecule; 2) structural changes to the heme ring; 3) although some aggregation occurs there is reversible denaturation; 4) the oxidation of oxymyoglobin to metmyoglobin is decreased by increasing pressure in high pH products; and, 5) the oxidation of
nitrosylhemochrome is protected by pressure. Although these are generalizations made about the
myoglobin molecule across various studies using whale, dog, and horse, similar attributes were
noted by researchers studying the effects of HPP on fresh beef products (Carlez et al., 1995).
Carlez et al. (1995) showed that beef processed as low as 200 MPa showed an increase in
lightness (L* values), while a* values, indicating redness, remained unchanged up to 400 MPa.
The authors attributed these findings to the oxidation of ferrous iron to ferric iron contributing to
the increase of metmyoglobin and the increase of a more greyish-brown color. It was also noted
that the removal of oxygen, by an oxygen scavenger prevented the formation of metmyoglobin.
Although the prior discussed changes were noted in fresh beef products, the curing of
beef products using 1% NaCl and 100-200 ppm NaNO2, followed by high pressure processing
stabilized redness values (a* values) up to 500 MPa (Carlez et al., 1995). The authors did
however report an increase in the L* values, stating that all pressurized samples were pink and a
whitening effect was present and not possible to prevent. Rubio et al. (2007) reported that in a
dry cured beef product known as “Cecina de Leon” that there was no change due to HPP in any
of the measurements for color (L*, a*, or b*) when pressurized at 500 MPa for 5 mins and
subsequent storage up to 210 days. Feiner (2006) attributed the protection of cured meat color to
26 the denaturation of the nitrosylmyochromogen, to nitrosylhemochrome and the subsequent
stability of the latter molecule. It is evident that there are changes in color due to HPP, however,
the color of cured meat products has continually been reported as stable in the sparse amount of
literature available.
Lipid stability is often reported as being negatively affected by increasing the pressure
and hold times during HPP for fresh meat products such as beef, pork, chicken, and fish
(Ohshima et al., 1993; Cheah and Ledward, 1996; Ma et al., 2007; McArdle et al., 2010).
Additionally, there has been shown to be a degree of further lipid degradation in dry-cured
Iderian hams upon the application of high pressure (Andres et al., 2006). However, other studies
have reported no difference in lipid stability from products when the oxygen has been removed,
such as those in vacuum packaging (Cheah and Ledward, 1996). Marcos et al. (2007) performed
a study evaluating the lipid stability of low acid fermented sausages and found no difference
between pressurized and non-pressurized samples, and attributed the lack of differences to the
late ripening stages of sausage completing any sort of lipid degridation prior to treatment.
Although there are reports of HPP having an influence on the lipid stability, there is also
documentation that HPP may not have an effect on the stability of fermented and long dried
salami products, especially when high pressure processed and stored in oxygen excluded environments.
Conclusions
It is clear from a thorough review of literature that texture is extremely important to the production of fermented and (semi) dry sausage products. A multitude of processing parameters
including, blending time, raw material temperature, knife blade sharpness, vacuuming the batter
27 prior to stuffing, fermentation degree and temperature, as well as the degree of drying and its schedule play an intricate role in the development of the textures of these products. The literature also indicated that traditional manufacturing techniques used to produce these products are inadequate in the control of Escherichia coli O157:H7 and the non-O157 STECs of concern.
There are also knowledge gaps due to the methods of enumeration used for the target organisms in the current literature which need further clarification. Namely, the greatest concern is that the reductions of shiga-toxin producing E. coli may be over represented or poorly depicted.
Therefore, the evaluation of HPP in the production of low acid low temperature fermented and dried sausages and salamis containing beef should be further investigated, for not only their impacts on the textural properties that are so crucial to the identity of these products, but also the safety with regards to the various shiga-toxin producing E. coli serotypes of concern in comminuted ready-to-eat beef products produced in the United States.
28
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38
CHAPTER 3: TEXTURE OF FERMENTED SUMMER SAUSAGE WITH DIFFERING pH,
ENDPOINT TEMPERATURE, AND HIGH PRESSURE PROCESSING
39 Macc Rigdon, Harshavardhan Thippareddi, Robert W. McKee, Chevise L. Thomas, Alexander M. Stelzleni To be submitted to Meat and Muscle Biology Abstract:
The objective was to evaluate the quality and texture of all-beef summer sausages produced with varying degrees of fermentation, endpoint cooking temperatures, and high pressure processing
(HPP) hold times. Across three replications, sausages were fermented and cooked to: pH 4.6 and thermally processed to 54.4ºC with smokehouse chilling (A), pH 5.0 and thermally processed to
54.4ºC with smokehouse chilling (B), pH 5.0 and thermally processed to 54.4ºC with rapid ice bath chilling (C), pH 5.0 and thermally processed to 48.9ºC with rapid ice bath chilling (D), and pH 5.0 and thermally processed to 43.3ºC with rapid ice bath chilling (E). After chilling, the sausages were sliced, layered, vacuum packaged, and subjected to HPP at 586 MPa for 0, 1, 150, or 300 s. Post HPP, the sausages were evaluated for objective color (n=9), lipid oxidation (n=9), water activity (n=9), texture profile analysis (n=15), sensory analysis (n=9), and proximate analysis (n=9). Neither process (combination of pH and endpoint temperature) or HPP affected lipid oxidation (P=0.45 and P=0.69, respectively). Process A resulted in a lighter color (P<0.01)
compared to the other process treatments. Additionally, process A was less red (P<0.01) than all other process treatments, and processes D and E were the most red (P<0.01). The texture profile analysis and trained sensory analysis indicated that as endpoint temperature increased, so did sample hardness (P<0.05). Springiness, cohesiveness, and gumminess decreased (P<0.05) as the endpoint temperature decreased. Although springiness and gumminess increased (P<0.05) with longer HPP hold times, the panelists were unable to detect differences between the samples with longer hold times. The use of HPP at 586 MPa for up to 300 s may be incorporated into process validation for semi-dry beef summer sausages without causing adverse effects on color or texture.
Keywords: Beef, Texture, Sausage, Sensory, Quality, Shelf-stable
40
INTRODUCTION
Consumers in the United States are seeking specialty food products and traditionally
processed foods (Ilbery and Kneafsey, 1999; Guerrero et al., 2009) that historically have not
been processed at high temperatures. However, the Escherichia coli O157:H7 outbreak in
commercially produced dry-cured salami (CDC, 1995) caused industry representatives to outline
a course of action aimed at mitigating the E. coli O157:H7 risk in such products (Reed, 1995).
This team of representatives, known as The Blue Ribbon Task Force of the National Cattlemen’s
Beef Association, recommended several methods aimed at eliminating further foodborne
illnesses associated with dried and fermented beef products (Nickelson II et al., 1996) by
reducing Eschrichia coli O157:H7 by 5-logs. The majority of the methods developed centered
around thermal processing (Nickelson II et al., 1996), which deviated from traditional production methods, in which low temperature fermentation and drying were a customary practice.
Alternatively, The Blue Ribbon Task Force also suggested that hurdle technology could be used in leu of, or in combination with, thermal processing for the reduction of pathogens (Nickelson II et al., 1996).
High pressure processing (HPP) is a technology that can aid in the reduction of pathogenic bacteria (Patterson et al., 1995; Cheftel and Culioli, 1997) and may be a viable option to meet the food safety performance standards requested by regulatory agencies (USDA, 2017) without requiring prior approval for use (USDA, 2012). However, inspection program personnel must verify that the hazard analysis supports the use and parameters of HPP (USDA, 2012). The performance standards suggest that establishments producing dry, fermented, and salt cured products containing beef have scientific documentation demonstrating a 5D process for E. coli
O157:H7 (USDA, 2017). High pressure processing subjects the food substrate to extreme
41 pressures (200 - 700 MPa; Campus, 2010) through forced water displacement (Cheftel and
Culioli, 1997). Since pressure is created from forced water, the distribution of that pressure is isostatic, pseudo-instantaneous, and should not cause gross deformation, provided there is not a significant amount of gas present in the food or package (Cheftel and Culioli, 1997). Although
there should be no product deformation, the pressures achieved during HPP are enough to
influence molecular changes and interactions including weak hydrostatic interactions, hydrogen
bonding, and hydrophobic bonds; as well as, increasing protein denaturation, aggregation, and
gelation (Messens et al., 1997; Campus, 2010). While the pressures created through HPP have
been shown to reduce pathogens, including in dried and fermented meat products (Hugas et al.,
2002; Morales et al., 2006; Omer et al., 2010; Scheinberg et al., 2014), little is known about the
influence of HPP on the texture and color of fermented dry and semi-dry meat products containing beef. High pressure processing has been shown to variably alter meat texture, tenderness, color, and oxidation stability dependent on rigor state, pressure setting, use of nitrate/nitrite, cooked state, and packaging type (Simonin et al., 2012). Therefore, the objective of this study was to investigate the influence of HPP in combination with total production process parameters (pH and endpoint cooking) on the texture and color of an all beef fermented, semi-dry sausage product.
MATERIALS AND METHODS
Beef Trimmings Procurement and Batter Processing
For each of three replicates, 20 kg of beef trimmings were procured from the University of Georgia Meat Science Technology Center (MSTC), ground through a 12.7 mm plate, and
42 blended to a target fat percentage of 10%. After blending, the trimmings were ground (4.76 mm)
and placed into a reverse action mixer (Model A-80, Koch, Kansas City, MO). The ground
trimmings were subsequently mixed for 1 min with a typical summer sausage seasoning blend
including 2% salt (Mortons, Chicago, IL), 0.8% or 0% dextrose (to achieve target pH values of
4.6 or 5.0, respectively, after fermentation), 2.25% spices typical to summer sausage flavoring,
156 ppm sodium nitrite, and 551 ppm sodium erythorbate. The batter was then inoculated with
10 g of thawed Pediococcus acidilacti starter culture (Kerry, Rochester, MN) diluted in 236 ml
of deionized water (23 ± 2ºC) and mixed for an additional 2 min. The prepared batter was then
placed into a vacuum stuffer (Vemag Robot 500, Reiser, Canton, MA), stuffed into 5.08 cm dia.
(11 chubs) fibrous mahogany casings (Visko Teepak, Kenosha, WI), and clipped. After stuffing,
5 chubs were allocated to texture profile analysis, 3 chubs were allocated for color and lipid
oxidation, and 3 chubs were allocated for sensory analysis. All chubs were then hung on a smoke
cart, and placed in an Alkar smokehouse (Model 8770-4-12000, Lodi, WI). Sausages were allowed to ferment until the target endpoint pH was achieved. The fermentation step was 43.3ºC dry bulb with an 85% relative humidity (RH). After fermentation, the dry bulb temperature was increased to 62.8ºC with a relative humidity of 85% for 30 mins and then increased again to
73.9ºC with 95% RH for the remainder of the cooking cycle and the sausages were cooked to an internal temperature of 43.3ºC, 48.9ºC, and 54.4ºC followed by ice bath chilling. Two processing treatment groups, one from pH 4.6 and one from pH 5.0, were cooked to 54.4ºC and cooled using a smokehouse cold water shower for 10 min followed by refrigerated chilling methods to simulate industrial chilling. The endpoint pH and cooking parameters were arranged into 5 different total processes including: fermented to pH 4.6 and thermally processed to 54.4ºC with smokehouse chilling (A), fermented to pH 5.0 and thermally processed to 54.4ºC with
43 smokehouse chilling (B), fermented to pH 5.0 and thermally processed to 54.4ºC with rapid ice
bath chilling (C), fermented to pH 5.0 and thermally processed to 48.9ºC with rapid ice bath
chilling (D), and fermented to pH 5.0 and thermally processed to 43.3ºC with rapid ice bath
chilling (E). These 5 total processes follow the NCBA Blue Ribbon Task Force’s “Option 4”
with regard to total process validation, in which the total process is the treatment applied to each
group of sausage samples (Nickelson II et al., 1996). After chilling, samples from all treatment
groups were packaged and exposed to various HPP hold times at 586 MPa. High pressure
processing typically ranges from 300-600 MPa for meat pasteurization dependent on the time
and temperature during pressurization and the organism being targeted (Aymerich et al., 2008;
Omar et al., 2010). Pressures close to 600 MPa have been shown to be the most effective against
pathogenic E. coli spp. (Gill and Ramaswamy, 2008; Omer et al., 2010; Simonin et al., 2012;
Hygreeva and Pandey, 2016) and are commonly used for meat products in current industrial
practices (B. Cook, personal communication, 2017).
High Pressure Processing
After thermal processing and chilling, the sausages destined for texture profile analysis
3 2 (TPA) were cut to 5-cm lengths and vacuum packaged (B-620 series; 30–50 cm O2/m /24 h/101,325 Pa/23°C; Cryovac Sealed Air Corporation, Duncan, SC). The remaining chubs were sliced to 3.18 mm using a Hobart meat slicer (Model HS9, Hobart, Troy, Ohio), shingle packed, and vacuum sealed (Cryovac Sealed Air Corporation, Duncan, SC). The packages were then
transported (0 ± 2°C; 170 km) to Universal Pasteurization (Villa Rica, GA) and high pressure
processed at 4±2°C and 586 MPa for 0, 1, 150, and 300 s.
Proximate Analysis
44 Samples from different HPP hold times were not different (P > 0.05) from each other for proximate analysis, therefore, HPP hold time was composited by process treatment within each replicate to determine moisture and crude protein for the determination of the moisture to protein ratio (M:P). Total lipids were determined from the raw meat block. All analyses were performed in duplicate (C.V. < 10).
Moisture was determined using disposable aluminum pans which were dried at 100ºC in a forced-air oven (Fisher Scientific, Pittsburg, PA) overnight and equilibrated for 10 min in a desiccator. Pans were weighed and 2.0 ± 0.1 g of homogenized sample was dried in duplicate at
100ºC for 18 h (Soderberg, 1991). Samples were removed from the oven and allowed to cool for
10 min in the desiccator. Percent moisture was calculated following:
% moisture = [1-(dry sample weight/wet sample weight)] x 100%
Crude protein was determined using a Nitrogen auto-analyzer (Leco FP-528 Nitrogen analyzer,
Leco Company, St Joseph, MI) for the determination of N content (0.1 ± 0.05 g) and was expressed as percent crude protein (N content x 6.25). Total lipid content of the raw meat block was analyzed using wet tissue lipid extraction as outlined by Folch et al. (1957). Additionally, water activity was measured using an Aqualab water activity meter (Aqualab 4TE, Pullman,
WA) immediately upon returning from high pressure processing. Sausage pH was measured immediately after the fermentation step and was performed using a 1:10 dilution method in deionized water with an Oakton pH meter (Vernon Hills, IL; AMSA, 2012).
Lipid Oxidation
Lipid oxidation was performed using the rapid, wet method of thiobarbituric acid reactive species (TBARS) following the methods of Sinnhuber and Yu (1958) and Buege and
45 Aust (1978), as outlined by AMSA (2012). Lipid oxidation was expressed as mg of
malonaldehyde/kg meat.
Objective Color
Subsequent to HPP treatment, product was transported to the MSTC under refrigeration and vacuum-packaged slices were removed from the package and stacked 5 slices thick, making
3 stacks. Objective color was preformed using a Hunter-Lab MiniScan EZ (Hunter Associates
Laboratory, Reston, Virginia) with illuminant A and a 10° viewing angle with a 32 mm aperture.
Prior to use, the colorimeter was standardized with white, black, and saturated red tiles.
Commission Internationale de l’Eclairage (CIE) L* (Lightness), a* (Redness), and b*
(Yellowness) were measured in triplicate and averaged. Cure color fading values were calculated
using the reflectance at isobestic wavelength ratios of R570:R650 (AMSA, 2012).
Sensory
Trained sensory analysis was approved and conducted under Institutional Review Board
no. STUDY00005493. An 8-member trained panel (AMSA, 2012) was used to evaluate the
organoleptic properties. Samples were removed from the vacuum package, and six slices were
cut into fourths and served on coded serving plates for consistent mastication orientation. Each
panelist received 3 slices of each sample. A maximum of 16 samples were served each day
across two sessions, with 3 h between the start of each session. Sliced quarters were served with
a glass of deionized water and salt-free soda crackers to cleanse panelist palates between
samples. Panelists sampled and recorded traits in a dark room with positive air flow and
illuminated with red lighting to mask color. Once plated, samples were given to all panelist at the
same time through a breadbasket with individual walls separating each panelist.
46 Slices were evaluated on the textural descriptors firmness, cohesiveness, springiness, and
gumminess using a 15-cm line scale with anchors at 0, 7.5, and 15 (0 indicates the least intense
and 15 is the most intense). The lines were anchored on both ends using various food products as
a reference points for the 0 and 15 cm points. The center of each line scale was anchored at 7.5
cm with a commercially available all beef summer sausage. The extremity anchors for hardness,
springiness, and cohesiveness were bologna, Now and Later candies, and SweeTARTS candies,
respectively, for 0 cm, and beef jerky, marshmallows, and Now and Later candies, respectively,
for 15 cm. The benchmarks for gumminess were SweeTARTS candies for 0 cm and refrigerated
gummy bears for 15 cm. The panelists were comprised from a standing trained beef sensory
panel. Panelists were further screened across 4 training sessions for their ability to identify and
calibrate themselves to the standard anchors. After panelist screening and calibration, two
additional sessions directed toward the measurement of various commercial meat products were
included to ensure that panelists were accurately identifying each attribute consistently.
Texture Profile Analysis (TPA)
Chilled samples (4ºC) were removed from the package and a hand-held coring device was used to extract a 1.27-cm diameter by 1.27-cm long core from the geometric center of each
sausage chub. Each core was centered on the platform of a TA-XT Texture Analyzer (Texture
Technologies Corp., Hamilton, MA) and compressed to 50% of its original height by a 75 mm
compression probe and a 25 kg load cell with a crosshead speed of 3.33 mm/s and a trigger force
of 5 g. A 2-cycle sequence was used with a 5 s pause between compressions to allow for sample
recovery. The TPA values were obtained from the graphed force-time curve output by Exponent
Connect (Texture Technologies Corp., Hamilton, MA) for hardness, cohesiveness, springiness, gumminess, and chewiness.
47 Statistical Analysis
Data were analyzed using Proc Mixed of SAS (version 9.3) as a randomized split plot
design, where total process batch (pH and cooking temperature combination) was the whole-plot and chub within process treatment batch was the sub-plot. High pressure processing times were included as fixed effects and replication was included as a blocking factor. Chub within process batch was considered the experimental unit and slice(s) or core was considered the observational unit. Means were separated using the LSmeans pdiff option for main effects and the interaction of process treatment by HPP hold time. Means were considered different at α < 0.05.
RESULTS AND DISCUSSION
Proximate Analysis
The descriptive data for the sausage treatments are presented in Table 3.1. The percent fat
in the raw meat block was not different between fermentation endpoints or replicates (P = 0.17
and P = 0.32, respectively). As expected, due to target endpoint pH and dextrose in the
formulation, differences between fermentation endpoint pH for the treatments were achieved (P
< 0.01). There were no differences for the moisture to protein ratio (MPR) attributed to process
treatment or HPP (P = 0.68, P = 0.63, respectively). High pressure processing times did not
affect the water activity (P = 0.60) of the product. Both process treatments cooked to 54.4ºC and traditionally chilled (A and B) had similar (P = 0.51) water activities. However, Processes B, C, and D were also similar to each other (P ≥ 0.06) with Process C and D having less (P ≤ 0.05) water activity than Process A. Process E had a greater (P ≤ 0.01) water activity than all other
48 processes. Although there were differences among processes for water activity all processes were
between 0.96-0.97 with minimal variation among replicates.
All treatments met USDA-FSIS requirements for a summer sausage product with pH 5.0
or less and MPR of 3.1 or less (USDA, 2011). Similar to the differences in the water activity
reported in the current project, Porto-Fett et al. (2010) described the water activity of fermented,
cooked, and HPP semi-dry fermented meat, with differences of 0.06 as subtle. The small
magnitude of differences in water activity combined with similar product MPR would not be
expected to affect overall summer sausage quality.
Lipid Oxidation
Process treatment nor HPP hold time impacted lipid oxidation (P = 0.23, P = 0.53 respectively). Due to the lack of differences for lipid oxidation, data is not presented in tabular form however, samples ranged from 0.41 ± 0.01 to 0.45 ± 0.01 with an average of 0.4 mg
malondialdehyde/kg sausage. Previous research has shown that high pressure processing can
have a negative effect on lipid oxidation and lipid stability of meat and meat products. The effect
of high pressure processing on lipid oxidation has been reported to be dependent on pressure
setting, temperature during high pressure processing, lipid amount and saturation index, product
packaging, and whether the meat product was further processed (ready-to-eat, cooked, cured,
dried) or fresh (Campus 2010; Simonin et al., 2012; Hygreeva and Pandey, 2016). High pressure
processing can have a negative effect on the lipid stability of fresh meat samples including beef,
pork, and chicken (Cheah and Ledward, 1996; Ma et al., 2007; McArdle et al., 2010) both
immediately after HPP and continuing through post processing storage; however, in these studies
the HPP parameters included temperatures in excess of 20°C which was approximately 16°C
above those used in the current study. Maintaining product in vacuum packaging or modified
49 atmosphere packaging after HPP has been suggested to decrease the impact of HPP on lipid oxidation. Sun et al. (2017) reported no difference in lipid oxidation when vacuum packaged beef steaks were subjected to HPP at 450 and 600 MPa for up to 15 min. Utama et al. (2017) found that vacuum packaged beef steaks subjected to HPP at 600 MPa had greater lipid oxidation than steaks exposed to HPP at lower pressures, and that lipid oxidation potential increased out to 6 d postprocessing. Contrary to the current research, Banerjee et al. (2017) reported differences in lipid oxidation between untreated mutton patties and patties processed at
200 and 400 MPa. Beltran et al. (2004) reported that samples subjected to pressurization under elevated temperatures had greater lipid oxidation than non-pressurized samples that were heated and refrigerated over an extended shelf life. Additionally, there was no difference in lipid oxidation between pressurized and non-pressurized samples on day 1, indicating similar findings as the present study where no difference was found in pressure treated samples (Beltran et al.,
2004). However, after 6 and 9 days of storage pressure treated thighs had higher amounts of oxidation products present. In the current study, the use of nitrite in the formulation as a lipid stabilizer prevents any further lipid oxidation during storage (Freybler et al., 1993).
Objective Color Analysis
There was not a process by HPP time interaction (P ≥ 0.08) for any objective color measure (L*, a*, b*, and R570:R650 for cured color fading); therefore, the main effects of process and HPP time are shown in Tables 3.2 and 3.3, respectively. For L* values, Process A was lighter (P < 0.05) than all other treatment processes which were similar to each other (P ≥ 0.17).
High pressure processing time did not impact sausage lightness (P = 0.29). Measurements of a* showed Processes D and E, while similar to each other (P = 0.47) were more red (P < 0.05) than all other treatments. Treatments cooked to pH 5.0 and 54.4ºC (Processes B and C), regardless of
50 chilling, were similar in redness (P = 0.97) but redder (P < 0.01) than Process A. High pressure processing times indicated that as time at 586 MPa increased, redness decreased (P < 0.05).
However, the difference between 0 and 300 s was only 0.56 units.
The treatments cooked to a lower degree of doneness (Processes D and E) were similar to
each other (P = 0.64) and more yellow (P < 0.01) than the other processes which were similar (P
≥ 0.77) to each other. Samples processed under high pressure processing for any amount of time,
while similar to each other (P ≥ 0.06), had lower b* values than the non-HPP controls (P < 0.05).
Cured color fade (R570:R650) was greater (P < 0.01) in the sausages from Process A than all others, which were similar to each other (P ≥ 0.06). Furthermore, sausages subjected to HPP for
300 s had a more faded color (P < 0.01) than sausages that were not subjected to HPP. Sausages that underwent HPP for 1 or 150 s were similar (P ≥ 0.18) to those that were exposed to HPP for
0 and 300 s, respectively. Although there were differences regarding cured color fading, it is important to note that the greatest magnitude of difference was 0.01 units and likely not discernable to the naked eye.
It is likely that the majority of the overall color differences are due to the manufacturing process (pH and endpoint temperature) rather than the effects of HPP, as indicated by the differences found for L* between the processes but not the HPP times. Additionally, for a* there was a greater magnitude of difference objectively observed as a result of process compared to the slight differences noted as a result of HPP. Although admittedly HPP potentially imparts differences in redness as (indicated by its main effect of a*) the difference is minimal and likely undetectable in subjective observation. Banerjee et al. (2017) found no difference between pressurized and non-pressurized mutton patties for the color parameters L*, a*, and b*, further
substantiating the impact of cooking treatments over HPP on color parameters. Beltran et al.
51 (2004) reported changes in the redness of minced chicken thighs; when pressure (500 MPa) was applied, the sample redness decreased. In addition, the authors reported a decrease in yellowness, although the decrease in yellowness was less remarkable than the decrease in redness. Although differences were unable to be detected in a* values with magnitudes greater than the current study (1.4 - 3.4 units), it is also important to note the elevated temperatures (5°C - 50°C) of the environment in which their chicken patties were processed compared to those of the current study (Beltran et al., 2004).
Sensory Analysis
An interaction for process by HPP time (P ≥ 0.58) was not observed for any of the texture attributes evaluated by the trained sensory panelists. The panelists were able to distinguish process effects for firmness (P < 0.01), springiness (P < 0.01), cohesiveness (P <
0.01), and gumminess (P < 0.01; Table 3.4). As expected, there was a stepwise increase in the firmness and gumminess detected by panelists as temperature and fermentation intensity increased where Process A was the firmest and gummiest (P < 0.01) product. The sausages fermented to pH 5.0 and cooked to 54.4ºC, regardless of chilling method, were similar in firmness and gumminess to each other (P = 0.40). However, only the rapidly chilled samples were similar to the treatments cooked to 48.9ºC (P = 0.15). Processes D and E were also similar to each other (P = 0.08), but Process E was less firm and gummy than Process C (P < 0.01).
Panelists also noted a stepwise increase in springiness as fermentation and cooking intensity increased (Table 3.4). Processes A and B were similar to each other (P = 0.09).
However, Process A was springier than C, D, and E (P < 0.05). Process B was similar to C and D
(P ≥ 0.31) but springer than E (P < 0.01). Finally, Processes C, D and E were all similar to each other (P ≥ 0.05). The sausages fermented to pH 4.6 and cooked to 54.4°C were rated the most
52 cohesive (P < 0.01), followed by the treatments fermented to pH 5.0 and cooked to 54.4°C and
48.9°C, regardless of chilling method, which were similar to each other (P ≥ 0.20). The sausages
fermented to pH 5.0 and cooked to 43.3°C were the least cohesive but similar (P > 0.39) to the
sausages fermented to pH 5.0 and cooked to 48.9°C. The trained panelists were unable to detect
difference in texture attributes in the sausages exposed to different HPP times (P ≥ 0.46; Table
3.5).
Others have shown that increased temperatures were associated with a less tender and
firmer product (Mathevon et al., 1995; Pohlman et al., 1997). Other attributes evaluated by the
panelists in the current study showed similar results to that of firmness, where an increased
cooking intensity led to increased springiness, cohesiveness, and gumminess. Additionally,
Marcos et al. (2007) reported that trained sensory panelists, when evaluating the hardness and
gumminess of low acid fermented sausages, were unable to distinguish differences between
pressurized and unpressurized fermented sausages. Mor-Mur and Yuste (2003) performed
triangle tests comparing pressurized fermented sausages to heat treated fermented sausages, finding that in all cases panelists preferred the pressurized samples over the heat-treated samples,
describing them as less grainy and more uniform in texture. Although only a small amount of
literature is present, the inability of trained sensory panelists to discern textural differences
among HPP processing times should give processors complete confidence that selecting longer
HPP times would not affect sensorial perceptions.
Texture Profile Analysis
Similar to the trained sensory analysis, there was not a process by HPP time interaction
(P = 0.47) for TPA. The main effects for TPA due to cooking treatment and HPP time are
presented in Tables 3.6 and 3.7, respectively. The differences among processes for hardness of
53 the sausages when measured by TPA followed a similar trend as did the sensory firmness.
Process A was harder (P < 0.01) than the other processes. The treatments fermented to pH 5.0 and cooked to 54.4ºC, regardless of cooling method, were similar (P = 0.47) in hardness;
however, only Process C was similar (P = 0.13) to D. Additionally, Processes D and E were similar (P = 0.17), even though E was less hard (P < 0.01) than C. Similar to sensory analysis, hardness as measured by TPA, was not impacted by HPP time (P = 0.10).
The sample recovery in height after the first compression and before the second compression is an indication of springiness. The treatments cooked to 54.4ºC, regardless of pH or cooling method, were similar to each other (P ≥ 0.08) but springier (P < 0.01) than those cooked to 43.3ºC. Processes A, C, and D were also similar (P ≥ 0.09) in springiness but springier
(P < 0.01) than Process E. Unlike sensory analysis, HPP hold time did influence springiness (P <
0.05). Hold times of 300, 150, and 0 s were similar to each other (P ≥ 0.19), while samples held
for 300 and 150 s were springier (P < 0.01) than those held for 1 s. There was not a difference (P
= 0.09) in sample springiness between 0 and 1 s hold times.
The sausages from Process A were more cohesive (P < 0.01) than the others, and all other
processes were similar to each other (P ≥ 0.18). Cohesiveness was not affected by HPP time (P =
0.62), again following the same trend as recorded from sensory analysis.
Gumminess, a product hardness as it relates to its ability to stay together, was influenced
by both cooking treatment and HPP hold time (P < 0.01). The sausages fermented to pH 4.6
(Process A) were the gummiest (P < 0.01), while the products fermented to 5.0 and cooked to
54.4ºC, regardless of cooling method, were similar to each other (P = 0.20) but gummier (P <
0.05) than the sausages cooked to 43.3ºC. Processes C and D were similar (P = 0.10); however, only Process D was similar to E (P = 0.55). High pressure processing hold times of 300 and 0 s
54 were similar to each other (P = 0.17). The treatments receiving 150 s of hold time during HPP
were less gummy (P < 0.05) than those receiving 300 s of hold time, even though 150 s of hold
time was similar (P > 0.06) to both 0 and 1 s.
Chewiness showed that there was both a process and HPP hold time main effect (P <
0.01). The measurements for chewiness followed the same trend for process as those for gumminess, where Process A was the chewiest (P < 0.01) and Processes D and E were similar to each other (P = 0.14) and less chewy (P < 0.05) than the other treatments. Furthermore,
Processes B and C were similar to each other (P = 0.06). Treatments receiving 300 s HPP were chewier (P < 0.05) than the other treatments. Sausages subjected to high pressure processing for
0 and 150 s were similar (P = 0.68) in chewiness and chewier (P < 0.05) than samples processed for 1 s.
Hardness results performed as expected; as fermentation and degree of doneness
increased, so did the hardness of the sausage. These results were in agreement with sensory
panelist observations of hardness/firmness, springiness, cohesiveness, and gumminess.
Furthermore, the instrumental texture analysis agreed with sensory panelist evaluations, such that
HPP hold times did not have an impact on hardness and cohesiveness. Mor-Mur and Yuste
(2003) also reported no difference between HPP for 300 s at 500 MPa and non-pressurized
controls in texture measurements of hardness. However, it was also reported that there was no
difference in the springiness of the same samples. The findings were attributed to the industrial
cooking process causing gelation within the sausage, and the subsequent pressure processing
having only the ability to induce exudation, minimally effecting texture. Interestingly enough,
pressurization parameters used in the study included temperatures of 65°C, well above the
heating parameters used in the current study (Mor-Mur and Yuste, 2003). This indicates that a
55 further increase of protein gelation and subsequent hardening may have occurred during the pressurization process. Marcos et al. (2007) also reported an increase in cohesiveness, chewiness, and springiness during the pressurization of low acid fermented sausages; however, the parameters used (400 MPa at 17°C for 10 mins) were above the temperatures used during the ripening process, indicating an increase in the ultimate temperature of the sausage. As noted in this study by the difference, between treatments C, D, and E, the ultimate temperature end point does have an effect on sausage texture. Although there are very few manuscripts regarding the texture of low acid fermented sausages subjected to HPP, the data shows that while there may be an effect on sausage texture, the main focus is on the product ultimate temperature.
Conclusions
As expected, summer sausage products that received less rigorous fermentation and thermal processing were less firm, springy, cohesive, and gummy than summer sausage products fermented and cooked to industry validated endpoints (pH 4.6 and thermally processed to
54.4°C). Interestingly, the use of high pressure processing at 586 MPa for up to 300 s did not influence the ability of trained sensory panelists to differentiate among the products even though there were slight difference for springiness, gumminess, and chewiness when evaluated by objective texture profile analysis. Although there were some differences for objective color attributed to cooking treatment and high pressure processing hold times, the differences were minimal and likely would not influence consumer preference. The current study suggests that high pressure processing at 586 MPa for up to 300 s may be incorporated into a food defense plan without impacting sensory and texture attributes.
56 Acknowledgments
This work was supported by grant no. 2012-68003-30155 and grant no. 2011-68003-
30012 from the USDA National Institute of Food and Agriculture, Agriculture and Food
Research Initiative Foundational Program.
57
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63 Table 3.1: Least squares means (± SEM) for proximate analysis parameters of all beef summer sausage fermented and cooked to varying degrees of doneness Water Activity by Cook Temperature Moisture:Protein by Cook Temperature Fat Content pH 54.4°C T1 54.4°C 48.9°C 43.3°C 54.4°C T1 54.4°C 48.9°C 43.3°C (%) High 0.96 ± 3.0 ± 4.60 ± 0.02b 8% ------Acid 0.001c 0.07 Low 0.96 ± 0.97 ± 0.97 ± 0.97 ± 3.0 ± 3.0 ± 3.1 ± 3.1 ± 5.03 ± 0.02a 11% Acid 0.001bc 0.001b 0.001b 0.001a 0.07 0.07 0.07 0.07 abcMeans within a heading with different superscripts differ; α < 0.05.
1T indicates traditional smokehouse and cooler chilling methods. All remaining samples were chilled using rapid ice water chilling.
64 Table 3.2: Least squares means for objective color scores for all beef summer sausage cooked to
varying degrees of doneness
Process1 L a* b* Fade2 Process A 53.21a 23.83c 14.58b 0.28a Process B 52.24b 24.28b 14.57b 0.27b Process C 52.43b 24.27b 14.56b 0.27b Process D 51.96b 24.62a 14.89a 0.27b Process E 52.12b 24.54a 14.92a 0.27b Standard Error 0.24 0.08 0.04 0.002 abcMeans within a column with different superscripts differ at α < 0.05.
1Processes: A – pH 4.6 at 54.4°C with traditional smokehouse chilling, B – pH 5.0 at 54.4°C with traditional smokehouse chilling, C – pH 5.0 at 54.4°C with rapid ice bath chilling, D - pH
5.0 at 48.9°C with rapid ice bath chilling, E - pH 5.0 at 43.3°C with rapid ice bath chilling.
2Values determined by the following equation using isobestic wavelengths: fade = 570 nm / 650
nm (AMSA, 2012).
Table 3.3: Least squares means for objective color scores for all beef summer sausage high pressure processed at 586 MPa for varying hold times
High Pressure L* a* b* Fade1 Hold Time (sec) 0 52.13 24.56a 14.84a 0.27b 1 52.27 24.38ab 14.71b 0.27ab 150 52.48 24.24bc 14.67b 0.27ab 300 52.69 24.05c 14.60b 0.28a Standard Error 0.22 0.07 0.04 0.002 abcMeans within a column with different superscripts differ at α < 0.05.
1Values determined by the following equation using isobestic wavelengths: fade = 570 nm / 650
nm (AMSA, 2012).
65
Table 3.4: Least squares means for the sensory analysis of all beef summer sausage cooked and fermented to varying degrees of doneness
Process1 Firmness2 Springiness2 Cohesiveness2 Gumminess2 Process A 8.4a 8.0a 8.6a 8.5a Process B 7.8b 7.7ab 8.1b 7.9b Process C 7.7bc 7.6bc 8.1b 7.9bc Process D 7.4cd 7.5bc 7.9bc 7.5cd Process E 7.1d 7.2c 7.8c 7.3d Standard Error 0.1 0.1 0.1 0.1 abcMeans within a column with different superscripts differ at α < 0.05.
1Processes: A – pH 4.6 at 54.4°C with traditional smokehouse chilling, B – pH 5.0 at 54.4°C with traditional smokehouse chilling, C – pH 5.0 at 54.4°C with rapid ice bath chilling, D - pH
5.0 at 48.9°C with rapid ice bath chilling, E - pH 5.0 at 43.3°C with rapid ice bath chilling.
2Firmness, springiness, cohesiveness, and gumminess were measured on a 15 cm line scale with anchors at 0, 7.5, and 15 cm indicating least intensity, average intensity and greatest intensity, respectively.
66 Table 3.5: Least squares means for the sensory analysis of all beef summer sausage high pressure processed at 586 MPa for varying hold times
High Pressure Hold Firmness1 Springiness1 Cohesiveness1 Gumminess1 Time (sec) 0 7.6 7.6 8.0 7.7 1 7.6 7.5 8.1 7.7 150 7.7 7.7 8.1 7.9 300 7.8 7.6 8.2 8.0 Standard Error 0.1 0.1 0.1 0.1 1Firmness, springiness, cohesiveness, and gumminess were measured on a 15 cm line scale with anchors at 0, 7.5, and 15 cm indicating least intensity, average intensity and greatest intensity, respectively.
67 Table 3.6: Least square means for the instrumental texture analysis of all beef summer sausage cooked and fermented to varying degrees of doneness
Process1 Hardness (N) Springiness (%) Cohesiveness Gumminess Chewiness Process A 66.3a 63.5ab 0.324a 21.1a 1340a Process B 55.4b 65.0a 0.292b 16.1b 1053b Process C 54.0bc 63.8ab 0.286b 15.4bc 986bc Process D 50.9cd 62.7b 0.286b 14.4cd 907cd Process E 48.2d 60.2c 0.296b 14.1d 854d Standard Error 1.6 0.6 0.006 0.4 30 abcMeans within a column with different superscripts differ at α < 0.05.
1Processes: A – pH 4.6 at 54.4°C with traditional smokehouse chilling, B – pH 5.0 at 54.4°C with traditional smokehouse chilling, C – pH 5.0 at 54.4°C with rapid ice bath chilling, D - pH 5.0 at 48.9°C with rapid ice bath chilling, E - pH 5.0 at 43.3°C with rapid ice bath chilling.
68
Table 3.7: Least squares means for the instrumental texture analysis of all beef summer sausage high pressure processed at 586 MPa for various hold times
High Pressure Hardness (N) Springiness (%) Cohesiveness Gumminess Chewiness Hold Time (sec) 0 55.7 62.9ab 0.296 16.aab 1040a 1 52.5 61.8b 0.293 15.3c 949b 150 54.9 63.4a 0.295 16.1bc 1025a 300 56.8 64.0a 0.302 17.1a 1098a Standard Error 1.5 0.5 0.013 0.4 27 abcMeans within a column with different superscripts differ at α < 0.05.
69
CHAPTER 4: MEETING THE PERFORMANCE STANDARD FOR ESCHERICHIA COLI
O157:H7 AND SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) LETHALITY IN
BEEF SUMMER SAUSAGE USING HIGH PRESSURE PROCESSING
70 Macc Rigdon, Alexander M. Stelzleni, Chevise L. Thomas, Sanjay Kumar, Harshavardhan
Thippareddi To be submitted Meat and Muscle Biology Abstract:
The USDA-FSIS performance standards require that manufacturers of fermented beef sausages validate their processes to achieve a 5-log reduction of Escherichia coli O157:H7 and Shiga toxin producing E. coli (STEC). Most processors rely on fermentation to a low pH and thermal
treatment to achieve the lethality performance standard. However, this process alters the
sensorial characteristics of traditional fermented sausages. An alternative method to achieve the
lethality treatment, using high pressure processing (HPP) during the manufacture of summer
sausage with higher pH (5.0) and with minimal thermal treatment was evaluated. Sausages
inoculated with greater than 9.1 log CFU/g of E. coli O157:H7 and 8.9 log CFU/g of STEC,
were fermented to pH values of 4.6 or pH 5.0. Subsequently, fermented sausages were heated to
endpoint temperatures of 54.4°C, 48.9°C, or 43.3°C to the total process treatments of (A) pH 4.6
thermally processed to 54.4°C with smokehouse chilling, (B) pH 5.0 thermally processed to
54.4°C with smokehouse chilling, (C) pH 5.0 thermally processed to 54.4°C with rapid chilling,
(D) pH 5.0 thermally processed to 48.9°C with rapid chilling, and (E) pH 5.0 thermally
processed to 43.3°C with rapid chilling. The product was processed (HPP) at 586 MPa for a hold
time of 1s, 150 s, or 300 s, along with a non-treated (no HPP) control. All treatments subjected to
HPP for 150 and 300 s reduced (P≤0.05) E. coli (O157:H7 and STEC) populations by >5.0 log
CFU/g and >7.5 log CFU/g, respectively. The use of HPP allows for the production of more mild
beef summer sausage (pH 5.0 and a mild thermal treatment of 43.3°C) while still achieving
USDA-FSIS performance standards for lethality.
Keywords: Beef, Safety, Sausage, Escherichia coli, High-pressure, Shelf-stable
71 INTRODUCTION
Historically, dried and fermented meat products were regarded as safe for human consumption due to their use of multiple biological hurdles such as acidification, salt, competitive exclusion, nitrite/nitrate, drying, and sometimes even thermal treatment. However,
after a multistate food-borne outbreak of Escherichia coli O157:H7 in California and
Washington, which was associated with dried and fermented salami (CDC, 1995), the National
Cattlemen’s Beef Association (NCBA) assembled a team of industry representatives to address
the control of E. coli O157:H7 and prevent future outbreaks associated with the organism
(Nickelson II et al. 1996). This team of representatives, known as the Blue Ribbon Task Force,
recommended 5 options to prevent future outbreaks. Option 4 was described specifically, as any
combined process that demonstrates a collective 5D control of E. coli O157:H7 with precise
documentation. Subsequently, Levine et al. (2001) reported that USDA FSIS sampled over 3,400
samples of dried and fermented meat products for E. coli O157:H7 from March 1995 to
December 1999, none tested positive for the pathogen. There is no literature evaluating the
reduction of E. coli O157:H7 in non-thermally processed mildly fermented sausages (pH > 4.99)
that successfully met the guidelines set forth by the Blue Ribbon Task Force and USDA FSIS.
Processors of products, using similar parameters (pH > 4.9 with a low degree of thermal
processing or no thermal processing) currently operate under the in-plant validations described
by Option 4 due to the lack of performance in the scientific literature.
Currently there are studies that include mild fermentation and cook and hold type
procedures along with aggressive fermentation to a low ultimate pH in conjunction with the use
of high pressure processing (HPP; Calicioglu et al., 1997; Porto-Fett et al., 2010). While these
methods are effective at creating a 5D process, consumers desire specialty food products (Ilbery
72 and Kneafsey, 1999; Guerrero et al., 2009) such as more traditional mildly fermented and low
temperature processed products, whose processes do not meet the precise documentation
required by Option 4. High pressure processing is a technology currently being used in food
production to improve the safety of raw fruit juices, deli meats, and other high water activity
foods. During HPP, food products are subjected to 500 – 700 MPa of pressure by forced water
displacement with the objective of eliminating pathogenic organisms. High pressure processing
inactivates potential pathogens through oxidative burst and increasing pathogen sensitivity to
reactive oxygen (Aertsen et al, 2005). Reactive oxygen species are produced by the organism in
times of stress (Wuytack et al., 2003), such as during fermentation and drying in salt cured
products. Therefore, the objective of this study was to evaluate the fate of pathogenic strains of
E. coli O157:H7, O26, O45, O103, O111, O121, and O145 in mildly fermented, low temperature
thermally processed beef summer sausages using HPP.
MATERIALS AND METHODS
Culture Preparation
Across 3 replications, five rifampicin resistant (100 µg/ml; Sigma Chemical Co., St.
Louis, MO) strains of E. coli O157:H7 (USDA-FSIS 011-82, ATCC 43888, ATCC 43889,
ATCC 43890 and USDA-FSIS 45756) and 6 kanamycin resistant (100 µg/ml; Sigma Chemical
Co., St. Louis, MO) serotypes of non-O157:H7 Shiga-toxin producing E. coli (STEC; H30
[serotype O26:H11], B395 [serotype O111:H7], CDC 96-3285 [serotype O45], CDC 90-3128
[serotype O103:H2], CDC 97-3068 [serotype O121], and CDC 83-75 [serotype O145:HNM]) were used. Each isolate was maintained in glycerol stock solution at -80°C prior to use. Each isolate was individually incubated on Sorbitol-MacConkey agar with its respective antibiotic at
37°C for 20 ± 2 h and maintained at 4°C until needed. Prior to incubation each serotype was
73 confirmed by polymerase chain reaction and gel electrophoresis. One loopful of each isolate was
transferred into individual 10 mL tubes of tryptic soy broth (TSB) plus 2.5% glucose with 100 ppm of the respective antibiotic and incubated at 37°C for 24 ± 2 h. After the initial incubation, 1 ml of culture was aseptically transferred into individual 1 L TSB solutions with 2.5% glucose and its respective antibiotic. After incubation at 37ºC for 18 h, inoculated strains were individually collected by centrifugation at 7200 x g and 4ºC for 10 min. After centrifugation, the
supernatant was decanted, and cells were re-suspended in 10 mL of sterilized deionized (DI)
water. Isolates were combined into two separate solutions of O157 and non-O157 strains. Two
DI water washes were used to remove the residual antibiotic from the cultures. After the final
wash step, the pellet was resuspended in 70 mL of DI water prior to overnight storage (4 ± 2 ºC)
and inoculation.
Beef Trimmings Procurement and Batter Processing
Sausage production was performed following the methods outlined in Chapter 3 with
modifications. Each replicate consisted of 4.5 kg of beef trimmings blended to 10% fat and
ground (Biro Model G58483, St. Louis, MO) through a 4.76 mm grind plate. After grinding, the
meat was placed into a reverse action mixer (Model A-80, Koch, Kansas City, MO) and mixed with a seasoning block consisting of 2% salt (Mortons, Chicago, IL), 1% or 0.4% dextrose (for a targeted pH of 4.6 or 5.0, respectively), 2.25% spices typical to summer sausage flavoring, 156 ppm sodium nitrite, and 551 ppm sodium erythorbate. A commercially available Pediococcus acidilacti starter culture (Kerry, Rochester, MN) was used for a natural fermentation. Ten grams of 12 log CFU/g starter culture was diluted in 236 ml of distilled water and mixed with the meat and seasoning for a total of 3 min. The prepared batter was then placed in plastic bags and transported to a BSL 2 laboratory for inoculation and stuffing.
74 Inoculation and Processing
Prior to inoculation, one chub from each treatment was stuffed (Mighty Bite, LEM West
Chester, Ohio) into 5.08 cm fibrous mahogany casings (Visko Teepak, Kenosha, WI) and tied closed for proximate analysis. The remaining batter (approximately 2.25 kg) was spread out in plastic trays lined with wax coated butcher paper approximately 1-2 cm thick. Both inoculum
were dropped onto the flattened meat batter using a pipette, and hand mixed using double latex
gloves and following aseptic guidelines. Following the pipetting of both O157:H7 and non-
O157:H7 cocktails the batter was placed into the stuffer and one chub per treatment was stuffed
and hung on a smoke cart and placed in an Alkar smokehouse (Model 8770-4-12000, Lodi, WI).
Sausages were allowed to ferment until end point pH was reached with a dry bulb temperature of
43.3ºC and 95% relative humidity (RH). The remainder of the cooking schedule and parameters followed the procedures outlined in Chapter 3. Breifly, dry bulb temperatures were increased to
62.8ºC with a relative humidity of 85% for 30 mins followed by a dry bulb temperature of
73.9ºC with 95% RH for the remainder of the cooking cycle. Each total process treatment was removed from the smokehouse and placed in an ice water bath for chilling after they reached their endpoint cooking temperatures of 43.3ºC, 48.9ºC, and 54.4ºC. One additional treatment cooked to 54.4ºC was removed from the smokehouse and placed into a cooler with ice to simulate industrial chilling in a cooler. These parameters were arranged into 5 treatment groups:
pH 4.6 heated to 54.4ºC with industrial chilling simulation (process A), pH 5.0 heated to 54.4ºC
with chilling simulation, 54.4ºC with rapid chill, 48.9ºC with rapid chill, and 43.3ºC with rapid
chill (processes B, C, D, and E respectively). After chilling, samples were processed for high
pressure processing.
75 High Pressure Processing
After thermal processing and chilling each inoculated sausage chub was cut so that
approximately 6 cm was removed from each end then, 5 slices were hand cut to approximately 3
mm and the slices were individually vacuum packaged for high pressure processing. One slice
from each treatment was assigned to one of 4 HPP times: 0, 1, 150, or 300 s with one slice being
sampled immediately for post-thermal enumeration. Vacuum packaged samples were then
bagged together by HPP time, vacuum sealed, and placed in a third bag which was also sealed.
All four sample bags were then shipped (4 ± 2°C) overnight to the University of Nebraska,
Lincoln to be high pressure processed at 4 ± 2°C and 586 MPa for their designated time and then returned overnight. Pressures up to 600 MPa are commonly used in the industry (B. Cook, personal communication, 2017), as they have been shown to be effective against pathogenic E. coli spp (Gill and Ramaswamy, 2008; Omer et al., 2010; Simonin et al., 2012; Hygreeva and
Pandey, 2016).
Microbial Sampling
Upon return, the samples were unpackaged from their HPP bags, individually placed in sterile filter stomacher bags with 25 mL of 0.1% peptone water (Difco, BD, Sparks, MD), and stomached for 90 s. After stomaching 19.8 mL was removed from the stomacher bag and placed in sterile dilution tubes with 9.9 mL aliquoted to 2 tubes: 1 tube for rifampicin resistant O157 strains and 1 tube for kanamycin resistant non-O157 strains. The following procedure was performed for both rifampicin and kanamycin for their respective organisms: serial dilutions
(1:10) were performed using 0.1% peptone water containing 100 ppm of antibiotic in the final concentration. Samples were plated in duplicate on 3M aerobic plate count film (APC) with 1
76 mL aliquots and incubated at 37°C for 48 h. Raw counts were log transformed for statistical
analysis and reporting.
Proximate Analysis
Moisture was determined from pre-inoculated samples using a forced air oven (Fisher
Scientific, Pittsburg, PA). Aluminum pans were placed in a forced air oven at 100ºC for 48 h and then placed in a desiccator until used. Two gram from each sample was measured in duplicate and placed into one of the aluminum pans. The pans were then placed in the forced air oven at
100°C for 18 h (Soderberg, 1991). After oven drying, the samples were moved to a desiccator and allowed to equilibrate and cool for 10 min. Percent moisture was calculated as follows:
% moisture = (wet sample weight - dry sample weight/wet sample weight) x 100
Crude protein was determined using a nitrogen auto-analyzer (Leco FP-528 Nitrogen analyzer,
Leco Company, St Joseph, MI) for the quantification of N content (0.1 ± 0.05 g) and multiplied by 6.25 to be expressed as percent crude protein.
Water activity was read on each total process by HPP hold time treatment combination using an Aqualab water activity meter (Aqualab 4TE, Pullman, WA) upon return from HPP.
Sausage pH was taken immediately after the fermentation step by directly probing the sausage for pH measurement and again after the product cooled using a 1:10 dilution method in deionized water using a Hanna Instruments Edge pH meter with a general-purpose glass body bulb probe (Hanna Instruments, Smithfield, RI; Koniecko, 1979).
Statistical Analysis
A completely random block experimental design was used to evaluate the populations of
Escherichia coli O157:H7 and STECs during the processing of summer sausage to varying pH
(2), thermal processing parameters (3), and HPP time (4) and their effects on the reductions
77 during the production process. For analysis, the total process was evaluated, which combined
final pH and thermal processing/cooling parameters. Microbial sampling was performed on the
uninoculated meat batter, inoculated meat batter, fermented sausage, after chilling prior to
shipment, and after HPP. Microbial counts were transformed and reported as log CFU per gram of sample. Data were analyzed using analysis of variance by PROC GLM of SAS v. 9.4 (SAS
Institute, Cary, NC). Means were separated using Tukey’s studentized range test and means were considered different at α < 0.05.
RESULTS AND DISCUSSION
Sausage Proximate Analysis
After sausage thermal processing, neither water activity readings, nor moisture to protein ratio values for the finished product treatments were different (P = 0.06 and P = 0.38, respectively; Table 4.1). Sausage pH immediately after the fermentation step as measured by direct probing was 4.63, and 5.00 for high dextrose (1%) and low dextrose (0.4%) sausages, respectively. However, during the initial cooking step, the pH continued to drop, yielding a difference in ultimate pH that was slightly lower than what was targeted for all cooking treatments (P < 0.01; Table 4.1). Although treatment combinations were originally described as
4.6 and 5.0, it should be noted that the ultimate pH of the treatments were 4.5 and 4.8 respectively. In a similar study conducted by Calicioglu et al. (1997), summer sausage products were produced to target pH of 4.6 and 5.0; however, in their study, the ultimate pH ended at 4.5 and 4.9, respectively. It is common during the fermentation of products to surpass the target due to continued fermentation during the come up heating step. In the current study, the lactic acid bacterial culture was not killed until it reached a temperature of approximately 130ºC.
78 Microbial Sampling of Escherichia coli O157:H7
The raw meat blocks were sampled prior to inoculation, and no rifampicin resistant organisms were detected by direct plating. The subsequent inoculation yielded 9.1 log CFU/g of
E. coli O157:H7 in the process A batter (Table 4.2). After fermentation to pH 4.6 populations of
E. coli O157:H7 decreased 1.6 log CFU/g. Although these reductions were associated with the post fermentation pH values, it is important to note that the total fermentation was unable to be recorded, as the pH continued to drop after the fermentation step and into the cooking steps.
While it is common to associate fermentation to a particular pH with an approximate log reduction in microorganisms, this is typically not an appropriate assessment of the effectiveness of the total fermentation process, which can be difficult to assess entirely. Thermal process A finished with an ultimate pH of 4.50 and 54.4°C thermal endpoint, which had a total reduction of
6.3 log CFU/g (P < 0.01), exceeding the 5D process required by USDA FSIS. Further safety could not be ensured by the use of HPP for hold times of 150, and 300 s yielding additional reductions of 1.7 log CFU/g, and 2.0 log CFU/g numerically, although not statistically different
(P > 0.11). Combining total process A with HPP for 150 or 300 s does; however, show total reductions of 7.1 log CFU/g and 8.1 log CFU/g, respectively, achieving greater reductions than that suggested by USDA FSIS.
Total processes B – E batters yielded 9.1 log CFU/g of E. coli O157:H7 after initial inoculation. These batters were fermented to pH 5.0 and E. coli O157:H7 populations decreased
0.6 log CFU/g through fermentation (P = 0.57). Through subsequent thermal treatment and chilling process B finished with a pH of 4.84 and 54.4°C thermal endpoint with simulated industrial cooling, reducing E. coli O157:H7 populations more than 5.0 log CFU/g. Additional control against E. coli O157:H7 can be ensured with the use of HPP for 1, 150, or 300 s (P <
79 0.01). These HPP hold times increase the reductions of target organisms by 1.5 log CFU/g, 1.7
log CFU/g, and 2.8 log CFU/g, respectively. Consequently, the total reductions of E. coli
O157:H7 for total process B with pressurization at 586 MPa for 1, 150, and 300 s were 6.6 log
CFU/g, 6.8 log CFU/g, and 7.9 log CFU/g, respectively. Reductions noted from total process C were not different (P ≥ 0.37) than those in total process B for process reductions or for subsequent HPP hold times (P ≥ 0.85) indicating the method of chilling did not affect the reduction of the pathogen.
The pH of sausage produced in process D was 4.82 after thermal processing at 48.9°C.
This yielded a process reduction of 1.2 log CFU/g prior to high pressure processing (P > 0.27).
Pressurization of process D to 586 MPa for 150 and 300 s showed a total decrease of 6.6 log
CFU/g and 7.8 log CFU/g in populations of E. coli O157:H7, respectively (P < 0.01). Holding samples for 1 s at 586 MPa did improve reductions (P < 0.05) of O157:H7 serotypes to 3.7 log
CFU/g, but did not meet USDA FSIS guidelines.
Sausages cooked to 43.3°C and fermented to pH 4.84 (process E) resulted in 0.6 log
CFU/g reductions for the total process prior to HPP. Escherichia coli O157:H7 populations on the day of chilling and after shipment were not different (P = 0.54); however, after 1 s of HPP, populations had a total process reduction of 1.6 log CFU/g (P > 0.10). Longer hold times of 150 and 300 s reduced populations of E. coli O157:H7 in total process E, 6.1 and 8.0 log CFU/g, respectively (P < 0.01).
Microbial Sampling of Non-O157:H7 Escherichia coli
Initial inoculation of STECs yielded 8.9 log CFU/g for total process A and 9.0 log CFU/g for processes B – E (Table 4.3). After fermentation, process A fermented to pH 4.6 and achieved
80 a 1.5 log CFU/g reduction (P = 0.18) while processes B – E fermented to pH 5.0 and reduced 0.6 log CFU/g of STECs (P = 0.56).
As previously mentioned, total process A fermented to a final pH of 4.5 and cooked to
54.4°C, which reduced a total of 6.3 log CFU/g of STEC on the day of sampling (P < 0.01).
After shipment and return of samples, process A continued its lethality and reduced a total of 7.6
log CFU/g organisms. After HPP hold times of 1, 150, and 300 s reductions, below detectable
limits were achieved (> 8.3 log CFU/g; P < 0.01).
Total process B reached a final pH of 4.84 and was cooked to 54.4°C with a total process
reduction of 4.9 log CFU/g on the day of chilling (P < 0.01); however, reductions observed
before and after shipment were not different (P = 0.86) for process B, yielding reductions less
than 5.0 log CFU/g. High pressure processing sausages from this process for 1 s reduced 7.8 log
CFU/g of non-O157 STEC (P < 0.01). Continuing to hold the pressure for 150, or 300 s reduced
populations of non-O157 STEC below detectible levels for a total reduction of greater than 8.3
log CFU/g (P < 0.01). Fermentation to a final pH of 4.84 and cooking to 54.4°C with rapid ice water chilling did not create differences (P < 0.05) from total process B on the day of sampling or after shipment of the product without HPP. After HPP for 1 s, total process C reduced 6.4 log
CFU/g of STEC (P < 0.01), while holding for 150, and 300 s reduced the populations below detectible levels, reducing greater than 8.3 log CFU/g of the target organism (P < 0.01).
Total process D was fermented to a final pH of 4.82 and thermally processed to 48.9°C.
This process caused a reduction of 1.2 log CFU/g on the day of chilling (P = 0.28) and the reduction did not change (P = 0.96) after shipment. High pressure processing sausages from process D for only 1 s had a total process reduction of the pathogen 3.2 log CFU/g (P < 0.01); however, after 150 s, HPP was able to reduce 7.4 log CFU/g (P < 0.01). Continuing to hold the
81 sausage at 586 MPa for 300 s reduced the populations of the pathogen below detectible levels,
reducing greater than 8.3 log CFU/g (P < 0.01).
After fermentation, continued cooking to 43.3°C allowed the pH to drop to 4.84 and
yielded a reduction of 0.7 log CFU/g (P = 0.49). However, after shipment of the samples total
process E is the only sample that did not numerically decrease after shipment, increasing only
0.4 log CFU/g. High pressure processing total process E for 1 s yielded a total decrease of non-
O157 STEC of 1.6 log CFU/g (P = 0.12). However, holding sausages from process E for 150 or
300 s created reduced populations by 5.6 log CFU/g and 8.1 log CFU/g, respectively (P < 0.01).
Discussion
Benito et al. (1999) suggested that pressure resistant strains of E. coli O157:H7 can be isolated from clinical patients of food-borne illness, and that those strains were also heat resistant, giving rise for concern that the use of HPP in conjunction with low temperature cooking may not be effective. Additionally, several studies have shown that the growth phase of cells during HPP can have an effect on the baroprotective properties of microorganisms
(Baccuss-Taylor, 2015; Cheftel, 1995; Benito et al. 1999). These baroprotective properties can be due, in part, to an increase in heat shock proteins over that of exponential phase cells (Aertsen et al, 2005); this is important due to the extended time of natural fermentation. Aertsen et al
(2005) states that this phenomenon can be important in the production of high pressure processed foods in conjunction with mild heat treatment. Contrarily, the data presented in the current study indicate that any baroprotective properties that may have been present in these cultures are not sufficient enough to overcome the pressures encountered for the HPP hold times evaluated.
Omer et al. (2015) reported that the use of HPP at 600 MPa for 10 min on sausages fermented to pH 4.7 and dried to a water activity of 0.85 obtained an approximate 2 log CFU/g
82 reduction attributed to the fermentation and drying alone, while pressurization attained an additional 3 log CFU/g reduction of E. coli O103. These researchers showed a 5 log CFU/g total process reduction from a drier product than that of the current study. Porto-Fett et al. (2010) also reported reductions of E. coli O157:H7 as high as 6 log CFU/g attributed to pressurization alone for genoa salami with pH values ranging from 4.6 – 4.86 and a water activity ranging from 0.88
– 0.94 units. This indicates that although the use of HPP on some products may not be effective, the pressurization of sausage products in the presence of small amounts of acid drastically increases the effectiveness, even at lower water activities, reaching and exceeding the suggested guidelines set by USDA FSIS (2017).
Similar findings to the current study were noted when sausages were fermented to a target of pH of 4.6 and 5.0 and then cooked to 54.4°C, showing that the product continued to ferment during the cooking stages to a final pH of 4.5 and 4.9, respectively (Calicioglu et al.,
1997). Calicioglu et al. (1997) reported reductions of E. coli O157:H7 populations for those parameters to be similar to those in the current study, achieving fermentation reductions of 1.3 log CFU/g and 0.3 log CFU/g for pH declines to 4.5 and 4.9, respectively. Reductions reported by Calicioglu et al. (1997) for sausages fermented to pH 4.9 and then cooked to 54.4°C and chilled were less than those in the current study; however, the current study demonstrated reductions at or below the required 5D for all thermal treatments fermented to pH 4.8. The use of
HPP at 586 MPa for as little as 150 s on sausages fermented to a pH as high as 4.8 and thermally processed as low as 43.3°C exceeded the USDA FSIS requirement for the 5D reduction of E. coli O157:H7 and achieved a 6D or greater process. Sausages produced using any of the total processes described in this study with the use of 300s of HPP at 586 MPa ensures further safety, exhibiting an 8D process. These parameters can be used in a complete process following Option
83 4 of the Blue Ribbon Task Force (Nickelson II et al., 1996) to achieve a validated process of
production under USDA-FSIS inspection.
Conclusions
Collectively, the findings presented in the current study provide a method for processors of low temperature thermally processed, and mildly fermented summer sausage, to ensure
microbiological safety against pathogenic E. coli. These results indicate that thermal processes as
low as 43.3°C and fermentation endpoints as high as pH 4.8, in conjunction with HPP at 586
MPa for 150 s or greater, can meet a 5D process suggested by USDA FSIS.
Acknowledgments
This work was supported by grant no. 2012-68003-30155 and grant no. 2011-68003-
30012 from the USDA National Institute of Food and Agriculture, Agriculture and Food
Research Initiative Foundational Program.
84
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injury induced in Salmonella enterica serovar Typhimurium by heat and by different
nonthermal treatments. Journal of Food Protection 66(1):31-37.
87 Table 4.1: Least square means of proximate analysis of summer sausage products
Cooking Treatment1 Process A Process B Process C Process D Process E SEM P-Value pH 4.50b 4.84a 4.84a 4.82a 4.84a 0.03 <0.01 Titratable Acidity (%) 1.66a 1.22b 1.18b 1.22b 1.19b 0.05 <0.01 Water Activity 0.96 0.97 0.97 0.97 0.97 0.001 0.06 Moisture:Protein 2.7 2.8 2.9 2.9 3.0 0.11 0.38 abcMeans within an attribute with differing superscript differ; α < 0.05.
1Cooking treatment endpoint parameters are as follows: A – pH 4.50, 54.4°C with industrial chilling simulation; B – pH 4.84, 54.4°C with industrial chilling simulation; C – pH 4.84, 54.4°C with rapid ice bath chilling; D - pH 4.82, 48.9°C with rapid ice bath chilling;
E - pH 4.84, 43.3°C with rapid ice bath chilling.
88 Table 4.2: Means of Escherichia coli O157:H7 populations (CFU/g) in all beef summer sausage. High Pressure Hold Time Inoculation Post Fermentation After Heating Control 1s 150s 300s to (pH) to (temp1) 9.17 ± 0.06 7.56 ± 0.92 (4.5) (54.4°C T) 2.83 ± 2.46z 2.82 ± 1.72z 2.92 ± 1.28z 2.06 ± 1.82 1.01 ± 1.76 9.13 ± 0.30 8.49 ± 0.04 (4.8) (54.4°C T) 4.04 ± 1.11az 3.83 ± 0.90az 2.53 ± 2.22abz 2.28 ± 1.99ab 1.22 ± 2.11b (54.4°C RC) 5.05 ± 0.93az 4.84 ± 0.89az 2.77 ± 2.44abz 2.38 ± 2.08b 1.23 ± 2.12b (48.9°C RC) 7.88 ± 0.76ax 7.74 ± 0.90ax 5.37 ± 1.81bx 2.44 ± 2.12b 1.26 ± 2.19b (43.3°C RC) 8.53 ± 0.05ax 8.47 ± 0.05ax 6.67 ± 1.06ax 3.02 ± 0.58b 1.10 ± 1.91b abcMeans with different superscript within a total process differ; α < 0.05. yzMeans with different superscript within a sampling time differ; α < 0.05.
1Chilling method denoted by T – industrial chilling simulation or RC – rapid ice-water chilling.
89 Table 4.3: Least square means of non - O157 shiga toxin producing Escherichia coli populations (CFU/g) in all beef summer sausage High Pressure Hold Time Inoculation Post Fermentation After Heating Control 1s 150s 300s to (pH) to (temp1) 8.94 ± 0.15 7.42 ± 0.86 (4.6) (54.4°C T) 2.63 ± 2.31az 1.30 ± 2.25abz <0.58bz <0.58bz 0.87 ± 1.51b 9.04 ± 0.20 8.38 ± 0.24 (4.8) (54.4°C T) 4.10 ± 0.82az 4.00 ± 0.88ay 1.15 ± 2.00byz <0.58bz 0.65 ± 1.13b (54.4°C RC) 5.00 ± 1.04az 4.92 ± 0.94ay 2.62 ± 2.27ay <0.58bz <0.58b (48.9°C RC) 7.82 ± 0.86ay 7.75 ± 0.59ax 5.82 ± 1.54ax 1.68 ± 2.91byz <0.58b (43.3°C RC) 8.26 ± 0.32ay 8.54 ± 0.04ax 7.36 ± 0.86ax 3.37 ± 1.04by 0.92 ± 1.59c abcMeans with different superscript within a total process differ; α < 0.05. xyzMeans with different superscript within a sampling time differ; α < 0.05.
1Chilling method denoted by T – industrial chilling simulation or RC – rapid.
90
CHAPTER 5: TEXTURE OF LOW TEMPERATURE FERMENTED SALAMI WITH
DIFFERING WATER ACTIVITIES, AND HIGH PRESSURE PROCESSING TIMES
91 Macc Rigdon, Harshavardhan Thippareddi, Chevise L. Thomas, Robert W. McKee, Alexander M. Stelzleni To be submitted to Meat and Muscle Biology Abstract:
The objective was to evaluate the textural and quality parameters of low temperature fermented
Italian style salami with pH 5.0 and dried to varying water activities in combination with post-
manufacture high pressure processing for varying times. Over three replications, salami was
fermented at 23°C to a minimum of pH 5.0 and dried to a water activity of 0.92, 0.90, or 0.85.
After drying, salami was cut, vacuum packaged, and subjected to 586 MPa for 0, 1, 150, or 300 s. After pressurization the salami was evaluated for texture profile analysis (n = 9), proximate analysis (n = 9), objective color (n = 9), and lipid oxidation (n = 9). Texture analysis indicated differences in water activity treatments for hardness, cohesiveness, gumminess, and chewiness
(P < 0.01). Salami dried to water activity 0.85 were harder, more cohesive, more gummy, and more chewy than those dried to 0.92. Objective color scores for lightness (L* values) showed an incremental darkening of the salami as water activity decreased (P < 0.01). High pressure processing (HPP) regardless of hold time, had no effect (P > 0.51) on salami color. Salami hardness, cohesiveness, springiness, and gumminess remained unaffected by HPP regardless of hold time (P > 0.06). The use of HPP in commercial salami production can be used in a variety of product categories with little or no impact on their rheological properties.
Keywords: Beef, Texture, Salami, Quality, Shelf-stable
92 INTRODUCTION
Traditional food products make up a large part of European culture and heritage, which has a substantial effect on American niche markets of consumers seeking traditional European style foods (Ilbery and Kneafsey, 1999; Guerrero et al., 2009). Salami is an example of one of these niche markets where consumers are seeking traditional Italian style, mildly fermented, and dried foods. However, in 1994 a foodbourne outbreak of Escherichia coli O157:H7 associated with salami products (CDC, 1995) initiated regulatory changes outlining a suggested course of action aimed at mitigating the risk of E. coli O157:H7 in these products (Reed, 1995).
Representatives of the beef, and salami industries (National Cattlemens Beef Association’s Blue
Ribbon Task Force) conviened with the intensions of outlining several propositions for eliminating the organism of concern and preventing further outbreaks (Nickelson II et al., 1996).
Some of these options included a test and hold program, or a thermal treatment listed in 9 CFR
318.17 (145°F for 4 min). Thermal processing of these types of products is strictly against traditional processing techniques as it alters the rheological properties associated with traditional
Italian salami. One option in NCBA’s outline was the use of a validated total process that exhibits a 5D reduction of the target organism.
High pressure processing (HPP) is a processing technology currently being used in the production of various clean label food products for the reduction of pathogenic organisms. This technology is a practical method of reducing pathogenic organisms through forced water displacement in a closed system (Patterson et al., 1995; Cheftel and Culioli, 1997). The elevated pressures of HPP (500 - 700 MPa) inactivate organisms through oxidative stress increasing the organisms sensitivity to oxygen radacals (Aertsen et al., 2005). These pressures do not effect low energy state molecules such as those present in protein covalent bonding or long chain fatty
93 acids, but do have an effect on high energy state molecules, such as Van der Waals interactions or other ionic bonding (Cheftel and Culioli, 1997). High pressure has been shown to be effective in reducing up to 6 log CFU/g of E. coli O157:H7 and other Shiga toxin producing E. coli in the processing of summer sausage style products cooked as low as 43.3°C and fermented to pH 5.0
(Chapter 4). Additionaly, it has also been shown that texture and quality characetistics of pressurized beef summer sausage remains unchanged after extended holding at 586 MPa (up to
300 s; Chapter 3). Although, the use of HPP is well documented for summer sausage products, little is known about the influence HPP has on the texture and quality characteristics of fermented and dried salami. Therefore, the objective of this research was to evaluate the textural and quality characteristics of low temperature fermented Italian style salami produced with beef and processed with the use of high pressure processing.
MATERIALS AND METHODS
Beef Trimmings Procurement and Batter Processing
Boneless beef chuck rolls were purchased from a local commercial purveyor and delivered to the University of Georgia Meat Science Technology Center (MSTC) and frozen (-
20°C) until needed. On the day of production, for each of three replications, one chuck roll was ground to 2.54 cm (Biro Model G58483, St. Louis, MO) and sufficient beef back fat was added to the ground chuck roll to target 20% fat in the meat batter. Ground chuck and back fat were subsequently ground together to 4.76 mm and placed into a reverse action mixer (Model A-80,
Koch, Kansas City, MO). A seasoning blend containing 0.2% dextrose, 0.75% spices typical to a salami flavoring, 250 ppm cure #2, and 551 ppm sodium erythorbate was added to the mixer, and spices were blended for 30 s. During this time, 15 g of a starter culture blend consisting of
Pediococcus, Lactobacillus, Micrococcus, and Staphylococcus species (SAGA AF5, Kerry
94 Industries, Rochester, MN) was diluted in 35 ml of deionized water and added to the batter.
Subsequent to mixing, 2.5% salt (Mortons, Chicago, IL) was added to the batter and mixed for
an additional 30 s, for a total mixing time of 1 min. The batter was then vacuum stuffed (Vemag
Robot 500, Reiser, Canton, MA) into 47 mm collagen middles approximately 54 cm long and
clipped.
Fermentation and Drying
After stuffing, the salami chubs were hung on sticks and placed into a Stagionello drying
cabinet (Stagionello Maturmeat 100+100kg twin, Arredo USA, Crotone, Italy) and inoculated
with a diluted solution of Penicillium nalgiovense mold (M-EK-4, Chr. Hansen, Hoersholm,
Denmark). Sausages were then allowed to rest for 12 h at 10ºC and 65% relative humidity (RH).
Subsequent to the resting period, the salami was fermented at 23ºC and 90% RH for 96 h. Salami
pH was monitored daily for the duration of the fermentation cycle by direct probing with a spear
tip pH meter (Oakton, Eutech Instruments, Vernon Hills, IL.). Upon the completion of fermentation, a sample was taken for the measurement of pH by homogenizing 10 g of sample in
100 ml of distilled water to confirm direct pH measurement (Koniecko, 1979) prior to the natural
pH buffering which took place through continued drying. The following steps were used as
guidelines to maintain approximately 1-2% loss per day until target water activities of 0.92, 0.90,
and 0.85 were met: 19ºC 85% RH for 48 h; 18ºC 90% RH for 81 h; 17ºC 85% RH for 48 h; 16ºC
85% RH for the remainder of drying. During the drying stages, samples were periodically
collected for the determination of water activity (aw) using an Aqualab water activity meter
(Aqualab 4TE, Pullman, WA). Upon the completion of each of the three aw treatment
parameters, the salami were removed from the cabinet, vacuum packaged (B-620 series; 30–50
3 2 cm O2/m /24 h/101,325 Pa/23°C; Cryovac Sealed Air Corporation, Duncan, SC, USA) and
95 stored (2 ± 2°C) until all samples were completed. After the completion of the final treatment, salami was allowed to rest in a cooler (2 ± 2°C) for 7 d prior to high pressure processing (HPP).
High Pressure Processing
After the completion of the rest period the sausages were opened and cut into 4 pieces and randomly assigned to HPP hold times of 0, 1, 150, or 300 s and vacuum packaged (Cryovac
Sealed Air Corporation, Duncan, SC, USA). The packages were then transported (2 ± 2°C; 170 km) to Universal Pasteurization (Villa Rica, GA) and high pressure processed at 4 ± 2°C and 586
MPa for their respective treatment time point allocations, as advised for the replication of current industrial practices (B. Cook, personal communication, 2017).
Proximate Analysis and Lipid Oxidation
Initial analysis of HPP treatment effects on percent moisture and percent protein yielded no differences (P > 0.05), therefore HPP hold times of 0, 1, 150, and 300 s were combined within each aw treatment and replication for the determination of percent moisture and percent crude protein. Moisture and crude protein were used for the determination of moisture:protein.
Forced-air ovens (Fisher Scientific, Pittsburg, PA) were used for the determination of percent moisture. Samples were weighed in duplicate (2.0 ± 0.1 g) for each treatment and placed in pre-dried pans. Samples were dried for 18 h at 100ºC (Soderberg, 1991). After oven drying, the samples were placed in a desiccator and allowed to cool to room temperature. The following equation was used for the calculation of percent moisture:
% moisture = [1-(dry sample weight/wet sample weight)] x 100
The determination of protein content was done in duplicate for samples using a Nitrogen Auto-
Analyzer (Leco FP-528 Nitrogen analyzer, Leco Company, St Joseph, MI) and was expressed as the percent crude protein (N content x 6.25).
96 Thiobarbituric acid reactive species (TBARS) were measured to determine lipid
oxidation due to the effects of HPP following the methods of Sinnhuber and Yu (1958) and
Buege and Aust (1978), as outlined by AMSA (2012). Lipid oxidation was expressed as mg
malonaldehyde/kg meat.
Objective Color
Color was measured on the whole salami chub prior to TPA analysis. One cut face was made prior to reading color using a Hunter-Lab MiniScan EZ (Hunter Associates Laboratory,
Reston, Virginia). Three readings were taken with a 32 mm aperture using illuminate A and 10º viewing angle and averaged for analysis. Prior to reading samples, the colorimeter was standardized using black and white tiles. Commission Internationale de l’Eclairage (CIE) L*
(Lightness), a* (Redness), and b* (Yellowness) were measured and isobestic wavelength ratios of R570:R650 were used for the determination of cure color fading values (AMSA, 2012).
Texture Profile Analysis (TPA)
Texture analysis was performed in duplicate on each sample chilled to 4ºC. Samples were
sliced to 1.27 cm long, and two cores were removed using a hand held coring device measuring
1.27 cm in diameter. Each core was individually analyzed on a TA-TX, Texture Analyzer
(Texture Technologies Corp., Hamilton, MA) using a double compression method, compressing
75% of the sample original height with a 25 kg load cell. Cross head speed during testing was
3.33 mm/s with a trigger force of 10 g and a 5 s pause between compressions for sample recovery. Exponent Connect (Texture Technologies Corp., Hamilton, MA) was used to analyze the force time curves for the determination of hardness, springiness, cohesiveness, gumminess, and chewiness. Hardness was defined by the peak force during the first compression, while
97 springiness was the difference in height of the sample between the first and second compression.
Cohesiveness was determined by the ratio of positive force used during the second compression
compared to that of the first compression. Gumminess was the product of hardness and
cohesiveness, while chewiness was the product of hardness, cohesiveness and springiness (Claus,
1995).
Statistical Analysis
Data were analyzed using Proc Mixed of SAS (version 9.3) as a completely randomized
split plot design. Water activity treatment was applied to the sausage chub and was considered
the whole-plot. The chubs were then section into four pieces that received varying HPP hold times designating the sections as the sub-plot. The three replications were considered a blocking
factor, and each chub section was considered the observational unit. Means were considered
different at α < 0.05.
RESULTS AND DISCUSSION
Proximate Analysis
Water activity treatments of 0.92 reached 0.917, while the target treatments of 0.90
reached 0.899 and the target treatments of 0.85 reached 0.854, meeting the parameters of the
study treatments. Percent moisture remained unaffected by HPP hold time (P = 0.89) and there
was no aw by HPP hold time interaction (P = 0.10). Percent moisture did decline along with aw
(P < 0.01; Table 5.1), as would be expected during the drying of salami products.
Percent protein remained unaffected by HPP hold time (P = 0.96), and there was no
interaction between water activity and HPP hold time (P = 0.79). Percent protein was affected by
water activity (P < 0.01) where treatments dried to aw of 0.85 had the greatest percent protein (P
< 0.01), while treatments dried to aw of 0.90 and 0.92 were similar in percent protein (P = 0.07).
98 The combination of moisture and protein for moisture to protein ratio (MPR) was
unaffected by HPP hold time (P = 0.83). High pressure processing hold time and water activity treatments did not have an interaction on MPR (P = 0.16), but aw did have a treatment effect (P <
0.01). Treatments dried the least (aw = 0.92) had the greatest MPR (P < 0.01) while treatments dried the most (aw = 0.85) had the lowest MPR (P < 0.01). Although there were differences in
MPR for water activity treatments, all products produced met the USDA FSIS food standards and labeling guidelines to be called dry salami (USDA, 2011).
Lipid oxidation was also unaffected by HPP hold time (P = 0.66) and there was no
interaction between aw and HPP hold time for lipid oxidation (P = 0.99). Water activity also had
no effect on lipid oxidation (P = 0.17).
Percent moisture of the sausages declined as expected for a dried product as this is the
primary method for controlling the aw. These trends of decreased moisture as aw decreases
continue into MPR and percent protein as the percent protein becomes a greater proportion of the
whole product as the product is dried to reduce the aw.
High pressure processing has been continually reported as having a negative effect on
lipid stability in fresh meats such as fish, pork, beef and chicken (Ohshima et al., 1993; Cheah
and Ledward, 1996; Ma et al., 2007; McArdle et al., 2010). These studies indicate that the effect
of pressure on lipid oxidation is greater at higher pressures and longer hold times. However, the
current study indicates that the direct effects of HPP on beef salami are negligible. Cheah and
Ledward (1996) also reported results congruent with the current study, stating that the absence of oxygen leads to no increase in lipid oxidation. Marcos et al. (2007) found results similar to the current study that lipid oxidation was not affected by HPP for low acid fermented sausages, which was attributed to the late ripening stages of the sausage production, stating that lipid
99 oxidation had already occurred, although it is more likely that the inclusion of nitrate and nitrite
inhibited the oxidation of lipids for the duration of the ripening stages. Furthermore, Zanardi et
al. (2002) also reported TBARS values similar to that of the current study, stating that there was
no correlation of TBARS and sensory scores, indicating sensory pannelists were unable to detect
lipid oxidation in Milano-style sausages ripened for 42 days.
Objective Color Analysis
Objective color analysis for values indicating salami lightness (L* values) were not
affected by HPP hold time, nor was there an HPP hold time by aw interaction (P = 0.51; P =
0.83). Subsequent reduction of aw decreased the lightness of the salami (P < 0.01; Table 5.2)
where sausages dried to an aw of 0.85 were the darkest (P < 0.01), while sausages dried to aw of
0.90 and 0.92 were similar to each other (P = 0.19). Salami aw and HPP had no interaction (P =
0.95), nor was there an HPP hold time or aw main effect for salami redness (P > 0.37; a* values).
Objective color scores describing yellowness (b* values) were not affected by HPP hold time nor
was there an aw by HPP hold time interaction (P > 0.70). However, objective color scores
describing yellowness were affected by aw (P < 0.01). Salami dried to 0.92 was the most yellow
(P < 0.05), followed by salami dried to 0.90, which was more yellow than treatments dried to
0.85 (P < 0.01). Cure color fading was not affected by HPP hold time (P = 0.70), nor was there a
HPP hold time by drying treatment interaction (P = 0.85). However, aw did have an effect on
cure color fading (P < 0.01). Salami dried to 0.92 and 0.90 were similar in cure fading (P = 0.73)
but were both less faded than salami dried to aw 0.85 (P < 0.01).
Salami lightness as indicated in the current study is directly related to the extent of
drying. Although there are other factors that affect the overall color of salami, such as starter cultures and fermentation temperatures, the concentration of solids during the dehydration stages
100 is specifically effective in darkening the color (Ruiz et al., 2014). Ruiz et al. (2014) reported
similar L* values as the current study for Italian salami containing 20% beef. Redness values (a*
values) were also similar to the current study, where the authors showed no difference between
a* values in sausages dried to water activities of 0.89 and 0.84. Although the values reported by
Ruiz et al. (2014) were less red, this is likely due to the greater inclusion of beef in the current
study. Zanardi et al. (2002) reported that visual color stability declined with storage time, which
was explained by transient color fading, which is similar to the findings in the current study
where a greater amount of cure color fading was found in sausages dried to a lower aw. A reduction in the yellowness values (b* values) was previously reported by Ruiz et al. (2014) and was explained by the elimination of oxygen by the lactic acid starter culture, and the subsequent reduction of oxymyoglobin.
As previously described, the color variation in salami products is heavily dependent on the physiochemical characteristics of the sausage itself; however, the use of HPP in the production of these products has been shown to have no effect on salami color. The decline of color in fresh meat products that are processed above 400 MPa is well documented and attributed to the denaturation of the globin molecule and the release of iron (Carlez et al., 1995). However,
Tornberg (2013) stated that the greatest potential application of HPP in commercial meat plants is for sliced high value salami, due to the stability of shelf life attributes.
Texture Profile Analysis
Salami hardness remained unaffected by HPP hold time (P = 0.10; Table 5.3), and there was not a HPP hold time by aw interaction (P = 0.64). Decreasing the water activity of all beef salami caused the hardness to increase (P < 0.01; Table 5.4). Salami dried to 0.92 was the softest salami (P < 0.01), and salami dried to 0.85 was the hardest (P < 0.01). Cohesiveness also
101 remained unaffected by HPP hold time (P = 0.20) and did not have any HPP hold time by aw interactions (P = 0.72). Drying treatment did have an effect on the cohesiveness of salami (P <
0.05). Salami dried to an aw of 0.85 was more cohesive than salami dried to 0.92 (P < 0.01) but was similar to 0.90 (P = 0.09). Salami dried to a aw of 0.92 and 0.90 were also similar in cohesiveness (P = 0.30). Springiness remained unaffected by aw and HPP hold time and there was no aw by HPP hold time interaction (P = 0.98, P = 0.68, P = 0.95, respectively). Gumminess, as calculated by the hardness multiplied by the springiness, was affected by the water activity (P
< 0.01), but not the HPP hold time (P = 0.06), nor was there a water activity by HPP hold time interaction (P = 0.40). Sausages with the lowest water activity (0.85) were also the gummiest sausages (P < 0.01), while sausages dried the least were the least gummy (P < 0.01). Chewiness, a product of hardness, springiness and cohesiveness, was affected by both water activity and
HPP hold time (P < 0.01, P < 0.05, respectively), but there was no interaction between the two treatment parameters (P = 0.73). Salami dried to aw 0.92 was the least chewy of all the samples
(P < 0.05), and an increase in chewiness occurred as samples reached lower water activities.
Samples dried to aw 0.90 were chewier than 0.92 samples (P < 0.05), but less chewy than those dried to aw 0.85 (P < 0.01). Salami that was not HPP were less chewy than salami that was held for 300 s during HPP (P < 0.01). Holding salami during HPP for 1s, and 150 s were similar to each other (P = 0.93) and were also similar to both 300 s and no HPP (P > 0.09).
Texture of beef salami is dynamic through the drying or ripening stages. In the current study the hardness of salami is dramatically increased as the salami is dried to a lower water activity. Spaziani et al., (2009) reported similar findings of increased hardness during the later drying stages, which were attributed to the fact that the product shrinkage was directly proportional to the water loss. It was also noted by Spaziani et al. (2009) that the decrease of pH
102 by starter cultures increased the hardness of the sausage due to the gelation of the meat during
the acidification stages. Gonzalez-Fernandez et al. (2006) also stated that the major influence on
hardness of salami and other rheological properties is the later drying stages. As the salami dries,
the solids in the meat become concentrated and compacted, making the product denser and
subsequently harder.
Cohesiveness of salami in the current study was shown to increase as the degree of
drying increased. Herrero et al. (2007) analyzed the rheological properties of commercial salami
in Spain and determined that there was a significant correlation between cohesiveness of salami
and the aw of the product. The research indicated that various styles of dried and fermented
products were able to be grouped by rheological properties based on their physiochemical
properties, which supports the current work where it is shown that different water activities have
an impact on the cohesiveness and hardness of the sausage.
Bruna et al. (2001) evaluated dried fermented sausages for differences in the texture of
salami inoculated with different molds and showed a decrease in gumminess and chewiness in
finished products. These results were attributed to the proteolytic and lipolytic properties of the
mold, indicating that a longer exposure to these enzymes may lead to a decrease in gumminess
and chewiness. However, in the current study, we show that despite exposure to mold and the associated enzymes during the ripening stages, the gumminess and chewiness continues to
increase. This indicates that the likely cause of increases in gumminess and chewiness is due to
the dehydration, although admittedly the differences in hardness between mold inoculated and non-inoculated samples of the same aw were not tested in the current study. Additionally, Ruiz et
al. (2014) also reported that the texture of salami with lower water activity was more dehydrated,
producing a tougher product that was less desirable to consumers than one with a greater water
103 activity. The increase in toughness also negatively affected the percentage of consumers with the intent to purchase the product. Salami with a softer less chewy texture is also considered a defect in other types of products (Gimeno et al., 1996).
The texture findings are consistent with much of the current literature showing that there is great deal of variability between the rheological properties when salami is produced with varying physiochemical properties. It should be noted that the use of HPP at 586 MPa for up to
300 s did not have an effect on the hardness, cohesiveness, springiness, or the gumminess of salami products, regardless of degree of drying. The findings of the current study are encouraging for the use of HPP in a multitude of dried and fermented product categories, having little or no effect on the overall texture of the product.
Conclusions
Softness of salami is considered a desirable attribute in certain sausage categories but can be considered a quality defect in other varieties. With producers of dried fermented sausages attempting to manufacture more authentic southern European style sausages with more mild textures, texture and color results from the current study indicate that the use of HPP does not affect the texture for a variety of sausage types and can potentially be applied to all salami products regardless of aw.
Acknowledgments
This work was supported by grant no. 2012-68003-30155 and grant no. 2011-68003-
30012 from the USDA National Institute of Food and Agriculture, Agriculture and Food
Research Initiative Foundational Program.
104
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109 Table 5.1: Least squares means for proximate analysis parameters of all beef salami dried to varying water activities
Water Activity Trait 0.92 0.90 0.85 SEM P-Value Moisture, % 42.96a 38.03b 30.08c 0.01 <0.01 Protein, % 23.35a 25.37a 28.98b 0.75 <0.01 Moisture:Protein 1.84a 1.52b 1.05c 0.05 <0.01 Malonaldehyde, mg/kg tissue 0.71 0.72 0.64 0.03 0.17 abcMeans within a heading with different superscripts differ; α < 0.05.
110 Table 5.2: Least squares means for objective color scores for all beef salami dried to different water activities
Water Activity Trait 0.92 0.90 0.85 SEM P-Value L* 53.18a 52.47a 46.31b 0.38 <0.01 a* 18.20 17.88 17.71 0.25 0.37 b* 13.04a 12.71b 11.72c 0.11 <0.01 Fade1 0.36a 0.36a 0.33b 0.01 <0.01 abcMeans within a column with different superscripts differ; α < 0.05.
1Values determined by the following equation using isobestic wavelengths, where lower values indicate more fading: fade = 570 nm/650 nm (AMSA, 2012).
111 Table 5.3: Least square means for the texture profile analysis of all beef salami dried to varying water activities High Pressure Processing Hold Times, s Trait 0 1 150 300 SEM P-Value Hardness, N 9446 10643 9505 10713 470 0.10 Cohesiveness 0.28 0.28 0.30 0.29 0.01 0.20 Springiness, % 40.49 40.96 42.83 42.50 1.59 0.68 Gumminess 2621 2956 2868 3121 130 0.06 Chewiness 1036a 1198ab 1206ab 1354b 70 0.02 abcMeans within a column with different superscripts differ; α < 0.05.
112 Table 5.4: Least square means for the instrumental texture analysis of all beef salami dried to varying degrees Water Activity Trait 0.92 0.90 0.85 SEM P-Value Hardness, N 6815a 8435b 14980c 407 <0.01 Cohesiveness 0.274a 0.284ab 0.300b 0.01 0.02 Springiness, % 41.62 41.54 41.92 1.38 0.98 Gumminess 1855a 2361b 4458c 112 <0.01 Chewiness 757a 982b 1856c 61 <0.01 abcMeans within a column with different superscripts differ; α < 0.05.
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CHAPTER 6: CONCLUSIONS
The culmination of the work presented indicate that the use of high pressure processing
(HPP) of fermented beef sausages increases safety, with little or no change to sausage texture in
low temperature cooked summer sausage and low temperature fermented salami. These results
confirm the use of low temperature processing of these sausage products for the production of
traditional Eastern European, namely Italian, style salami products where low temperatures are used throughout the manufacture, fermentation, and drying process. While American consumers are looking for these more traditional products, the lack of literature validating the use of HPP on low temperature fermented products makes these findings novel. The USDA FSIS lethality standards recommend a 5 log CFU/g reduction of 7 Escherichia coli serotypes during the production of ready to eat comminuted beef products. The current work indicates that HPP in combination with temperatures as low as 43.3ºC and fermentation to pH 5.0 was sufficient to kill greater than 6.5 log CFU/g of all 7 serotypes. Texture, being one of the most important factors for traditionally fermented products, remains unchanged with the addition of HPP to the production process. This result was supported by the inability of trained sensory panelists to detect textural differences between HPP hold times of 300 s and a non-pressurized control.
Summer sausage color values for redness and cure color fading did show differences statistically,
although the differences were small in magnitude and likely would be undetectable to the
consumer. Lipid oxidation also remained unchanged for HPP summer sausage cooked to low
temperatures, potentially indicating no changes in off flavors due to aging lipids or degradation
114 products of lipid oxidation during HPP. Salami texture also remained largely unaffected by the
HPP process. The current body of work indicates that there is no change in hardness, springiness, cohesion, or gumminess when salami is fermented to pH 5.0 and dried between aw 0.92 and 0.85 and pressurized for up to 300 s. Slight differences were discovered in the chewiness of the same salami; however, these differences are likely due to the multiplicative properties of each of the parameters included in the calculation amplifying the numerical differences into significance.
The lack of definitive textural changes, in conjunction with a lack of differences for HPP treated salami in lipid oxidation and color, show that there is a possibility to use this processing step as a microbial intervention to potentially improve safety without detrimental effects on salami quality. Therefore, these data support the use of HPP in low temperature cooked, low acid fermented summer sausage for the reduction of all 7 serotypes of E. coli without detriment of color or texture. Additionally, the use of HPP for low temperature fermented, low acid salami can be used without detriment to texture, color, or lipid oxidation.
115