EFFECTS OF COMPATIBLE SOLUTES ON COLD TOLERANCE OF PROPIONIBACTERIUM FREUDENREICHII AND THE SIGNIFICANCE OF PROPIONIBACTERIUM COLD TOLERANCE IN SWISS CHEESE MANUFACTURING
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the
Graduate School of The Ohio State University
By
Corunda T. Pruitt, M.S.
* * * * *
The Ohio State University 2005
Dissertation Committee:
Dr. W. James Harper, Adviser Approved By
Dr. Polly D. Courtney
Dr. Mike Mangino ______Adviser Dr. Ahmed Yousef Graduate Program in Food Science and Technology
ABSTRACT
Propionibacterium freudenriechii is one of the bacteria utilized in Swiss cheese
starter cultures. Its carbon dioxide production is responsible for the development of the
eyes commonly associated with Swiss cheese. The bacteria must endure high cook
temperatures and low storage temperatures during ripening. Many bacteria are capable of
synthesizing compatible solutes in response to different stresses. The primary focus of
the present study was to determine the growth capabilities and transport of P.
freudenreichii strains in the presence of exogenous glycine betaine, proline, and
glutamate at low temperatures and evaluate the effects of storage temperature on eye
formation in Swiss cheese manufactured with these dairy P. freudenreichii strains
differing in low temperature growth abilities.
Four P. freudenreichii strains were grown in chemically defined media with the
addition of 1 mM or 100 mM of glycine betaine, proline, or glutamate. The cultures were
anaerobically incubated at 30, 22, 10, 7.2, and 4°C and spectrophometrically monitored
to determine growth capabilities. Strains capable of growing at 10, 7.2, and 4°C were
characterized as cold-tolerant and strains not able to grow at these temperatures were
characterized as cold-sensitive. Compatible solute transport was assessed using 14C- labeled amino acids added to a cell suspension with a final concentration of 1 mM. The growth rate at the lowest temperature decreased and the lag phase increased for strains
ii P843 and P57 and were therefore considered cold sensitive. At the same temperature the
growth rate for strains P728 and P873 increased with a decreased lag phase. These
strains were considered cold tolerant. All strains transported proline most abundantly at
all temperatures with its cryoprotective effects observed at 22°, 10°, and 7.2°C by
increased growth rates. Glycine betaine and glutamate were not transported as greatly as
proline until a decrease in temperature (7.2°C). All solutes increased the maximum growth rate of all strains at this lower temperature indicated they exert a cryoprotective at this temperature. These strains were also subjected to several freeze-thaw cycles to deterimine if glycine betaine exterts a cryoprotective effect at temperatures below refrigeration. Cells were frozen for 24 hours and thawed 30 minutes at 30°C. After four days a decrease in cell viability was observed as well as after five days. There was a difference between all strains suggesting that freeze-thaw tolerance is strain dependent in propionibacteria. Strain P572 was the most freeze-thaw tolerant with a 1.01-fold reduction after four cycles.
Swiss cheese was manufactured with a cold-tolerant strain (P873), a cold- sensitive strain (P572) and an intermediate strain (P196). All cheese blocks experienced
identical warm room treatment (21°C for 17 days) but differed in cool ripening storage.
The blocks were stored at 0, 4, or 7.2°C. Samples were taken at days 0, 30, 60, and 90.
Counts of viable propionibacteria cells remained constant throughout the ripening treatment with a range of 108-9. The twenty most common amino acids were quantified in
addition to citrulline during each ripening phase from water soluble cheese extracts.
Differecnes were observed among each strain but only differences were observed
between strains after 90 days at 4°C and 7.2°C. Total concentrations were higher in
iii strains P196 and P873 after these ripening periods. Digital images of day 60 and 90
samples were analyzed for splits. More eyes were seen in cheeses stored for ninety days
at 7.2°C. More splits were also seen at this time and temperature. Fewer eyes and splits were observed in cheeses stored for sixty days at 0°C. Cheeses manufactured with strain
P196 possessed the most eyes after sixty days at 4°C and cheeses manufactured with strain P873 possessed the most splits after ninety days at 7.2°C.
Cold tolerance varies among dairy propionibacteria and the role of glycine
betaine, proline, and glutamate as compatible solutes may contribute to cryoprotection of
some Propionibacterium strains. Strain selection, ripening temperature and ripening time
may have an affect on split occurrence often seen in Swiss cheeses.
iv
Dedicated to my parents
v ACKNOWLEDGMENTS
I would like to acknowledge all of those who have helped me along the way in
this long and difficult journey I call advanced education. These people made great
contributions, large and small, in helping me fulfill the requirements through their
knowledge, time, and support.
I thank my past and present advisers, Dr. Polly D. Courtney and Dr. W. James
Harper for guiding me through this entire process. I thank them for providing me with an
unforgettable experience in a scientific environment of learning, independence, and
enjoyment. I would like to extend my appreciation to Dr. Mike Mangino and Dr. Ahmed
Yousef for serving on my dissertation committee and offering extensive advice on my
project. I would like to all of my lab mates who helped create a tolerable work environment through their laughter and assistance.
I would like to express my appreciation to the Swiss Cheese Consortium and The
Ohio State University College of Food, Agriculture and Environmental Science for their financial support.
Finally I would like to thank my family and friends who supported and encouraged me during some difficult times and the one guy who has my heart. I thank
you all and love you.
vi VITA
July 15, 1977…………………………………………… Born – Dallas, Texas
1995- 1999………………………………………………B.S. Biology Xavier University New Orleans, Louisiana
2000-2002………………………………………………M.S. Biology Jackson State University Jackson, Mississippi
2002-present…………………………………………….Graduate Research Associate The Ohio State University Columbus, Ohio
PUBLICATIONS
Research Publication
1. Dave, S., M. K. Pangburn, C. Pruitt, and L. S. McDaniel. 2004. Interaction of human factor H with PspC of Streptococcus pneumoniae. Indian Journal of Medical Research 119(Suppl): 66-73.
Research Abstract
1. Pruitt, C.T., O. Anggraeni, and P.D. Courtney. 2004. Cold tolerance and compatible solute uptake in dairy Propionibacterium freudenreichii. Abstract 99D-13. Institute of Food Technologists Annual Meeting, Las Vegas, NV.
FIELDS OF STUDY
Major Field: Food Science and Technology
vii TABLE OF CONTENTS
Page
Abstract……………………………..……………………………………………………..ii
Dedication…………………………………………………………………………………v
Acknowledgments……………….……………………………………………………….vi
Vita…………………………..………………………………………………………..…vii
List of Tables………………………………………………………………….………….xi
List of Figures………………………………………………….………………………..xiii
Chapters:
1. Literature Review…………………………………………………………………1
1.1 Introduction……………………………………………………………………1
1.2 Cheese Manufacture…………………………………………………………...2
1.2.1 Swiss Cheese Manufacture………………………………………….4
1.2.2 Swiss Cheese Eye Formation……………………………………….5
1.2.3 Split Defects………………………………………………………...7
1.3 Compatible Solutes as Osmoprotectants and Cryoprotectants……………9
1.3.1 Listeria monocytogenes...... 14
1.3.2 Other Bacteria……………………………………………………...17
1.4 Effects of Membrane Composition on Compatible Solute Transport……….19
1.4.1 Compatible Solute Transport Systems………………………….…20
viii 1.4.1.1 Listeria monocytogenes………………………….………….….21
1.4.1.2 Other Bacteria…………………………….……………….……24
1.5 Conclusion…………………………………………………………….……26
2. Cold Tolerance and Compatible Solute Uptake of Dairy Propionibacterium
freudenreichii…………………………………………………………………………..28
2.1 Introduction………………………………………………………………...28
2.2 Materials and Methods…………………………………………….……….31
2.3 Results……………………………………………………………….……..34
2.4 Discussion…………………………………………………………….……38
3. Determination of Freeze-Thaw Resistance in Dairy Propionibacterium freudenreichii
3.1 Introduction………………………………………………………….…….57 . 3.2 Materials and Methods…………………………………………….………59
3.3 Results…………………………………………………………….……….60
3.4 Discussion…………………………………………………………………61
4. The Effects of Storage Temperature on Split Defects in Swiss Cheese Manufactured
with Dairy Propionibacterium freudenreichii Differing in Low Temperature Growth
Capabilities……………………………………………………………………………66
4.1 Introduction……………………………………………………………….66
4.2 Materials and Methods……………………………………………….…...67
4.3 Results……………………………………………………………….……71
4.4 Discussion………………………………………………………….……..77
Conclusions…………………..………………………………………………………88
ix References………………………………………………………………………………..90 Appendices:
Appendix A: Graphs displaying cold tolerance of Propionibacterium strains….98
Appendix B: Bacteria enumeration and digital images of Swiss cheese manufactured with different Propionibacterium strains……………………..…103
x LIST OF TABLES
Table Page
2.1 Effects of glycine betaine, proline, and glutamate on the maximum specific growth rate (µmax) and the lag phase of P. freudenreichii strain P843 at various temperatures……………………………………………………………………..44
2.2 Effects of glycine betaine, proline, and glutamate on the maximum specific growth rate (µmax) and the lag phase of P. freudenreichii strain P572 at various temperatures……………………………………………………………………..45
2.3 Effects of glycine betaine, proline, and glutamate on the maximum specific growth rate (µmax) and the lag phase of P. freudenreichii strain P728 at various temperatures……………………………………………………………….….…46
2.4 Effects of glycine betaine, proline, and glutamate on the maximum specific growth rate (µmax) and the lag phase of P. freudenreichii strain P873 at various temperatures…………………………………………………………….……….47
3.1 The effects of cyclic frozen storage of four P. freudenreichii strains grown in chemically defined media with glycine betaine………………………..………..68
4.1 Make day composition of Swiss cheese manufactured in with different P. freudenreichii strains……………………………………………………………82
4.2 Amino acid concentrations of cheeses prepared with P. freudenreichii strain P572……………………………………………………………………….…….83
4.3 Amino acid concentrations of cheeses prepared with P. freudenreichii strain P196……………………………………………………………………………..84
4.4 Amino acid concentrations of cheeses prepared with P. freudenreichii strain P873…………………………………………………………………………….85
4.5 Digital analysis of eyes during cool ripening for sixty and ninety days at 0, 4, and 7.2°C…………………………………………………………………………….86
xi 4.6 Digital analysis of splits during cool ripening for sixty and ninety days at 0, 4, and 7.2°C……………………………………………………………………………..86
B.1 Viable cell counts of lactic acid bacteria used to manufacture Swiss cheese in triplicate with different P. freudenreichii strains…………………………...…..104
xii LIST OF FIGURES
Figure Page
1.1 Common compatible solutes synthesized or imported by bacteria…………....…11
1.2 Two-step oxidation process of glycine betaine from choline……………………13
2.1 Maximum growth rates (umax) and lag phase for P. freudenreichii strain P843…48
2.2 Maximum growth rates (umax) and lag phase for P. freudenreichii strain P572....49
2.3 Maximum growth rates (umax) and lag phase for P. freudenreichii strain P728....50
2.4 Maximum growth rates (umax) and lag phase for P. freudenreichii strain P873…51
2.5 Transport of glycine betaine, proline, and glutamate by P. freudenreichii strains at 30°C……………………………………………………………………………...52
2.6 Transport of glycine betaine, proline, and glutamate by P. freudenreichii strains at 22°C……………………………………………………………………………...53
2.7 Transport of glycine betaine, proline, and glutamate by P. freudenreichii strains at 10°C……………………………………………………………………………...54
2.8 Transport of glycine betaine, proline, and glutamate by P. freudenreichii strains at 7.2°C……………………………………………………………………………..55
2.9 Transport of glycine betaine, proline, and glutamate by P. freudenreichii strains at 4°C………………………………………………………………………………56
4.1 Digital images of cheeses manufactured with different P. freudenreichii strains ripened at different temperatures………………………………………………..87
xiii A.1 Effects of various concentrations of glycine betaine, proline and glutamate on the
growth of P. freudenreichii strains P843, P572, P728, and P873 at 30°C……...99
A.2 Effects of various concentrations of glycine betaine, proline and glutamate on the
growth of P. freudenreichii strains P843, P572, P728, and P873 at 22°C……..100
A.3 Effects of various concentrations of glycine betaine, proline and glutamate on the
growth of P. freudenreichii strains P843, P572, P728, and P873 at 10°C……..101
A.4 Effects of various concentrations of glycine betaine, proline and glutamate on the
growth of P. freudenreichii strains P843, P572, P728, and P873 at 7.2°C…….102
B.1 Digital images of P. freudenreichii strain P572 (cold-sensitive) at 0, 4, and 7.2°C for 60 days and 0, 4, and 7.2°C for 90 days……………………………………105
B.2 Digital images of P. freudenreichii strain 196(intermediate) at 0, 4, and 7.2°C for 60 days and 0, 4, and 7.2°C for 90 days………………………………….……106
B.3 Digital images of P. freudenreichii strain P873 (cold-tolerant) at 0, 4, and 7.2°C for 60 days and 0, 4, and 7.2°C for 90 days……………………………….…..107
xiv CHAPTER 1
LITERATURE REVIEW
1.1 Introduction
Cheese is produced through the method of controlled fermentation in which a starter culture, a large number of specific microorganisms, is added to raw materials and the conditions are adjusted for the growth of theses microorganisms. The distinct characteristics of various cheese types are attributed to the use of a starter culture that ferments the milk sugar lactose, a disaccharide comprised of glucose and galactose.
These starter cultures are subjected to high temperature shifts (up to 55°C for 30-60 minutes) followed by a long cooling period (20-24 hours) (Boyaval et al., 1999). Swiss cheese starter cultures contain three different bacteria, Lactobacillus helveticus,
Streptococcus thermophilus, and Propionibacterium freudenreichii. L. helveticus and S. thermophilus are classified as lactic acid bacteria (LAB), and P. freudenreichii are known as propionic acid bacteria (PAB). Some of the LAB characteristics are gram-positive, salt tolerant, acid tolerant, thermophilic, catalase negative, and non-sporeforming.
Lactobacilli undergo cell lysis shortly after cheese manufacture which results in a large production of free amino acids. The growth of PAB is highly dependent on the ability of lactobacilli to produce these amino acids regardless of the lactobacilli strain (Baer and
1 Ryba, 1999). PAB are competitive inhibitors of other microorganisms due to the
production of propionic acid and other antifungal and antibacterial substances such as
bacteriocins (Meile et al., 1999).
Lactic acid bacteria cannot respire due to an incomplete Krebs cycle and electron
transport system and therefore generate adenosine triphosphate (ATP) by fermentation.
There are two main glucose fermentation pathways, homolactic and heterolactic. In the
homolactic pathway, fermentation of glucose results in lactic acid and ATP. In the
heterolactic pathway, fermentation of glucose results in lactic acid, ethanol, carbon
dioxide, and ATP.
Amino acids are precursors to flavor and aroma compounds. LAB require some
amino acids from the environment but cannot make all of the amino acids required for
synthesis of cellular proteins. The bacteria must degrade proteins to obtain the amino
acids required for growth. The breakdown of the protein matrix causes cheese curds to
knit together which softens the texture. There must be a balance in proteolysis. If too
much proteolysis occurs then there is a decrease in cheese yield and off flavors result. If
there is too little proteolysis then there is slow culture growth, slow acid production and a
bitter flavor may be present.
1.2 Cheese Manufacture
There are basic steps in cheese manufacture that are the same among all cheese
types. These steps include setting the milk, cutting the curd, cooking the curds, draining
the whey, salting the curds, pressing and ripening. Setting or ripening the milk involves inoculating the milk with a starter culture at a certain temperature to start acid production.
2 It also involves adding a coagulant such as rennet to aid in coagulation. Once
coagulation has ensued and the curd has been set it is then cut into small cubes to initiate whey expulsion. The firmness of the curd when cut and the amount of whey removed determine if a soft or hard cheese will result. The more whey that is expelled the harder the cheese that will result. A hard cheese is formed from a soft set curd that has been cut into small cubes. These conditions allow for more moisture loss. A soft cheese is formed from a firm set curd that has been cut into large cubes and it is under these conditions that less moisture is loss.
The curd particles are cooked in the whey. The amount of whey expelled and the rate of acid production are influenced by the cooking temperature. More moisture will be loss at a higher temperature and less will be loss at a lower temperature. A temperature closer to the optimum growth temperature of the cultures will result in faster acid production. After cooking, the whey is drained by separating the particles from the whey. It is during this time that the lactic acid production increases while the pH rapidly decreases. The curds are pressed in a shaped mold and pressure is applied usually for 12-
14 hours. This aids in more whey expulsion while providing shape. Once the cheese has been pressed the solid mold is brined in a salt solution. Brining stops lactic acid production, adds flavor, and inhibits the growth of some spoilage microorganisms. The cheese is then ripened in an environment where the temperature and humidity are regulated.
Cheese ripening involves changes in the microbial population and chemical reactions. These changes are influenced by temperature, moisture, salt, pH, and oxygen availability. The change in microbial population includes the gradual death of the starter 3 culture bacteria, addition of other bacteria, yeasts or molds, and unintentionally added bacteria. Some of the chemical changes that take place during ripening include the degradation of carbohydrates, proteins, and lipids. These changes influence flavor, texture, and appearance.
1.2.1 Swiss Cheese Manufacture
The manufacture of Swiss cheese follows the aforementioned process under
“Cheese Manufacture” with some variations. Raw milk is pasteurized and ripened by
adding a starter culture containing Lactobacillus helveticus, Streptococcus thermophilus,
and Propionibacterium freudenreichii and a coagulant to form a paracaseinate or enzymatic curd. L. helveticus and S. thermophilus are usually added in a 1:1 ratio
(Reinbold, 1972). The milk is allowed to set and then cut into curds the size of rice grains. After cutting the curd, the curd is continuously stirred before cooking. This is forework and it allows for the curds to shrink and whey expulsion to ensue. Cooking involves gradually raising the temperature to further induce necessary physical changes in the curd such as elasticity. During cooking the curd is continuously agitated to prevent curd accumulation and knitting. There is also rapid whey expulsion, an increase in acid production, and rapid curd syneresis thus producing smaller, firmer, and more elastic curd particles. Once the desired cook temperature has been reached, the curds are pumped under part of the whey into a pressing mold lined with cheesecloth in a vat for pressing. The curd mass is carefully handled to avoid disruption to any curds that have begun to knit together. The whey is drained and the curds pressed to induce a further decrease in pH. The curds are pressed overnight (12 to 14 hours) and the pH measured.
4 Once the desired pH is reached the cheese is brined in a 23% saturated salt solution. The surface of the cheese block is dried before wrapping to prevent rind rot and then pre- cooled at 45 to 55°F (7.2 to 12.7°C) for up to ten days. This period helps reduce the level of fermentable sugars to trace amounts and prevent further growth of LAB while the cheese matrix becomes more elastic thus beginning the ripening process. The autolysis of PAB may be important for the release of intracellular peptidases, which may influence ripening (∅stlie et al., 1999).
1.2.2 Swiss Cheese Eye Formation
The determination of U.S. grades of Swiss cheese is based on the rating of several quality factors including flavor, body, eyes and texture, finish and appearance, and color
(USDA). The interest of this study was the quality of eye formation, eye deformations and related texture. Swiss cheese has holes (eyes) that are developed through the fermentation of Propionibacteria. These holes or eyes should be smooth and shiny as opposed to the rough, uneven and dull appearance of the mechanical openings originating from incomplete knitting of curd particles within the cheese body (Reinbold, 1972).
Swiss cheese is ripened in two phases, a warm ripening and a cool ripening. Warm room ripening temperatures range between 72° to 76°F (22.2 to 24.4°C) and cool room ripening temperatures range between 36°to 55°F (2.2 to 12.7°C) however temperature of
40°F (4.4°C) or lower are recommended (Reinbold, 1972). Lactobacillus helveticus and
S. thermophilus provide a relatively slow development of lactic acid throughout the curdmaking process. Streptococcus thermophilus acts as the initial and primary lactic acid producer and L. helveticus acts as the secondary lactic acid producer producing lactic 5 acid during the period of the coagulum to brining and causing proteolysis in later ripening
as well as the formation of flavor compounds (White et al, 2003).
The lactic acid produced from these bacteria is metabolized by P. freudenreichii into
propionate, acetate, and CO2 when the cheese is stored at 68° to 77°F (20-25ºC) during
the warm ripening phase. The propionate and acetate contribute to the cheese’s flavor
and the CO2 contributes to eye formation. Propionbacterium freudenreichii growth is encouraged during this time because the texture of the cheese is more pliable and able to withstand gas production. The cheese’s pH at this stage (5.2-5.4) is crucial to ensure an elastic texture. Propionibacteria also possess probiotic properties because of their ability to produce vitamin B12 (Jore et al., 2001).
Reinbold (1972) states five purposes for cold storage following eye development: 1) to arrest eye development at the proper size; 2) to chill and firm the cheese so that it may readily be handled, stored and cut; 3) to inhibit bacterial growth other than, as well as, the propionbacteria; 4) to permit continued flavor development; and 5) to avoid onset of certain body and flavor defects.
Eye formation is determined by gas production, hole nucleation, and growth in a protein matrix with appropriate mechanical and physicochemical properties (Noël et al.,
1999). The high fracture strain and high fracture stress in Swiss type cheeses contribute to its high mechanical resistance. Gas diffuses within the cheese then outside. Gas holes are usually round and shiny, and a Grade A Swiss cheese should possess eyes 3/8 to
13/16 inch in diameter (USDA). There are certain conditions that should be consistent for proper eye formation. These include a high mechanical resistance of bonds between curd particles to support the internal pressures, the cheese matrix must be locally
6 saturated with gas allowing creation of local overpressure which would stress the curds around the gas pocket, and gas production when the mechanical, biochemical, and structural properties of the cheese are appropriate (Noël et al., 1999). The cold storage temperatures must also be consistent because the loss of elasticity and increase in gas pressure potential can produce splits and cracks if temperature fluctuations occur. This defect can be unappealing to consumers who desire pre-sliced prepackaged Swiss cheese.
1.2.3 Split Defects
Splits and cracks can be found most often in cheeses aging in the finishing cooler because cheese provides the proper degree of anaerobiosis for fermentation to continue
(Hettinga et al., 1974). The basic control of eye development depends upon the evolution of proper body and texture in the cheese curd, both initially and throughout making and curing (Reinbold, 1972). Proper body refers to sufficient elasticity to permit gradual expansion of eyes without roughness or other defects and proper texture refers to a sufficient number of faults, openings or areas of incompletely knit curd that will determine the number and location of the eyes.
There are many factors that can contribute to split defects, including late gas production, citrate fermentation, and changes in CO2 solubility related to changes in temperature. Splits and cracks can also be a result of secondary fermentation, which is accompanied by increased proteolysis of casein identified by high values of trichloracetic acid soluble nitrogen (Baer and Ryba, 1999). The intensity of secondary fermentation has been correlated with the amount of free amino acids in the cheese due to preference
7 of free amino acids to peptides by propionic acid bacteria suggesting that PAB prefer free amino acids for their growth in cheese.
The combination of an increase in internal pressure and low resistance to fracture can also contribute to split formation (Noël et al., 1999). If the cheese is too rigid, a spilt or crack will form from its inability to bend during gas production. This usually occurs after the warm room and during the cold room because the flexibility of the cheese changes during the cooler ripening phase when the cheese is stored at 39.2 to 45° F (4 to
7.2°C). The increase in pH from proteolysis during this time also contributes to the cheese’s decreased flexibility. According to White et al. (2003), cracks can be as small as one centimeter or extend over an entire 90 kilogram block of cheese. The split formation increases with time, usually between 60-120 days of storage (White et al.,
2003).
Splits can also be influenced by the rate of gas production. If gas production is too rapid the casein network becomes inflexible to gas pressure, and if it is too slow cheese will become underset with small eyes or no eyes. Starter and nonstarter propionibacteria that produce gas at 39.2 to 45°F (4 to 7.2°C), starter and nonstarter lactic acid bacteria metabolite stimulation of propionibacteria, and the presence and germination of Clostridium spores can also contribute to splits (White et al., 2003). The presence of Clostridium is usually found in cheese made with milk during the winter months. Hettinga et al. (1974) states that the type of wrapping material can affect the occurrence of splits and cracks. It was observed that cheese wrapped in highly permeable film is less likely to split as compared to cheeses wrapped in impermeable films.
8 Therefore cheese must be wrapped in a film that is selectively permeable to CO2 and impermeable to O2.
Reinbold (1972) states several factors that can help control the occurrence of split
defects: 1) selection of propionibacteria strains that do not grow readily at finished
cooler temperatures; 2) avoidance of fluctuation of temperatures over 36°F (2.2°C); 3)
cutting at a relatively young age (3 months if sufficient flavor has developed); 4)
avoidance of cultures containing S. diacetilactis and Leuconostoc spp.; 5) prompt cooling
of cheese following removal from the warm room to prevent overdevelopment of eyes; 6)
avoidance of too low pH values at pressing; 7) selection of a wrapper only impermeable
to mold growth; and 8) use of rigid boxes to prevent pressure during stacking and
vibration or shock during transport. The presence of splits may also be attributed to
continued gas production during the cooler ripening period which may be from cold
tolerance of the propionic acid bacteria provided by amino acids that serve as compatible
solutes.
1.3 Compatible Solutes as Osmoprotectants and Cryoprotectants
Compatible solutes are highly soluble, low molecular weight molecules that do
not carry a net charge at physiological pH. These compounds are considered compatible
because of their ability to accumulate at high concentration levels by de novo synthesis or
transport without interfering with vital cellular processes, such as DNA replication,
DNA-protein interactions, and metabolic machinery (Kempf and Bremer, 1998).
Compatible solutes aid bacterial proliferation in ecological niches that would otherwise
be considered inhibitory to their growth. They function as effective stabilizers of enzyme
9 function, providing protection against salinity, high temperature, freeze-thaw treatment,
and drying (Rosser and Muller, 2001 and Sleator and Hill, 2001). Small changes in
hydrostatic pressure can bring about changes in protein-protein and protein-ligand
interactions (Csonka and Hanson, 1991). In response to changes in the cellular
environment compatible solutes accumulate intracellularly and force proteins to remain in
their native folded state thus offering protein stability. The stabilizing effect compatible
solutes have on protein structure opposes protein unfolding and denaturation, acting as
salting-out ions of the Hoffmeister series (Yancy et al., 1982 and Bayles and Wilkinson,
2000). Therefore compatible solutes serve multiple roles in osmoregulating cells, restoring cell volume, and stabilizing protein structure (Sleator and Hill, 2001).
Compatible solutes can be in the form of sugars, polyols, or amino acids and their
derivatives. Some that have been identified include glycine betaine, proline betaine,
carnitine, glutamate, trehalose, and ecotine (Figure 1.1). Bacterial cells are required to maintain an intracellular osmotic pressure greater than that of the growth medium in order to generate cell turgor, generally considered to be the driving force for cell extension, growth, and division (Sleator and Hill, 2001). Compatible solutes have been found in Gram-positive and Gram-negative bacteria, and are known to provide osmoprotection through osmoregulation. This occurs during a change in osmotic stress, which results in the balance of the intracellular and extracelluar osmotic environments via high intracellular accumulation (Bayles and Wilkinson, 2000). This balance is due to an increase in the volume for free cytoplasmic volume, changes in the concentration of one or more solutes or the water concentration (Csonka and Hanson, 1991).
10 There are two types of osmoregulation phenomena: long-term or steady state responses that are manifested during the growth of organisms at a constant osmolality, and short-term or transient responses that occur soon after changes in the external
CH CH 3 3
+ - + - CH3 N CH2 COO CH3 N CH2 CH CH2 COO
CH3 CH3 OH CH2OH Glycine betaine Carnitine O
OH
OH OH CH2OH O - COO- OH COO O + N+ N OH OH CH CH HH 3 3 Proline Trehalose Proline betaine
FIGURE1.1 Common compatible solutes synthesized or imported by bacteria.
11 osmolality (Csonka and Hanson, 1991). The primary response to osmotic upshock is the
accumulation of potassium and glutamate with a secondary response leading to the
accumulation of more compatible solutes (Rosser and Muller, 2001). The composition of
the solute pool can vary in response to the growth phase and the growth medium. The
efficacy of solutes depends not only on their ability to replace potassium ions and
glutamate but also on their interactions with macromolecules (Csonka and Hanson,
1991).
Glycine betaine is one of the most widespread and most effective. It is a trimethyl amino acid and occurs at high concentrations in sugar beets and other food products of plant origin (Beumer et al., 1994). It is usually taken up from the environment due to the
inability of many organisms to synthesize it de novo. De novo synthesis of glycine
betaine is catalyzed in prokaryotes by oxidation of exogenous choline or methylation of
glycine (Rosser and Muller, 2001). Production of glycine betaine is a two-step oxidation
process with glycine betaine aldehyde as the intermediate (Figure 1.2). Glycine betaine
aldehyde is a highly reactive compound that can readily form Schiff bases with free
amino groups (Boch et al., 1996). Glycine betaine is synthesized both by halophiles that
are adapted to living in high salt concentrations and non-halophilic bacteria that are
subjected to osmotic stress (Cleland et al., 2004).
Glutamate and trehalose are two compatible solutes that can be synthesized
internally by members of the family Enterobacteriaceae (Csonka et al., 1994). Glutamine
can be a precursor of glutamate, a major anioic organic solute in bacteria. Two pathways
synthesize glutamate: biosynthetic glutamate dehydrogenase and glutamate synthetase in
conjunction with glutamine synthetase (Csnoka et al., 1994). Trehalose is synthesized 12
CH3
+ Choline CH3 N CH2 CH2 OH
CH3
Choline dehydrogenase
CH3
+ Glycine betaine CH3 N CH2 COH aldehyde
CH3
Glycine betaine aldehyde dehydrogenase
CH3
+ - Glycine betaine CH3 N CH2 COO
CH 3
FIGURE 1.2 Two step oxidation process of glycine betaine from choline.
13 constituitively in archaebacteria and spores of eubacteria (Csonka and Hanson, 1991).
Carnitine is widely distributed in nature and predominantly found in foods of animal
origin. It is transported from the external environment by most bacteria because it can not be synthesized endogenously. Carnitine has been found to provide osmoprotection to
Lactobacillus plantarum and Listeria monocytogenes cells grown in medium containing added salt (Sleator and Hill, 2001). Proline has also been shown to accumulate in various bacteria in high intracellular concentrations following exposure to osmotic stress with
Gram positive bacteria being able to synthesize it and Gram negative bacteria being able to transport it.
1.3.1 Listeria monocytogenes
Listeria monocytogenes is a gram positive, non-sporeforming, non-acid-fast rod that is catalase positive. It is ubiquitous in nature and often isolated from food processing plant environments. It grows over a wide range of temperatures 34 to 113°F (1- 45°C) and therefore considered a pyschrotroph. The principle compatible solutes in L. monocytogenes are glycine betaine and carnitine and are involved in osmoregulation. L. monocytogenes cannot synthesize glycine betaine or carnitine but accumulates the compounds from the environment by active transport (Dykes and Moorhead, 2001 and
Smith, 1996). The rate and the extent of their transport can be controlled by regulation of the synthesis of the transport systems and by their biochemical activation by osmotic gradients, temperature, and other factors (Angelidis et al., 2002). Sigma factor B is one regulatory factor that could coordinate both osmotic and low temperature stress response in L. monocytogenes, whose activity is stimulated in response to osmotic upshift and
14 temperature downshift (Becker et al., 2000). Osmolyte accumulation may vary with the phase of bacterial growth (Dykes and Moorhead, 2001). It is suggested that L. monocytogenes has independent pathways for the adaptation to growth at low temperatures: a sigma factor B independent pathway that can be entered into by log- phase cells and a sigma factor B dependent pathway that can be entered into by stationary-phase cells (Becker et al., 2000). Osmolyte uptake is also controlled at the protein level by a novel osmolyte sensing mechanism in which uptake of both betaine and carnitine is subject to inhibition by preaccumulated solute (Sleator et al., 2003).
Beumer et al. (1994) demonstrated that exogenous proline, glycine betaine, and carnitine stimulated the growth of L. monocytogenes in a minimal medium in the presence of high salt concentrations. Although proline aided in growth, in Gram-positive bacteria a higher concentration (up to 50 mM) is needed to produce a considerable osmoprotective effect in comparison to <1 mM needed in Gram-negative bacteria. It was also found that exogenous betaine reduced lag time, which also increased the growth rate of L. monocytogenes in the presence of high salt concentration. Proline was also found to play a minor role because of its availability in small amounts due to its liberation from proteins by the proteases of other bacteria in foods (Beumer et al., 1994).
Bayles and Wilkinson (2000) showed that other betaines and betaine analogs also have cyroprotectant as well as osmoprotectant properties in L. monocytogenes.
Osmoprotectants increased the growth rate of NaCl stressed cells up to 2.6 fold compared to stressed cells without an osmoprotectant. They demonstrated that glycine betaine stimulated growth rate at 4°C and 7°C, but not at 30°C, and at 5°C glycine betaine reduced the lag phase in minimal media. The growth rate of the low temperature stressed
15 cells was increased 1.3-1.9 fold by cryoprotectants (Bayles and Wilkinson, 2000). This
stimulated growth rate may be due to the positive influence compatible solutes have on
membrane fluidity at low temperatures.
Angelidis et al (2002) showed that glycine betaine and carnitine were the major
compatible solutes accumulated from cell extracts grown in milk whey at 7 and 30°C
with the amount of accumulation being higher at 7°C (Angelidis et al., 2002). In late log phase carnitine accumulation was greater than glycine betaine accumulation with carnitine being predominant in a mutant strain and glycine betaine being predominant in a wild type strain. They also observed that the amount of accumulated osmolytes was lower in the stationary phase than in exponential phase but higher under chill stress than at 30°C, irrespective of growth phase (Angelidis et al, 2002). To confirm the observations from the growth assays done in whey, Angelidis et al. (2002) performed 13C
NMR spectroscopy and found that the concentrations of glycine betaine and carnitine
were several-fold higher in strains grown at 7°C than those strains grown at 30°C.
It is believed that chill stress affects protein structure and membrane fluidity
because cell kinetics are slow at low temperatures (Jaenicke, 1990 and Bayles and
Wilkinson, 2000). Ko et al. (1994) demonstrated that when the temperature is raised to
30°C, glycine betaine transport subsides and radioactivity measurements (counts per
minute) decreased indicating that glycine betaine is readily disposed when the cell is not
under chill stress and active transport of glycine betaine is stimulated by cold (Ko et al.,
1994). Gerhardt et al. (2000) observed transport of glycine betaine in whole L.
monocytogenes cells was stimulated at low temperatures in the absence of osmotic stress
as long as the membrane vesicles were energized by an exogenous energy source such as 16 ascorbate and phenzine methosulfate (PMS) when temperature was decreased to a range
of 4 to 15°C.
At low temperatures the strength of the hydrophobic effect is decreased and ionic interactions are increased, thereby destabilizing protein conformation, subunit
interactions and interactions with the membrane (Bayles and Wilkinson, 2002 and
Priovalov and Gill, 1988). Hydrophobic interactions weakened by low temperatures may influence the mechanism of cold activation of glycine betaine transport through changes
between membrane and protein.
1.3.2 Other Bacteria
Staphylococcus aureus is among the most osmotolerant of the nonhalophilic
eubacteria, growing at water activities as low as 0.86 (equivalent to 3.5M NaCl) (Graham
and Wilkinson, 1992. Glycine betaine, proline, taurine, as well has choline because of its
ability to be oxidized into glycine betaine have been observed as omoprotectants in S.
aureus with glycine betaine observed as being more effective and proline and taurine
playing secondary roles (Graham and Wilkinson, 1992). A direct proportionality had
been observed between the degree of osmotic stress and glycine betaine concentration.
Stimeling et al. (1994) observed S. aureus cells grown in the presence of glycine betaine accumulated significant pools of glycine betaine either under stressed or unstressed conditions exhibited lower rates of glycine betaine transport than those cells grown in its absence suggesting internal glycine betaine from the media inhibits the transport of external glycine betaine. This feedback also inhibits the transport of other potential
competitors such as proline by reducing their activity.
17 Glycine betaine and proline has displayed osmoprotective capabilities in
Lactobacillus acidophilus (Beumer et al., 1994). The transport of the solute by the bacteria requires a metabolic energy source such as glucose. It was observed that omission of the energy source abolished betaine uptake (Hutkins et al., 1987). Boyaval et al. (1999) observed that trehalose was the most abundant compatible solute in stressed and unstressed P. freudenreichii cells, whereas glutamate and alanine were present only in cells that were grown under hyper-osmotic conditions suggesting that these compounds may serve to alleviate the growth inhibition caused by salt.
Glycine betaine uptake was activated in Lactococcus lactis by hyper-osmotic stress; with an instantaneous efflux via channel-like activities being observed once cells were washed with hypo-osmotic solution (Heide and Poolman, 2000). In L. lactis, proline transport is inducible and therefore dependent on energization of cells with an energy source but betaine uptake was found to be independent of cell energization and effectively inhibited proline transport (Molenaar et al., 1993). Sigma factor B is known in
Bacillus subtilis to control transcription of a large general stress regulon, whose products include different classes of proteins that alleviate the physiological consequences of environmental and nutritional stress conditions (Becker et al., 2000). Glycine betaine has also been observed to be a better osmoprotectant than carnitine in Eschericia coli because the longer carbon chain length of carnitine decreases its osmoprotective function (Becker et al., 2000). Lamosa et al. (1998) examined and compared the compatible solutes and strategies of thermoadaptation and osmoadaptation of genera Pyrococcus and
Thermococcus, thermophiles and hyperthermophiles respectively. It was found that
Pyrococcus species accumulated two compatible solutes, mannosylglycerate (MG) and
18 di-myo-inositol-1,1’(3,3’)-phosphate (DIP). The concentration of MG increased as the salinity of the medium increased and the concentration of DIP increases sharply in response to supraoptimum temperatures. Thermococcus species showed increased levels of MG, aspartate, and β-galactopyranosyl-5-hydroxylysin (GalHL) in response to salt stress and increased levels of DIP in response to temperature stress.
1.4 Effects of Membrane Composition on Compatible Solute Transport
Fluidity and permeability of the membrane are vital parameters to maintain cell homeostasis and effective transmembrane processes, such as nutrient transport or solute gradients for energetic purposes (Guillot et al., 2000). There is an alteration in fatty acid content to control membrane viscosity when variation in temperature, pH, ethanol or osmolality occurs. Gerhardt et al. (2000) state that the temperature dependence of transport below 18°C is independent of the nature of the solute suggesting that transport may be influenced by the physical state of the membrane and the nature of the stressing solute (Gerhardt et al., 2000). Temperature regulates the extent of membrane desaturation. Bacteria increase the level of unsaturated fatty acids to maintain appropriate fluidity of membrane lipids when there is a decrease in ambient temperature
(Sakamoto and Murata, 2002). As previously stated L. lactis transports glycine betaine.
Guillot et al. (2000) observed that activation of betaine transport in L. lactis was always higher when there was an increase in the ratio of unsaturated fatty acids to saturated fatty acids in the membrane composition. This increase was a result of membrane alteration from change in salt concentration, decrease in temperature or addition of Tween-80. This
19 suggests that changes in membrane properties could be involved in the regulation of membrane embedded protein activity.
Cold shock proteins are induced in response to cold shock. Cold shock protein
A (CspA) is induced after a temperature shift and regulates other cold shock genes (Kim and Dunn, 1997). This study suggested that cold shock prior to freezing might improve cell viability. Cold induced expression of genes for fatty acid desaturases increases the
extent of desaturation of fatty acids at low temperatures (Sakamoto et al., 2001).
Therefore there may be a mechanism for the perception and transduction of cold signals
via a cold sensor such as histidine kinase.
1.4.1 Compatible Solute Transport Systems
Osmoprotectant transporters usually exhibit high affinity for their substrates with
Km values in the micromolar range, and their capacity is geared to permit high-level
compatible solute accumulation (Kempf and Bremer, 1998). Gram-positive and Gram-
negative bacteria differ in their compatible solute transport systems. Transport systems
have been identified for L. monocytogenes, S. aureus, Escherichia coli, Bacillus subtilis
and Cornyebacterium glutamicum with L. monocytogenes being the best characterized of
the Gram-positive bacteria. These transport systems serve as osmosensors, acting as
transducers of the resulting signal and the respondent, which mediates cytoplasmic
compatible solute accumulation (Wood et al., 2001). These transport systems are either
osmotically activated or chill activated, and are regulated at the level of gene expression
or transport activity. There are two responses, a primary and secondary response. The
primary osmoregulatory mechanism is a homeostatic control circuit that maintains turgor
20 within a range that can support cell growth (Csonka and Hanson, 1991). This mechanism
involves a stimulation of potassium uptake to counterbalance the negative charge from
hyper-osmotic shock because the concentration of potassium ions is the regulatory signal
for all other osmoregulatory responses. The secondary response, compatible solute
accumulation, may be induced by potassium ion uptake from the primary response.
Compatible solutes are capable of being discharged via carrier like systems opening
mechanosensitive efflux channels. These channels are activated by membrane stretch.
The review below will focus primarily on compatible solute transport systems in L.
monocytogenes as well as other bacteria.
1.4.1.1 Listeria monocytogenes
Listeria monocytogenes is incapable of synthesizing compatible solutes and must
transport them from the environment. Osmotically activated and chill activated transport
systems for this bacterium have been documented. There are two types of glycine
betaine transporters. Glycine betaine transporter I is a sodium-driven, osmotically
activated glycine betaine uptake system that is functional in membrane vesicles. It is
activated by osmotic stress and couples the transport of glycine betaine to sodium ion
influx. This transporter responds quickly to upshock to provide immediate protection and
can provide long-term protection for low levels of stress and appears to be responsible for
the majority of glycine betaine uptake following osmotic upshock (Mendum and Smith,
2002). Glycine betaine transporter II belongs to the family of ATP binding cassette
(ABC) transporters whose activation is dependent upon the presence of a solute gradient
21 or the presence of low temperatures (Gerhardt et al., 2000) and can therefore be activated
by osmotic stress as well as cold stress.
Glycine betaine transporters in L. monocytogenes have been specifically identified as BetL, OpuC, and Gbu. BetL is a secondary uptake system that couples betaine accumulation to a Na+ motive force and appears to be responsible for the majority of betaine uptake immediately after osmotic up-shock thus providing immediate protection
(Sleator et al., 2003). It is homologous to the betaine transporter OpuD of B. subtilis and
BetP of C. glutamicum (Wemekamp-Kamphuis et al., 2002). Glycine betaine also has significant similarity to the Gram-negative choline transporter BetT from E. coli (Sleator et al., 1999). These systems act as secondary transporters that transport an ion in symport with the compatible solute. BetL is highly specific for glycine betaine and fails to transport other trimethylammonium compounds such as carnitine or choline (Sleator et al., 1999), and classified as betaine porter I. Transcription of betL has been proposed to be under control of the L. monocytogenes general stress-related transcription factor sigma
B, whereas gbu appears not to be under its control (Angelidis et al., 2002). According to
Sleator et al. (1999), a sigma B knockout was affected in its ability to accumulate glycine betaine under high osmlarity conditions suggesting that high osmolarity may stimulate increased transcription of betL in addition to activating preexisting BetL proteins. As previously stated, glycine betaine transporter II can be activated by either osmotic or cold stress but does not appear to be under the control sigma factor B (Angelidis, 2002).The transport of carnitine is chill and osmotically activated, and its accumulation contributes to tolerance to both stresses (Angelidis et al., 2002). OpuC is primarily responsible for carnitine uptake but transport may also proceed through one of the other glycine betaine 22 porters, BetL or Gbu. Sleator et al. (2003) observed that mutating the opuC gene resulted in a reduction in carnitine uptake and decrease in carnitine utilization as an effective osmoprotectant. OpuB may also be responsible for carnitine accumulation based on the observed residual carnitine uptake with a triple mutant (∆betL, ∆gbu, ∆opuC). Angelidis et al. (2002) demonstrated that a single transposon insertion in the opuCB impaired both the chill-activated and osmotically activated transport of carnitine suggesting that the same transporter is responsible for transport under both stresses suggesting that the salt activated and the chill activated transporter are one in the same. Thus this transporter plays a significant role in the observed chill tolerance of L. monocytogenes in the absence of glycine betaine (Angelidis et al., 2002).
To evaluate osmolyte transport, studies were conducted using multiple deletion mutants with mutations in the known osomolyte transporter. The deletion of the betL gene lowered the specific growth rate of the bacteria at high osmolarities, and deletion of gbu also decreased the growth rate at high osmolarities as well as at low temperatures
(Wemekamp-Kamphuis et al., 2002). The growth rate of the double mutant betL and gbu was lower than the double mutant opuC and gbu showing the significance of the BetL and Gbu transporters. Betaine transport was reduced from 43 to 37 nmol mg of cell protein –1 min –1 when there was a disruption in opuC. Single mutants showed differences in betaine and carnitine accumulation. Disruption in betL and opuC resulted in about 70 µmol/g betaine accumulation and carnitine was undetectable (below
40µmol/g). Disruption in gbu resulted in a higher accumulation of carnitine than betaine,
300 µmol/g and 125 µmol/g, respectively. This suggests Gbu may be the main betaine
23 transporter. Gbu is chill responsive and primarily transports betaine and classified as betaine porter II (Sleator et al., 2003). The Gbu system is ATP-dependent, and plays a major role in long-term regulation. It is encoded by the gbuABC operon, which belongs to the binding protein dependent ATP binding cassette superfamily homologous to OpuA in B. subtilis. This operon contains three genes: gbuA encodes ATPase, gbuB encodes permease, and gbuC encodes a substrate binding protein (Wemekamp-Kamphuis et al.,
2002). Gbu and OpuC are activated by chill and BetL is not suggesting that activation by chill may involve a change in the organization of the subunits of multimeric transporters, perhaps mediated by changes in the physical state of the membrane (Angelidis et al.,
2002).
1.4.1.2 Other bacteria
Many other bacteria possess two or more glycine betaine transport systems. B. subtilis has three transport systems for glycine betaine: OpuD, OpuA, and OpuC. These osmotically controlled uptake systems allow glycine betaine to be accumulated directly from the environment into the bacteria or be synthesized from the precursor choline or glycine betaine aldehyde in the growth medium (Boch et al., 1996). OpuD is a secondary transport system, and OpuA and OpuC are two binding dependent transport systems.
These systems are related to the binding protein-dependent transport system ProU from
E. coli (Boch et al., 1996). Bacillus subtilis has the OpuE system for proline transport
(Rosser and Muller, 2001) and the OpuB system, which exhibits high substrate specificity for choline (Kempf and Bremer, 1998).
The genes gbsA and gbsB may be the only genes required by B. substilis for glycine betaine synthesis. Boch et al., (1996) found that the genes encoding the glycine 24 betaine biosynthetic enzymes are separated from the locus encoding the choline transport system. It was found that the combination of high medium osmolarity and the presence of choline in maximally stimulated the expression of the gbsAb gene, which suggests that these two environmental factors act concurrently to control the level of gbsAB transcription through a regulatory protein exists for the choline glycine betaine pathway of B. subtilis.
A constitutive low affinity ProP system and osmotically induced high affinity
ProU system are found in Salmonella typhimurium, E. coli and L. lactis. These systems transport proline with a low affinity and betaine with a higher affinity, and are activated by high osmolality at the levels of both transcription and catalytic rate (Molenaar et al.,
1993). The uptake of proline by these systems is inhibited by betaine. In S. typhimurium the steady state level of expression of the proU operon increases with increasing osmolality in cells that have achieved complete osmotic adaptation with their environment (Csonka et al., 1994). This suggests that a large solute pool is not necessary for induction of the operon and that levels of expression may not correlate with solute pool levels. A transcriptional silencer located downstream of the promoter provides a negative mechanism that mediates the osmotic control of proU expression.
The ProP transport system found in E. coli is an example of long-term regulation. This system is a permease for proline and glycine betaine and is synthesized at a nearly constitutive level but its activity is stimulated in cells growing exponentially in media of high osmolality (Csonka and Hanson, 1991). Salmonella typhimurium and E. coli also possess a third proline transport system, PutP. This system serves to transport proline
25 solely for use as a carbon or nitrogen source and has a very little role in osmoadaptation
(Sleator and Hill, 2001).
C. glutamicum has four secondary tranporters, BetP, PutP, ProP, and EctP, respond to osmotic upshock by accumulating potassium ions for proline transport
(Rosser and Muller, 2001). BusA has been identified as the glycine betaine transport system in Lactococcus lactis subspecies lactis but not subspecies cremoris (Obis et al.,
2001). BusA is an ABC transporter encoded by the busA operon. OpuA, OpuC, and
ProU couple hydrolysis of ATP to substrate translocation across the membranes, but
OpuD and BetP form single component mechanisms which couple proton motive force to solute transport across the membrane (Sleator et al., 1999).
1.5 Conclusion
Mechanisms that permit low-temperature growth of microorganisms include modifications in DNA supercoiling, maintaining membrane fluidity, regulating uptake and synthesis of compatible solutes, production of cold-shock proteins, modulating mRNA secondary structure, and maintaining the structural integrity of macromolecules and macromolecule assemblies such as ribosomes (Wouters et al., 2000). This review focused on uptake regulation and synthesis of compatible solutes. The role of compatible solutes in osmoregulation and osmoadaptation has been well established in both Gram positive and Gram negative bacteria yet their role in cold adaptation is not well defined.
Therefore their role was examined in dairy Propionibacteria at temperatures lower than
26 the optimum. Compatible solutes may have a positive effect in the ability of these bacteria to adapt to temperatures lower than the optimum which may result in continued fermentation.
27 CHAPTER 2
COLD TOLERANCE AND COMPATIBLE SOLUTE UPTAKE OF DAIRY PROPIONIBACTERIUM FREUDENREICHII
2.1 Introduction
There are two types of propionibacteria: classical and dairy. Dairy
propionibacteria are used in the manufacture of Swiss cheese. These bacteria are
responsible for the characteristic eyes associated with Swiss cheese. Their optimum growth temperature is 30°C and growth is strongly encouraged during the warm ripening period (20-24°C) (Boyaval et.al, 1999). After warm ripening, Swiss cheese undergoes further ripening at cooler temperatures (0-7.2°C). Propionibacteria must endure extreme conditions during Swiss manufacture such as increased cook temperatures, decreased ripening temperatures, and increase salt concentrations. The bacteria are capable of surviving these environmental changes through the aid of compatible solutes and other stress response mechanisms.
Compatible solutes are molecules that allow microorganisms to respond to
physiological changes in the environment. They have been shown to provide
osmoprotection in various microorganisms and plants during osmotic stress. When
bacteria are subjected to osmotic stress, organic solutes, which are often the
osmoprotectant molecules, accumulate intracellularly in high concentrations therefore
becoming major osmolytes allowing the organisms to re-establish osmotic balance
28 (Bayles and Wilkinson, 2000). Their compatibility lies in their ability to exist in high
concentrations but not interfere with cellular functions such as DNA replication, DNA-
protein interactions, and metabolic machinery.
Compatible solutes have demonstrated not only the ability to provide
osmoprotection during changes in osmolality, but cryoprotection during a decrease in
temperature as well (Bayles and Wilkinson, 2000). Cryoprotectants have excellent
colligative properties with water, which both retard the formation of intracellular ice
crystals and reduce the potential for osmotic injury during the freezing and thawing
processes (Cleland et. al, 2004).
Compatible solutes can be in the form of sugars, polyols, or amino acids and their
derivatives. Some that have been identified include glycine betaine, proline betaine,
carnitine, glutamate, trehalose, proline and ecotine. Glycine betaine, a choline derivative,
is the only compatible solute that has displayed both osmoprotective and cryoprotective
properties. These properties have been observed extensively in Listeria monocytogenes.
It is a trimethyl amino acid and occurs at high concentrations in sugar beets and other food products of plant origin (Beumer et al., 1994). It is usually taken up from the environment due to the inability of many organisms to synthesize it de novo. De novo
synthesis of glycine betaine is catalyzed in prokaryotes by oxidation of exogenous
choline or methylation of glycine (Rosser and Muller, 2001). It is synthesized in a two-
step oxidation process with glycine betaine aldehyde as an intermediate that can readily
form Schiff bases with free amino acids (Boch et.al, 1996 and Noël et al., 1999). Two
pathways synthesize glutamate: biosynthetic glutamate dehydrogenase and glutamate
synthetase in conjunction with glutamine synthetase (Csnoka et al., 1994). Proline has
29 been found in high concentrations in Gruyére and Emmental (Swiss) cheese and production of this amino acid is generally associated with growth of large numbers of
propionibacteria (Hettinga and Reinbold, 1972). Proline can be synthesized from
glutamate by three enzymes: γ-glutamyl kinase, γ-glutamyl phosphate reductase, and ∆1-
pyrroline-5-carboxylate reductase (Samaras et al., 1995).
Food provides a ready source of compatible solutes in products of animal and
plant origin (Dykes and Moorehead, 2001), with cheese being a good source of glycine
betaine and proline. Amino acids are not essential for Propionibacterium growth
whereas, certain vitamins, minerals, and unknown constituents of yeast extract are
required for growth and metabolism (Hettinga and Reinbold 1972). According to Baer
(1999), propionic bacteria prefer free amino acids to peptides for their growth in cheese, though this point has been disputed and it remains unclear whether propionibacteria prefer peptides or amino acids. Propionicbacteria contain the necessary peptidases that may be required for producing essential amino acids (Hettinga and Reinbold, 1972;
Limpisathian and Courtney, unpublished data). The break down of casein proteins during ripening provides the free amino acids, which may stimulate growth thus promoting cold tolerance of Propionibacterium strains.
The ability of compatible solutes to aid in cryoprotection of Propionibacterium is not clearly understood, thus the present study investigated the effects of glycine betaine, proline, and glutamate on the growth of P. freudenreichii strains and the transport of these compounds at temperatures lower than optimum.
30 2.2 Materials and Methods
Bacterial strains and growth media
Forty-three Propionibacterium freudenreichii strains were isolated from
commercial starter cultures or cheeses. The names of the cultures and companies were
coded for proprietary reasons. Strains were grown in complex or chemically defined
medium. The complex medium used was sodium lactate broth (SLB) (20 g L-1 Bacto tryptone, 5 g L-1 yeast extract, 5 g L-1 sodium lactate), and the chemically defined
medium (CDM) used was the minimal medium with additional methionine and cysteine
(MMC) described by Glatz and Anderson (1988). All CDM was filter sterilized. Strains
were grown in SLB and then transferred to CDM prior to growth and transport assays.
Growth of Propionibacteria in the Presence of Exogenous Glycine Betaine, Proline, and Glutamate
Four strains were selected based on their growth abilities in SLB. The strains
were grown in SLB at 4, 7.2, 10, 22, and 30°C, and the growth rate was evaluated to
determine cold tolerance which was used to characterize the strain as cold tolerant or cold
sensitive. The selected strains were P843, P572, P873, and P728. These four strains were
grown anaerobically to mid-log phase in CDM in the presence of the compatible solutes
glycine betaine, proline, or glutamate. Glycine betaine, proline, and glutamate were
added to final concentrations of 1 mM or 100 mM, and the media were filter sterilized. A
negative control was used for each strain to which no compatible solute was added to the
CDM.
According to Noël et al. (1999), the temperature range for Propionibacterium growth is 3-40°C, with an optimal value of 30°C. The strains were first grown at the
31 optimal growth temperature and monitored every day for fifteen days to establish the
maximum growth rates for each strain. The strains grown at 22°C, mimicking warm
room ripening temperature, were also monitored every day for fifteen days. The strains
grown at 10, 7.2, and 4°C, mimicking cool ripening and distribution, were monitored for
every five, seven, and ten days respectively for ninety days. Bacterial growth was
monitored spectrophotometrically by measuring absorbance at 600 nm (A600) (Spectronic
20 Genesys, Spectronic Instruments, Rochester, NY).
The maximum specific growth rate (µmax) and lag phase, defined here as time to reach µmax, were determined by fitting the growth curves to the Richards model with the
m parameter fixed to 2.0 as described by Dalgaard and Koutsoumanis (2001) using the
SigmaPlot 9.0 software package (Systat Software, Inc., Point Richmond, CA). Curves
that did not reach an A600 of 0.2 within 45 days could not be fit to the Richards model and
were labeled as NG (no growth). The means of the values from duplicate or triplicate
growth curves were subjected to statistical analysis as described below.
Glycine Betaine, Proline, and Glutamate Transport Assays
The four P. freudenreichii strains used in the growth assay were also evaluated for
their ability to transport exogenous glycine betaine, proline, and glutatmate. The cells
were grown anaerobically to mid-log phase in 5 ml of complex media containing bacto-
tryptone (20g/L), yeast extract (5g/L), and lactic acid or glucose (5g/L) (Difco
Laboratories, Detroit, MI). A 5% (v/v) aliquot was transferred to 10 ml of CDM and
grown anaerobically to mid-log phase. The 10 ml culture was equilibrated to the desired
assay temperature of 4, 7.2, 10, 22, or 30°C for three hours. The bacterial culture was
centrifuged for five minutes at 7,500 revolutions per minute (rpm). The bacterial pellet 32 was washed with 8 ml of 50 mM potassium phosphate buffer, pH 6.5. The pellet was
once again recovered and resuspended in 8 ml of 50 mM potassium phosphate buffer
containing 10 mM glucose. All buffers were at the same temperature as the assay. The
cells were energized for five minutes at the assay temperature. During this incubation period, 1 ml of the cells was taken for protein concentration determination by the Bio-
Rad assay utilizing a bovine serum albumin standard (Bio-Rad, Hercules, CA).
After the five minutes, 7 µl of 14C-labeled (10µCi/mmol) glycine betaine, proline,
or glutamate were added to the 7 ml cell suspension with a final concentration of 1 mM.
The radiolabeled amino acids were purchased from American Radiolabled Chemicals (St.
Louis, MO). Timing began immediately after addition of the labeled amino acid, and
1ml samples were taken at 0, 5, 10, 15, 30, and 60 minutes. The cells were recovered by vacuum filtration through a 0.45µm cellulose acetate-cellulose nitrate membrane filter
(Fisher, Pittsburgh, PA), and washed with 10 ml of 50 mM potassium phosphate, pH 6.5 at the assay temperature. The filter was placed in 10 ml of Scinteverse SX4-18 scintillation fluid overnight (Fisher, Pittsburgh, PA), and the intracellular radioactivity was read the next day using a TRI-CARB SERIES liquid scintillation analyzer (Packard
Bioscience, Downers Grove, IL).
Statistical Analysis
Mean values for µmax and lag phase were compared by one-way analysis of
variance (SigmaStat 3.1, Sysstat Software, Inc.). Dunnett’s post-hoc test was used to
compare the µmax and lag phase of test conditions to a control (sample lacking compatible
solute). Values with P < 0.05 were considered to be significantly different.
33 2.3 Results
Effects of exogenous glycine betaine, proline, and glutamate on the cold tolerance of
P. freudenreichii
Of the forty-three Propionibacterium freudenreichii strains evaluated for growth
at lower temperatures, fifteen were characterized as cold sensitive based on relatively
reduced growth rates at 4 and 7.2°C compared to other strains (Anggraeni and Courtney,
unpublished results). Strains P843 and P572 were characterized as cold sensitive and
P873 and P728 were characterized as cold tolerant in SLB. The growth properties for P.
freudenreichii strains P843, P572, P873, and P728 were further studied in CDM containing 1 mM or 100 mM concentrations of glycine betaine, proline, or glutamate.
The maximum specific growth rate and lag phase of each strain at 30, 22, 10, 7.2, 4°C
with each compatible solute concentration was compared to growth without the
compatible solute.
The maximum growth rates and lag phases for the four strains varied in CDM
with glycine betaine, proline, or glutamate are shown in Figures 2.1 - 2.4 and Tables 2.1
– 2.4. The umax decreased in strain P843 as temperature decreased while lag phase
increased for the control and each compatible solute (Figure 2.1, Table 2.1). This was
observed at all temperatures and concentrations except at 22°C with 1mM proline in
which the maximum growth rate was higher than the growth rate at 30°C suggesting
strain P843 grows better at 22°C with 1mM proline at 22°C than at the optimum
temperature without the compatible solute. The maximum growth rate of strain P843 was
significantly (P<0.05) increased in the presence of 100 mM proline at 30°C with a
significantly shorter lag phase, 1.559 d-1 and 4.127 d, respectively (Table 2.1). This 34 suggests that strain P843 grows best at optimum temperature in the presence of 100 mM
proline. The umax was significantly higher and lag phase significantly shorter with all
compatible solutes compared to the control for strain P843 at both 22 and 10°C with no
observed growth of the control at 10°C. This suggests that growth of P843 is improved at these temperatures with compatible solute, regardless the specific compatible solute or its concentration. There was also no observed growth of strain P843 at 7.2°C with or without
addition of compatible solutes suggesting that growth is hindered at this temperature in
the absence or presence of the amino acids.
The maximum growth rate for strain P572 was significantly lower for all compatible solutes compared the control (1.343 d-1) at 30°C except for 1mM glycine
betaine (0.988 d-1) suggesting that growth was not affected by this concentration and not
improved by the other compatible solutes at this temperature (Table 2.2). At 22°C, the
µmax increased with both concentrations of glutamate (1 mM or 100 mM) and the lag
phase decreased suggesting glutamate improves the growth of strain P572 at 22°C
(Figure 2.2, Table 2.2). At this same temperature the maximum growth rate decreased
with 1 mM proline indicating it does not improve growth. The µmax for strain P572 was
significantly higher and lag phase significantly shorter than the control at 10°C for all compatible solutes suggesting growth was improved by their presence at this temperature.
Once again there was no growth observed at 7.2°C suggesting this temperature also hinders the growth of strain P572 as previously observed in strain P843 (Table 2.1).
There was no significant difference in growth observed at 30°C for strain P728 with compatible solutes (Table 2.3). The maximum growth rate was increased and the lag
35 phase was decreased at all other temperatures for all compatible solute concentrations
versus the control indicating growth was improved by all compatible solute
concentrations at 22, 10, and 7.2°C. Results at 7.2°C were most dramatic in which no
growth was observed in the control but µmax was significantly increased and the lag phase was significantly shorter at all solute concentrations (Figure 2.3).
The maximum growth rate increased at all temperatures for all compatible solute concentrations for strain P873 (Figure 2.4, Table 2.4). The increase in µmax suggests that
growth of strain P873 is helped by all compatible solute concentrations at all
temperatures, especially at 7.2°C in which no growth was observed in the control. The
lag phase was decreased at 10°C with 1 mM glycine betaine (13.32 d) and 1 mM
glutamate (12.97 d) in comparison to the control (18.85 d) (Table 2.4). There was also an
increase in µmax at these concentrations at this temperature, suggesting 1 mM glycine betaine and 1 mM glutamate improve the growth of strain P873 by increasing the maximum growth rate and decreasing the time to reach that rate. At 7.2°C, strain P873 exhibited a significantly shorter lag phase at all solute concentrations versus the control in which no growth was observed. There was no growth observed for any strains at 4°C
(data not shown).
The dairy propionibacteria differ in their abilities to grow at lower temperatures.
In SLB, strains P843 and P572 exhibited sensitivity to lower temperatures while strains
P728 and P843 exhibited tolerance. In CDM, strains P728 and P873 were the most tolerant at the lowest temperature (7.2°C) while strains P843 and P572 were the most
36 sensitive. All strains showed improved growth at low temperatures when any of the three
compatible solutes were added to the medium.
Transport of Exogenous Glycine Betaine, Proline, and Glutamate
Based on the effects of exogenous glycine betaine, proline, and glutamate on growth of four P. freudenreichii strains at lower temperatures, the ability of the same strains to transport glycine betaine, proline, and glutamate at lower temperatures was also evaluated.
Glycine betaine and glutamate were minimally imported at 30°C while proline was transported the most (Figure 2.5). The degree of proline transport in relation to glycine betaine and glutamate was highest at this temperature but varied between strains.
Proline was transported the most by strain P572 and transported the least by strains P873 and P843.
There was minimal glycine betaine transport at 22°C (Figure 2.6). Glutamate was imported the most by strain P843 at 22°C while its importation by strains P572, P728, and P873 was minimal. The extent of proline transport by all strains was the greatest at this temperature. It was transported the most by strains P843 and P572 and the least by
P728 and P873. Proline was also the most transported solute at 10°C by all strains
(Figure 2.7). It was transported the most by strains P843 and P572 and the least by strains P728 and P873.
Transport of all solutes by all strains was observed at 7.2°C (Figure2.8). Strains
P873 and P728 exhibited the greatest degree of glycine betaine transport at this temperature. Strain P843 imported proline and glutamate more than the other strains but transported glycine betaine the least. Proline was transported the least by P572. Glycine 37 betaine was transported the most by strain P873 but its transport of glutamate was lower
than the other strains.
At 4°C, proline was the most transported solute and imported by all strains
(Figure 2.9). Its degree of transport was the greatest in strain P843. Glycine betaine was
transported the most by strain P843 at this temperature and minimally transported by
P572, P728, and P873. Glutamate was transported at the same extent by all strains.
Importation of glycine betaine, proline, and glutamate at low temperatures differs
among P. freudenreichii strains. The ability of dairy propionibacteria to transport proline is not affected by temperature but temperature does affect glycine betaine and glutamate transport. Proline was transported by all strains at all temperatures but at various degrees.
2.4 Discussion
The optimum growth temperature for Propionibacterium is 30°C with an
established range of 3-40°C (Noël et al., 1999) and 12-45°C (Parks et al., 1967). It has
been reported that excessive Propionibacterium growth after warm room ripening and in
later ripening causes splits or cracks, which are undesirable (Hammond et al., 1965 and
Parks et al., 1967). Hettinga and Reinbold (1975) found that CO2 production coincided
with growth rate and strains that are able to grow at temperatures lower than 10°C have a
greater enzyme activity. This is critical because pyruvate dehydorgenase is the enzyme
responsible for CO2 production and this production creates a predisposition for Swiss
cheese to split when stored at lower temperatures (Hettinga and Reinbold, 1975).
Compatible solutes added to the growth medium exerted protective effects
(Boyaval et al., 1999; present study). Dykes and Moorehead (2001) reported that 38 accumulation may vary with the phase of bacterial growth stating stationary phase
cultures contain lower total levels of compatible solutes than rapidly growing cultures.
Intracellular pools of compatible solutes can be raised by increased synthesis or uptake
from the medium (Glaasker et al., 1996). The initial response may be more rapid if the
solute can be taken up from the medium or released into the medium via semiconstitutive
transport systems (Poolman and Glaasker, 1998). Compounds that have osmoprotectant
activity also tend to have cryoprotective activity (Bayles and Wilkinson, 2000). Glycine
betaine is a choline derivative that commonly serves as an osmolyte promoting
osmoadaptation in most bacteria in response to osmotic stress by increasing cytoplasmic
volume and free water content of the cells permitting continued cell proliferation under favorable conditions (Kempf and Bremer, 1998). It is found in cheese at a level of 0.59 mg per 100 g of cheese (Zeisel et al., 2003). L. monocytogenes is the only microorganism in which the cryoprotective effects of glycine betaine have been extensively reported. Glycine betaine reduced the lag phase in L. monocytogenes when grown at 5°C in minimal media and stimulated the growth rate at 4°C and 7°C but not at
30°C (Bayles and Wilkinson, 2000). The growth rate of low temperature stressed cells was increased 1.3 to 1.9 fold by cryoprotectants (Bayles and Wilkinson, 2000).
Activation and deactivation of glycine betaine transport are affected by temperature changes, which were observed by Ko et al. (1994) when transport subsided and radioactivity lost in cells at 30°C but active transport was stimulated by cold. Glycine betaine also serves as an osmoprotectant in Bacillus subtilis with the presence of choline in the growth medium stimulating the expression of the genes required for its synthesis
39 (Boch et al., 1996). As previously stated, choline is the required precursor for glycine
betaine synthesis.
Although glycine betaine is the preferred and most effective compatible solute for
L. monocytogenes, carnitine, other betaines and betaine analogs can substititue for glycine betaine to some degree (Angelidis et al., 2002). Proline betaine has been found in osmoregulation of Staphylococcus species and Lactobacillus acidophilus (Beumer et al.,
1994). Proline is more abundant than glycine betaine in Swiss-type cheeses with concentrations reported to range from 150 mg to 215 mg per 100 g of cheese (Mitchell,
1981). This allows it to play a more pivotal role as a compatible solute. Beumer et al.
(1994) reported that gram-positive microorganisms needed higher concentrations of proline (50 mM) to produce a significant osmoprotective effect. In this study, there was more of an increase in growth rate of Propionibacterium at lower temperatures with 100 mM proline than with 1 mM.
Glutamate is an important compatible solute in the members of the family
Enterobacteriaceae (Csonka et al., 1994). In these microorganisms it is synthesized internally, but other compatible solutes are taken up from the medium under conditions of high external osmolality (Csonka et al., 1994). Glutamate is an essential amino acid for
Lactobacillus plantarum and has to be taken up (Glaasker et al., 1996). Glycine betaine was accumulated only when the compound was added to the growth medium, indicating
L. plantarum cannot synthesize this osmolyte (Glaasker et. al, 1996).
Dairy propionibacteria differ in their growth capabilities at temperatures lower than the optimum (30°C) in a defined medium with and without glycine betaine, proline, and glutamate. These differences were based on observed differences in maximum
40 growth rates and lag phases in the presence and absence of the amino acids. There was an observed decrease in the maximum specific growth rate and an increase in the time to
reach that rate as temperatures decreased. They also differ in their ability to transport
glycine betaine, proline, and glutamate at various temperatures. Proline was the most
abundantly transported solute at all temperatures including the optimum, suggesting that
accumulation might be to satisfy basic growth requirements for amino acids in addition to
cryoprotection.
There was little transport of glycine betaine and glutamate at the optimum
temperature suggesting this temperature affects their degree of transport or the need for
these compounds is minimal but these compounds increased the growth rate of strain
P873 at 30°C although it transported proline the most at this temperature. The growth of
strain P572 was not improved by any solute concentration at 30°C shown in growth rates
lower than the control but transported proline the best which may be due to its ability to
accumulate proline but not synthesize it at this temperature. Growth was also not
significantly affected by any solute concentration in strains P843 and P728 at 30°C
except in P843 at 100 mM proline. These strains imported proline at the same extent but
at a lower degree than strain P572. These findings suggest that the role of proline for
strain P843 at this temperature may not be for cryoprotection but is accumulated and
synthesized to meet nutrient requirements. All strains transported proline the best at
22°C. Strains P843 and P572 transported proline and glutamate to a greater degree than
glycine betaine at 22°C with P843 transporting both solutes better than P572. Both
solutes significantly increased the growth rate in strain P843 but in P572 there was an
observed decrease by proline and increase by glutamate. This suggests that proline and 41 glutamate are accumulated from the medium to serve a cryoprotective role in strain P843 but only glutamate has a cryoprotective effect on strain P572. Once again there was minimal transport of glycine betaine and glutamate by all strains at 10°C while proline was transported most abundantly. Growth of all strains was significantly enhanced by all solutes at 10°C. This suggests that the ability for glycine betaine and glutamate to be quickly imported from the environment is hindered at this temperature but can be synthesized and may provide a cryoprotective effect at this temperature over time.
All compatible solutes concentrations improved the growth of strain P873 at 30,
22, 10, and 7.2°C. Improvement in growth was shown by a significant increase in the maximum growth rate and decrease in the time to reach this rate. Transport of all solutes by this strain was only observed at 7.2°C and proline and glutamate at 4°C. The growth stimulation in this strain at the optimum is suggestive of the occurrence of protein synthesis while the growth at lower temperatures is suggestive of the solutes cryoprotective effect. It also can be implied that this strain is incapable of quickly importing glycine betaine below 7.2°C and all solutes above this temperature. It can also be implied that proline and glutamate are the most effective in protecting this strain at lower temperatures.
At the lowest temperature (7.2°C) all solutes were transported by all strains.
Althought glycine betaine was transported by all strains but it did not improve the maximum growth rate in strains P843 and P572 at this temperature. Glycine betaine did stimulate the growth rate in strains P728 and P873, which was indicated by a significant increase in the maximum growth rate. These observations indicate the glycine betaine provides a cryoprotective effect for strains P728 and P873 but not for strains P843 and 42 P572. These findings suggest that glycine betaine can be quickly imported from the
environment but this temperature may affect its ability to stimulate growth once
imported. Glutamate was well transported by strains P843 and P728, which increased the
growth rate for P728 indicating its role in cryoprotectivity for the strain. It was concluded that strains P843 and P572 are the most cold sensitive and strains P728 and
P873 are the most cold tolerant.
Eye formation in Swiss cheese is due to metabolic gas production during warm
room storage by P. freudenreichii, therefore their growth is strongly encouraged during
this ripening period. It has been demonstrated that excessive gas production may occur
during the subsequent cold room storage when the cheese’s protein matrix is less flexible
and incapable of withstanding further gas production, thus causing eye deformities such
as splits or cracks. Gas production exhibited at these lower temperatures may be due to
the continued growth of the propionibacteria. This continued growth and gas production
may occur if the amino acids present during ripening at lower temperatures are available
in substantial concentrations and serve as cryoprotectants. The occurrence of split
defects observed in Swiss cheese is a growing concern and continued propionibacteria
growth may contribute to these defects. The findings in this study can be utilized in
future studies to manufacture Swiss cheese with the differing Propionibacterium and
evaluate for splits during ripening at lower temperatures.
43
-1 a a µmax (d ) Lag phase (d)
CS (mM)b 30°C 22°C 10°C 7.2°C 30°C 22°C 10°C 7.2°C
0 0.789 0.323 NGc NG 6.442 10.85 NG NG
1 GB 0.946 0.541* 0.079* NG 5.430 7.118* 27.00* NG
100 GB 0.930 0.794* 0.106* NG 5.006 5.573* 26.84* NG
1 Pro 0.839 0.974* 0.047* NG 5.378 6.248* 28.07* NG
100 Pro 1.559* 0.621* 0.043* NG 4.127 7.006* 25.28* NG
1 Glu 0.929 0.643* 0.046* NG 5.884 6.091* 39.53* NG
100 Glu 0.811 0.522* 0.020* NG 4.832 6.401* 62.76* NG a Relative standard deviation (RSD) <15% (n = 2 or 3). b CS, compatible solute glycine betaine, proline, or glutamate added to chemically defined media to final concentrations of 1 or 100 mM. c NG, no growth observed.
*Values are significantly different (P < 0.05) from the control lacking, glycine betaine, proline or glutamate at the same temperature.
Table 2.1 Effects of glycine betaine, proline, and glutamate on the maximum specific growth rate (µmax) and the lag phase of P. freudenreichii strain P843 at various temperature.
44
-1 a a µmax (d ) Lag phase (d)
CS (mM)b 30°C 22°C 10°C 7.2°C 30°C 22°C 10°C 7.2°C
0 1.343 0.789 0.082 NGc 5.660 9.528 62.02 NG
1 GB 0.988 0.784 0.272* NG 4.601 9.531 22.13* NG
100 GB 0.925* 0.820 0.371* NG 4.172 11.30* 24.99* NG
1 Pro 0.802* 0.525* 0.418* NG 3.951 9.510 23.66* NG
100 Pro 0.652* 1.014 0.270* NG 4.232 10.19 26.02* NG
1 Glu 0.830* 1.220* 0.394* NG 4.146 7.630* 24.08* NG
100 Glu 0.346* 1.283* 0.423* NG 4.259 7.110* 24.17* NG a Relative standard deviation (RSD) <15% (n = 2 or 3). b CS, compatible solute glycine betaine, proline, or glutamate added to chemically defined media to final concentrations of 1 or 100 mM. c NG, no growth observed.
*Values are significantly different (P < 0.05) from the control lacking, glycine betaine, proline or glutamate at the same temperature.
Table 2.2 Effects of glycine betaine, proline, and glutamate on the maximum specific growth rate (µmax) and the lag phase of P. freudenreichii strain P572 at various temperatures.
45
-1 a a µmax (d ) Lag phase (d)
CS (mM)b 30°C 22°C 10°C 7.2°C 30°C 22°C 10°C 7.2°C
0 1.250 0.336 0.087 NG3 2.728 3.896 24.91 NG
1 GB 0.960 1.513* 0.249* 0.040* 2.778 2.494* 14.18* 73.08*
100 GB 1.222 1.640* 0.148 0.037* 2.910 2.559* 17.58* 77.64*
1 Pro 1.020 1.554* 0.184* 0.040* 2.815 2.358* 15.24* 73.10*
100 Pro 1.683 1.289* 0.237* 0.045* 2.476 2.781* 16.29* 78.31*
1 Glu 1.112 1.443* 0.260* 0.041* 2.879 2.699* 13.16* 82.84*
100 Glu 1.014 1.440* 0.119 0.037* 2.845 2.504* 17.93* 81.68* a Relative standard deviation (RSD) <15% (n = 2 or 3). b CS, compatible solute glycine betaine, proline, or glutamate added to chemically defined media to final concentrations of 1 or 100 mM. c NG, no growth observed.
*Values are significantly different (P < 0.05) from the control lacking, glycine betaine, proline or glutamate at the same temperature.
Table 2.3 Effects of glycine betaine, proline, and glutamate on the maximum specific growth rate (µmax) and the lag phase of P. freudenreichii strain P728 at various temperatures.
46
-1 a a µmax (d ) Lag phase (d)
CS (mM)b 30°C 22°C 10°C 7.2°C 30°C 22°C 10°C 7.2°C
0 0.658 0.439 0.047 NGc 3.419 4.676 18.85 NG
1 GB 1.214* 0.854* 0.294* 0.025* 3.060 4.611 13.32* 81.92*
100 GB 1.315* 0.943* 0.129* 0.036* 3.029 3.956 19.22 84.56*
1 Pro 1.455* 0.952* 0.141* 0.027* 2.491 3.816 18.61 84.96*
100 Pro 1.379* 0.974* 0.138* 0.031* 3.103 4.056 19.17 77.67*
1 Glu 1.326* 0.974* 0.227* 0.031* 2.850 4.213 12.97* 85.09*
100 Glu 1.440* 0.996* 0.131* 0.033* 2.560 4.270 19.11 76.94* a Relative standard deviation (RSD) <15% (n = 2 or 3). b CS, compatible solute glycine betaine, proline, or glutamate added to chemically defined media to final concentrations of 1 or 100 mM. c NG, no growth observed.
*Values are significantly different (P < 0.05) from the control lacking, glycine betaine, proline or glutamate at the same temperature.
Table 2.4 Effects of glycine betaine, proline, and glutamate on the maximum specific growth rate (µmax) and the lag phase of P. freudenreichii strain P873 at various temperatures.
47 1.8 1.6 1.4
) 1.2
1
-
d (
1.0 n
x o i a t 0.8 a m r
t µ 100 mM glu n 0.6 e 1 mM glu c 100 mM pro n 0.4 o c 1 mM pro e 0.2 100 mM GB t u 1 mM GB l o 0.0 0 s
30 e 22 10 l 7.2 b i t a Temperature (C) mp o
C
80
) d
( 60
e
s
a h
p n 40 o
g i t a a r L t * 100 mM glu n * 1 mM glu e 20 c * 100 mM pro n o 1 mM pro c * e * 100 mM GB t u 0 * 1 mM GB l * o 30 * 0 s 22 10 e 7.2 l b i t Tem a perature (C p ) m o C
Figure 2.1 Maximum growth rates (µmax) and lag phase of P. freudenreichii strain P843. * Indicates that no growth was observed, therefore lag phase is infinity.
48 1.8 1.6 1.4
) 1.2
1
-
d (
1.0 n
x o i a t 0.8 a m r
t µ 100 mM glu n 0.6 e 1 mM glu c 100 mM pro n 0.4 o c 1 mM pro e 0.2 100 mM GB t u 1 mM GB l o 0.0 0 s
30 22 e 10 l 7.2 b i t a Temperature (C) mp o
C
80
) d
( 60
e
s
a h
p n 40 o
g i t a a r L t * 100 mM glu n * 1 mM glu e 20 c * 100 mM pro n o * 1 mM pro c
e * 100 mM GB t u 0 * 1 mM GB l o 30 * 0 s 22 10 e 7.2 l b i t Tem a perature (C p ) m o C
Figure 2.2 Maximum growth rates (µmax) and lag phase of P. freudenreichii strain P572. * Indicates that no growth was observed, therefore lag phase is infinity.
49
1.8 1.6 1.4
) 1.2
1
-
d (
1.0 n
x o i a t 0.8 a m r
t µ 100 mM glu n 0.6 e 1 mM glu c 100 mM pro n 0.4 o c 1 mM pro e 0.2 100 mM GB t u 1 mM GB l o 0.0 0 s
30 22 e 10 l 7.2 b i t a Temperature (C) mp o
C
80
) d
( 60
e
s
a h
p n 40 o
g i t a a r L t 100 mM glu n 1 mM glu e 20 c 100 mM pro n o 1 mM pro c
e 100 mM GB t u 0 1 mM GB l o 30 * 0 s 22 10 e 7.2 l b i t Tem a perature (C p ) m o C
Figure 2.3 Maximum growth rates (µmax) and lag phase of P. freudenreichii strain P728. * Indicates that no growth was observed, therefore lag phase is infinity.
50
1.8 1.6 1.4
) 1.2
1
-
d (
1.0 n
x o i a t 0.8 a m r
t µ 100 mM glu n 0.6 e 1 mM glu c 100 mM pro n 0.4 o c 1 mM pro e 0.2 100 mM GB t u 1 mM GB l o 0.0 0 s
30 22 e 10 l 7.2 b i t a Temperature (C) p m o
C
80
) d
( 60
e
s
a h
p n 40 o
g i t a a r L t 100 mM glu n 1 mM glu e 20 c 100 mM pro n o 1 mM pro c
e 100 mM GB t u 0 1 mM GB l o 30 * 0 s 22 10 e 7.2 l b i t Tem a perature (C p ) m o C
Figure 2.4 Maximum growth rates (µmax) and lag phase of P. freudenreichii strain P873. * Indicates that no growth was observed, therefore lag phase is infinity.
51
3e+5
2e+5
1e+5 GlycineBetaine (nmol/mg of protein)
0 0 10203040506070
Time (minutes)
3e+5
2e+5
1e+5 Proline (nmol/mg of protein) (nmol/mg Proline
0 0 10203040506070
Time (minutes)
3e+5
2e+5
1e+5 Glutamate (nmol/mg of protein) (nmol/mg Glutamate
0 0 10203040506070 Time (minutes)
Figure 2.5 Transport of 14C-labeled glycine betaine, proline, and glutamate by P. freudenreichii strains at 30°C. Strains P843 (●), P873 (○), P572 (▼), and P728 (∆) were grown to mid-log phase in chemically defined media and assayed at 30°C.
52 1e+5
8e+4
6e+4
4e+4
2e+4 Glycine Glycine Betaine (nmol/mg of protein)
0 0 10203040506070
Time (minutes)
1e+5
8e+4
6e+4
4e+4
Proline of(nmol/mg protein) 2e+4
0 0 10203040506070
Time (minutes)
1e+5
8e+4
6e+4
4e+4
2e+4 Glutamate ofGlutamate (nmol/mg protein)
0 0 10203040506070 Time (minutes)
Figure 2.6 Transport of 14C-labeled glycine betaine, proline, and glutamate by P. freudenreichii strains at 22°C. Strains P843 (●), P873 (○), P572 (▼), and P728 (∆) were grown to mid-log phase in chemically defined media and assayed at 22°C.
53 1e+5
8e+4
6e+4
4e+4
2e+4 GlycineBetaine (nmol/mg ofprotein)
0 0 10203040506070
Time (minutes)
1e+5
8e+4
6e+4
4e+4
Proline (nmol/mg of protein) (nmol/mg Proline 2e+4
0 0 10203040506070
Time (minutes)
1e+5
8e+4
6e+4
4e+4
2e+4 Glutamate (nmol/mg of protein) (nmol/mg Glutamate
0 0 10203040506070 Time (minutes)
Figure 2.7 Transport of 14C-labeled glycine betaine, proline, and glutamate by P. freudenreichii strains at 10°C. Strains P843 (●), P873 (○), P572 (▼), and P728 (∆) were grown to mid-log phase in chemically defined media and assayed at 10°C.
54 25000
20000
15000
10000
5000 Glycine Betaine (nmol/mg of protein) of (nmol/mg Betaine Glycine
0 0 10203040506070
Time (minutes)
25000
20000
15000
10000
Proline (nmol/mg of protein) of (nmol/mg Proline 5000
0 0 10203040506070
Time (minutes)
25000
20000
15000
10000
5000 Glutamate (nmol/mg of protein) of (nmol/mg Glutamate
0 0 10203040506070 Time (minutes)
Figure 2.8 Transport of 14C-labeled glycine betaine, proline, and glutamate by P. freudenreichii strains at 7.2°C. Strains P843 (●), P873 (○), P572 (▼), and P728 (∆) were grown to mid-log phase in chemically defined media and assayed at 7.2°C.
55 10000
8000
6000
4000
2000 Glycine Glycine Betaine (nmol/mg of protein)
0 0 10203040506070
Time (minutes)
10000
8000
6000
4000
Proline (nmol/mg of protein) 2000
0 0 10203040506070
Time (minutes)
10000
8000
6000
4000
2000 Glutamate (nmol/mg of protein)
0 0 10203040506070 Time (minutes)
Figure 2.9 Transport of 14C-labeled glycine betaine, proline, and glutamate by P. freudenreichii strains at 4°C. Strains P843 (●), P873 (○), P572 (▼), and P728 (∆) were grown to mid-log phase in chemically defined media and assayed at 4°C.
56 CHAPTER 3
DETERMINATION OF FREEZE-THAW RESISITANCE IN DAIRY PROPIONIBACTERIUM FREUDENREICHII
3.1 Introduction
Propionibacteria are gram-positive, catalase-positive, non-sporforming, facultative anaerobic, rod-shaped bacteria (Hetting and Reinbold, 1972). Dairy propionibacteria are commonly used in the manufacture of Swiss cheese and represent the main ripening flora (Boyaval et al., 1999). According to Noël et al. (1999), the temperature range for Propionibacterium growth is 3-40°C, with an optimal value of
30°C. These bacteria must endure high cook temperatures (up to 55°C) as well as low storage temperatures (0-4°C). Storage at these low temperatures for extensive periods creates a stressful environment and under stressful conditions many bacteria are able to synthesis or transport compatible solutes in response to this stress. As part of the frozen starter culture they may also experience repetitive freezing at -20°C and thawing at 22-
30°C. If not protected, their metabolic properties might be lost.
Cryoprotectants have excellent colligative properties with water, which both retard the formation of intracellular ice crystals and reduce the potential for osmotic injury during freezing and thawing processes (Cleland et al., 2004). Cryoprotection is known to result from the inclusion of cryoprotectants in frozen bacterial suspension
57 (Panoff et al., 2000). Cryotolerance has been generated by incubation at suboptimal temperatures in Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii bulgaricus, or Enterococcus faecalis (Panoff et al., 2000). The role of compatible solutes as cryotoprotectants has been extensively studied in Listeria monocytogenes.
Compatible solutes are molecules that allow microorganisms to respond to
physiological changes in the environment. These compounds are considered compatible
because of their ability to accumulate at high concentration levels by de novo synthesis or
transport without interfering with vital cellular processes, such as DNA replication,
DNA-protein interactions, and metabolic machinery (Kempf and Bremer, 1998).
Compatible solutes aid bacterial proliferation in ecological niches that would otherwise
be considered inhibitory to their growth. They function as effective stabilizers of enzyme function, providing protection against salinity, high temperature, freeze-thaw treatment, and drying (Rosser and Muller, 2001 and Sleator and Hill, 2001). They are known to aid in osmotolerance in most bacteria, which helps balance the intracellular and extracellular osmotic environments (Bayles et al., 2000). Compatible solutes also confer increased tolerance to dessication, freezing, and elevated temperatures (Rosser and Muller, 2001).
Some accumulated compatible solutes include potassium ions, amino acids and their derivatives, and carbohydrates such as glycine betaine, proline betaine, carnitine, glutamate, trehalose, and ecotine. Glycine betaine and proline stabilize proteins in repeated freeze-thaw cycles (Rosser and Muller, 2001). The focus of this study was to evaluate the cryoprotective effects of glycine betaine on Propionibacterium freudenreichii cells during cyclic subzero freezing and thawing.
58 3.2 Materials and Methods
Four strains were selected based on their growth abilities in sodium lactate broth and chemically defined media (Anggraeni and Courtney, unpublished; and Pruitt and
Courtney, unpublished). The names of the cultures and companies were coded for proprietary reasons. The selected strains were P843, P572, P873, and P728. These four strains were grown anaerobically to mid-log phase in media containing 20g/L Bacto- tryptone, 5g/L yeast extract, and 5g/L glucose. Cells were transferred to chemically defined medium (CDM) and CDM containing 100 mM glycine betaine and anaerobically grown to mid-log phase at 28-30°C. The CDM used was the minimal media with additional methionine and cysteine (MMC) described by Glatz and Anderson (1988).
Cells were pelleted and resuspened in 1 ml of sterile phosphate buffered saline (PBS), pH 7.0. Serial dilutions were prepared in 0.1% peptone and plated onto agar plates containing 20g/L Bacto-tryptone, 5g/L yeast extract, 5g/L glucose, and 15g/L agar prior
to freezing to determine pre-freezing cell counts. The buffered sample was treated as
described by Elhanafi et al. (2004) with modifications. One freeze-thaw cycle consisted
of freezing at -20°C for 24 ± 2-hours and thawing in a 30°C water bath for 30 minutes,
and then returned to the freezer after a sample was removed for analysis. Serial dilutions
were prepared from thawed samples after designated number of cyles and plated as
previously mentioned. All plates were incubated anaerobically at 30°C for 3-4 days.
Freeze-thaw cycles were performed in triplicate for five days and samples were taken
after cycles four and five. Viable cells were calculated as colony forming units per
milliliter (CFU/ml). Log reductions were calculated by subtracting the log cfu/ml after
four or five freeze-thaw cycles from the log cfu/ml before freezing. 59 Statistical Analysis Mean values for the log reduction in viable cells were compared by one-way analysis of variance (SigmaStat 3.1, Sysstat Software, Inc.). Tukey’s post-hoc test was used to compare treatment with glycine betaine and treatment without glycine betaine. Values with P < 0.05 were considered to be significantly different.
3.3 Results Four P. freudenrecheii strains were selected based on their growth capabilities in chemically defined media at temperatures lower than optimum. The strains chosen were
P843, P572, P728 and P873. Strains P843 and P572 were characterized as cold sensitive and strains P728 and P873 were characterized as cold tolerant (Pruitt and Courtney, unpublished).
The decreased viability of bacteria caused by freezing and thawing can be described exponentially as log reduction in viable cells. The decrease in cell viability varied with each treatment and between strains. Differences in viability were observed after 4 and 5 freeze-thaw cycles with log reductions in all strains ranging from 1.01 to 9.96 after four cycles and ranging from 1.51 to 9.96 after five cycles. There was a significant (P<0.05) difference between all strains (Table 3.1), suggesting that freeze-thaw tolerance is strain dependent in propionibacteria. Strain P843 was affected the most by freeze-thaw cycles with a 9.17-fold reduction after four cycles. Strain P572 was the most freeze-thaw tolerant with a 1.01-fold reduction after four cycles. Additional loss of viability occurred with an additional freeze-thaw cycle at 5 cycles, except in strain P843 whose population was already undetectable after 4 cycles.
60 Growth with glycine betaine had no significant (P<0.05) effect on log reduction after
4 or 5 cycles in strain P843 or P873 (Table 3.1). This indicates that prior growth with
glycine betaine at 28-30°C does not protect these strains from freeze-thaw stress. Strain
P572 had a significantly larger log reduction when grown with glycine betaine, suggesting that growth with glycine betaine may sensitize this strain to freeze-thaw stress. Strain P728 was the only strain demonstrating a decreased log reduction when grown with glycine betaine, suggesting that prior growth with glycine betaine may help protect this strain from freeze-thaw stress. The effect of growing cells in glycine betaine at 28-30°C on subsequent freeze-thaw stress appears to be strain dependent. It provided a
cryoprotective effect on strain P728 and not strains P843, P873, and P572.
3.4 Discussion
The process of freezing and thawing induces injury to microorganisms through
membrane or cell wall disruption, leakage of intracellular constituents, and changes in
protein conformation (Yamamoto and Harris, 2001). Cells adapt to cold stress by
desaturation of fatty acids and unwinding of RNA (Panoff et al., 2000). Low
temperatures decrease the fluidity of the membrane in the absence of compensatory
changes in membrane lipid composition but compatible solutes may influence membrane
fluidity at low temperatures (Bayles et al., 2004). Freeze-thaw damage is frequently related to the fluidity of the cell membrane, which is determined by the unsaturation:saturation ratio of the lipids (Zavaglia et al., 2000). Glycine betaine is cited
as one of the most common compatible solutes produced by bacteria, synthesized both by
halophiles that are adapted to living in high salt concentrations and non-halophilic 61 bacteria that are subjected to osmotic stress (Cleland et al., 2004). Its cryoprotective effects have been demonstrated extensively in L. monocytogenes which adapts to low temperatures by accumulating glycine betaine in the cytosol (Dykes and Moorhead,
2001). Bayles et al. (2004) observed a stimulated growth rate at 4 and 7°C but not at
30°C. Glycine betaine has a stabilizing effect on protein structure opposing protein
unfolding and denaturation (Bayles et al., 2004). The mechanism of cold activation of
glycine betaine transport may involve changes in the hydrophobic interactions among
membrane proteins or between proteins and the membrane because hydrophobic
interactions are weakened at lower temperatures (Ko et al., 1994). Glycine betaine was
proven to be an effective cryoprotectant for freeze-drying, liquid-drying, and liquid
nitrogen freezing (Cleland et al., 2004).
Pruitt and Courtney (Chapter 2) characterized strains P843 and P572 as cold sensitive
when grown in the presence of compatible solutes and strains P728 and P873 as cold
tolerant. Glycine betaine was found to exert a better cryoprotective effect on strain P728
when subjected to multiple freeze thaw cycles. A greater log reduction was observed in
strains P873 and P843 after five cycles suggesting no protection was provided by glycine
betaine. These findings suggest the effects of glycine betaine providing cryoprotection
may be strain dependent and support the hypothesis that some compatible solutes
particularly glycine betaine serve as cryoprotectants. Strain differences in ability to
transport glycine betaine may play a role. In Chapter 2 it was reported that P873 and
P728 tranported glycine betaine better than other strains. However, these two strains
differ in the protective effect of glycine betaine on freeze-thaw stress. Therefore, other
physiological differences between these strains may be responsible for differences in 62 freeze-thaw protection by glycine betaine. Also, the temperature of growth in the present experiment (28-30°C) was not the optimum for glycine betaine uptake (Chapter 2). In future experiments, we recommend that the cells be grown at 7.2°C or in the presence of salt to maximize glycine betaine uptake prior to application of the freeze-thaw stress.
Cold shock response is a physiological response of living cells to temperature downshift and characterized by strong repression of the major metabolic activity of the cell (Phadtare et al., 2004). Lactic acid bacteria are mesophilic and thermophilic bacteria that are frequently subjected to cold environments and multiple freeze thaw cycles when used as starter cultures. Propionibacteria must endure these same stresses when used as
Swiss cheese starter cultures. It has been determined that cold shock proteins and/or cold acclimation proteins are synthesized in response to low temperatures in Lactococcus lactis, Enterococcus faecalis, and Lactobacillus acidophilus (Panoff et al., 2000) as well as Escherichia coli and Bacillus subtilis (Phadtare et al, 1999) indicating their presence in both Gram positive and Gram negative bacteria. Cold shock acclimation proteins are specifically synthesized during continuous growth at cold temperatures (Phadtare et al.,
1999). Strain P572 exhibited more freeze thaw resistance without glycine betaine. The cold shock proteins in the abovementioned bacteria may be synthesized and responsible for the observed differences seen in strain P572 grown with and without glycine betaine.
Kim and Dunn (1997) found nine proteins were overexpressed in L. lactis after a shift to cold temperatures indicating these bacteria can adapt rapidly to a temperature downshift.
Cryotolerance increases if the bacteria are cold shocked prior to freezing (Phadtare et al.,
1999). The medium in which the cells were frozen appears to play a role in protecting the cells during freezing (Kim and Dunn, 1997). This supports the difference seen in log 63 reduction between the strains when grown with glycine betaine and when grown without
glycine betaine. The lack of cryotolerance in strains P843, P572, and P873 may be a
result their growth phase. The induction of cryotolerance appears to be dependent on the
growth phase in which the cold shock takes place (Kim and Dunn, 1997).
Mechanisms that permit low temperature growth of microorganisms include
modifications in DNA supercoiling, maintaining membrane fluidity, regulating uptake
and synthesis of compatible solutes, production of cold shock proteins, modulating
mRNA secondary structure as well as maintaining the structural integrity of
macromolecules and macromolecule assemblies such as ribosomes (Wouters et al.,
2000). In this study the observed effects of glycine betaine were minimal suggesting its cryoprotective effect in some P.freudenreichii strains may be obsolete for freeze-thaw stress under these conditions. The findings in this study also suggest that some P. freudenreichii strains may synthesize cold shock/cold induced proteins in response to a rapid downshift in temperatures making them ideal for use in starter cultures. Further studies could be conducted to investigate the effects of glycine betaine on strains exhibiting different growth phases and grown under conditions promoting glycine betaine uptake. The effects on protection during extended frozen storage could also be explored.
64
P. freudenreichii strain Growth medium Log reduction 4 freeze-thaw cycles 5 freeze-thaw cycles P843 CDM 9.17f 9.17e,f CDM + GB 9.43f 9.43f
P572 CDM 1.01a 1.51a CDM + GB 1.92b 2.94b
P728 CDM 4.69d 5.67d CDM + GB 3.06c 4.09c
P873 CDM 6.90e 8.27f CDM + GB 9.96f 9.96f * Values represent the mean of two replications. Means in the same column with different superscript letters are significantly different (P<0.05).
# Log reductions were calculated by subtracting the log cfu/ml after four or five freeze-
thaw cycles from the log cfu/m before freezing. Detection limit was 10 CFU/ml (1.0
log10 CFU/ml).
Table 3.1 Log reductions in viable cell numbers in four P. freudenreichii strains grown in chemically defined medium with or without glycine betaine after four and five freeze-thaw cycles.
65 CHAPTER 4
THE EFFECTS OF STORAGE TEMPERATURE AND PROPIONIBACTERIUM FREUDENREICHII COLD TOLERANCE ON SPLIT DEFECTS IN SWISS CHEESE
4.1 Introduction
Dairy propionibacteria (PAB) are part of the starter cultures used in the manufacture of Swiss type cheeses. PAB obtain energy anaerobically from fermentation products that other bacteria produce through secondary fermentation (Madigan et al.,
2000). Propionibacterium freudenereichii utilize lactic acid produced by Streptococcus thermophilus and Lactobacillus helveticus, two common lactic acid bacteria (LAB) used in starter cultures. Pyruvic acid is formed from lactic acid through the action of lactate dehydrogenase, and pyruvic acid is then converted into propionic acid, acetic acid, and carbon dioxide (CO2) (Weinrichter et al., 2004). These PAB and LAB are commonly used in Swiss cheese starter cultures. The acids contribute to the cheese’s characteristic flavor and aroma, and the CO2 is responsible for the eyes commonly associated with
Swiss type cheeses. Other acids that contribute to flavor and aroma include n-butyric, isovaleric, and n-caporic (Ji et al, 2004).
Swiss type cheeses experience a two-step ripening process that involves a warm room treatment (WRT) and a cool room treatment (CRT). The cheese experiences a cooling period prior to WRT to slow the growth of the LAB and equilibrate the salt. The pliability of the cheese during WRT (20-24°C) is able to withstand CO2 production,
66 therefore growth of the PAB is encouraged for proper eye formation (Boyaval et.al,
1999). The number and presence of weak points or incomplete curd knitting permit and
encourage gas collection (Reinbold, 1972). The CRT (0-4°C) helps in flavor
development, arrests eye formation, firms the body and inhibits bacterial growth
(Reinbold, 1972). It is during CRT that the body becomes rigid and unable to withstand
CO2 pressure leading to defects such as splits and cracks. The focus of this study was to manufacture Swiss cheeses with PAB differing in their abilities to grow at cool ripening temperatures and evaluate the cheeses for common eye deformities (splits) during the cool ripening period.
4.2 Material and Methods
Starter Cultures
Three Propionibacteruim freudenreichii strains were selected based on their growth capabilities at low temperatures (4-10°C) (Pruitt, Anggraeni, and Courtney,
unpublished data). The names of cultures and company of origin were coded for
proprietary reasons. The strains chosen were P572, P196, and P873 and were characterized as cold sensitive, intermediate, and cold tolerant, respectively. Starter propionibacteria cultures were prepared by a commercial starter culture company.
Cultures were handled according to instructions provided by the culture company and maintained at temperatures of -20°C or below to ensure integrity. The cultures were partially thawed by immersing the containers in chlorinated water, and then the cultures were added to the cheese vat to continue thawing with agitation. Each starter culture contained the same LAB strains but contained one of the three different PAB strains. 67 Swiss Cheese Manufacture and Storage
Manufacture and Storage. Swiss cheeses were manufactured and stored by a commercial facility. The entire make procedure was not disclosed for proprietary reasons. Cheese was produced with starter cultures containing the same LAB strains but containing either PAB strain P572, P196, or P873. Each PAB strain was used to produce triplicate vats of cheese. The cheese milk was pasteurized, and the curds were cooked at
118-119°F to allow for kosher certification of the whey. The time lapse between setting the milk and pumping the curd was two hours and ten minutes. The curds were pumped into five universal presses forming blocks (1100 pounds). Each pressed block was divided into three 360 pound blocks that were wrapped and stored in plastic containers.
The cheeses were precooled at 4°C for 6 days prior to WRT. Each cheese experienced identical WRT at 21°C for 17-25 days. After WRT, cheeses experienced different CRT when one third of the blocks from each vat were stored at 0, 4, or 7.2°C for ninety days.
Cheeses stored at 7.2°C were placed at 4°C one week prior to cutting.
Cheese Sampling. Samples were taken at days 0, 30 60, and 90. Trier core samples were taken at days 0 and 30 and deli loaf samples were taken at days 60 and 90.
Samples taken at day 0 were analyzed for make day composition. Samples taken at day
30 were analyzed for salt, protein, and the presence of starter and nonstarter culture bacteria after WRT. Samples taken at day 60 and 90 were digitally analyzed for split defects. Samples taken at all time points were analyzed for total free amino acids, individual amino acid quantification, and enumeration of viable bacteria.
Analysis of Make Day Composition. The composition of Swiss cheese included fat on a dry basis (FDB), fat on cheese (FOC), salt, pH, moisture, and protein. These 68 components were measured on initial manufacture day. FDB, FOC, and moisture were
measured with an Instalab 600 NIR Product Analyzer (Data Specifics, Springfield, IL) by the commercial facility’s quality assurance component. Protein was measured at The
Ohio State University with a KJT-270 NIR Composition Analyzer (Kett US, Villa Park,
CA). Salt was measured with an M-926 Chloride Analyzer (Nelson-Jameson,
Marshfield, WI) and pH with a Sam Gray Gold Electrode (Nelson-Jameson, Marshfield,
WI) using the quinhydrone method. Results were expressed in percent cheese (w/w).
Enumeration of Viable Bacteria. One gram of grated cheese was homogenized in
a stomacher (Seward Stomacher Biomaster 80, Seward Co., Norfolk, UK) for 2 minutes
in 9 ml of 2% sodium citrate. Decimal dilutions for day 0 samples were plated onto
Rogosa SL agar plates (Difco, Becton, Dickinson, and Co., Sparks, MD) and incubated
anaerobically for 2 days at 37°C for L. helveticus enumeration. The same dilutions were
plated onto M17 agar (Difco) and 0.15% lithium chloride plates and incubated for 1 day
at 43°C for S. thermophilus enumeration. The PAB were enumerated at all time points on
lithium glycerol agar (LGA) plates and incubated anaerobically at 30°C for 8-10 days
(Madec et. al., 1996). Samples were plated in duplicate and colony forming units per
gram of cheese (cfu/g) were calculated.
Quantification of Free Amino Acids
Water soluble extractions were also used to profile the amino acid spectrum.
Water soluble extracts were prepared by homogenizing cheese in diethyl pyrocarbonate
(DEPC) treated water (1:2) and centrifuged to obtain the aqueous layer (Anderson et al.,
1991). The aqueous layer was filtered through an Ultra-Free Centrifugal 5000 NMWL
membrane (Fisher, Pittsburgh, PA). Samples were analyzed by the Laboratory for
69 Protein Chemistries at Texas A&M University (College Station, TX) using a Hewlett
Packard AminoQuant System. Samples were analyzed with a primary and secondary internal standard, norvaline and sacrosine, respectively. Each 40 µl standard was combined with 20µl of internal standard, and 1µl injected. Each 20 µl sample was combined with 20 µl of internal standard and 20µl of 0.4 M borate, and 1µl injected.
Samples were analyzed in duplicate and results expressed in micrograms per gram of cheese (ug/g).
Evaluation of Eyes and Splits
Two slices of uniform thickness (2mm) from standardized locations within the cheese block were evaluated for gas openings. Slices were approximately 4”L x 4”H.
Because triplicate cheeses per culture and cheese ripening temperature were prepared, a total of 6 slices were evaluated per treatment. Photographic technique and image analysis were performed according to Caccamo et al. (2004) with modifications. Slices were placed on a black background and an image taken using a digital camera (Nikon Coolpix
E5200, model no. 3005157, 5.1 Megapixel, Nikon Corporation, Tokyo, Japan). Each picture was taken under standardized conditions without a flash. The camera was set at macro in the auto function, 5 megapixel, and fine image quality. Each photograph had a resolution of 2592 x 1944 pixels and manually cropped to the dimensions of each slice.
The color of the cheese and openings were standardized for all pictures using
Adobe PhotoShop Elements (Adobe Systems Inc, San Jose, CA). The cheese was standardized to the following color settings: R=242, G=211, and B=154. The openings were standardized to the following color settings: R=3, G=3, and B=3. Each slice was
70 standardized in two formats: once with all openings visible, and a second time with all
visible splits and cracks filled with the same color as the cheese, but all eyes visible.
A collective digital image of all slices was analyzed using Paint Shop Pro
(Version 7.00, 2000, Jasc Software Inc.). Each collective image had a resolution of 8050
x 6219 with 225 pixels per inch. The images were analyzed using the Paint Shop Pro
histogram function. This function displays the grayscale value of 0 to 255 for each
manual selection in the user interface. Values in the range of 0 to 85 were considered
openings and values of 85 – 255 were considered non-openings. Each was analyzed for
total visible openings and total visible eyes (splits filled). Total splits were calculated by
subtracting the total eyes from the the total openings. Data are reported as percentage of
the surface area represented by eyes or splits.
Statistical Analysis
Mean values for amino acid concentrations, % eyes, and % splits were analyzed
by one-way analysis of variance (SigmaStat 3.1, Sysstat Software, Inc.). Tukey’s post-
hoc test was done to compare concentrations at each temperature and each ripening
period. Values with P< 0.05 were considered to be significantly different.
4.3 Results
The overall composition of the Swiss cheese was 38% moisture, 49% fat on a dry
basis, and 25% protein (Table 4.1). The composition was within the US requirements, a minimum milkfat content of 43 percent by weight of the solids and maximum moisture content of 41 percent by weight (FDA, 2004). Salt was analyzed after warm room and pH was analyzed after pressing. The salt was 0.39-0.61 % in the moisture phase and pH
71 was 5.21-5.22. The cheeses were graded during WRT. Most cheeses received a grade of
‘A’ (good for cheese slicing) and ‘Us’ (underset). Only one cheese received a grade of
‘Os’ (overset) and three cheeses received a grade of ‘C’ (defects in the cheese were very
little eyes in the middle of the block).
Bacterial Enumeration. LAB were enumerated the day after make day. Viable
cell numbers of L. helveticus were 108-109 CFU/g. Viable cell numbers of S.
thermophilus was 108-109 CFU/g. Propionibacteria were enumerated at all timepoints.
4 8-9 Propionibacterium viable cell numbers increased from 10 cfu/g before WRT to 10
after WRT. Counts remained at 108-9 throughout ripening.
Amino Acid Analysis. Cheeses were manufactured with P. freudenreichii strains
differing in their ability to grow at low temperatures. The cheeses were stored at 0, 4, and
7.2°C for sixty and ninety days after WRT. Samples were taken after make day, WRT,
and during CRT for each temperature. Aqueous extracts were made for amino acid
analysis. The twenty most common amino acids were reported in addition to citrulline for
each strain, P572, P196, and P873 after each sampling period. Total amino acid concentrations were also reported for each strain.
Fourteen amino acids showed a significant (P<0.05) increase in concentration between make day and WRT but no observed difference during CRT for strains P572 and
P196. This indicates that proteolysis does occur during warm ripening as expected.
Lysine was the dominant amino acid after make day but leucine dominated after WRT
through all sixty day treatments and ninety days at 0°C. There was no significant
difference in alanine, aspartic acid, cysteine, and methionine from make day to CRT for
these strains (Table 4.2). There was an increase in citrulline concentration for strain P572
72 from make day to completion of WRT but a significant decrease in concentration after
sixty days at 4°C and 7.2° C and 90 days at all temperatures. This may be due to
citrulline utilization by strain P572.
There were observed differences in proline and tryptophan concentrations for strain P572. Proline concentration increased during CRT with the highest concentration after ninety days at 4°C. Tryptophan concentrations were statistically the same in strain
P572 except after ninety days at 0°C in which the concentration increased (133.2 ug/g)
(Table 4.2). Leucine was the dominant amino acid after WRT and at all temperatures after sixty days. Total amino acid concentrations increased after make day but there was no significant difference among the concentrations after WRT. The highest total concentrations were observed after ninety days at 0°C and 4°C, 17659 and 13495 ug/g, respectively.
Citrulline, proline, and tryptophan showed significant differences after WRT and during CRT in strain P196 (Table 4.3). The citrulline concentration for strain P196 increased after WRT but significantly decreased during CRT. Glutamic acid was the dominant amino acid after WRT and after ninety days of ripening at 4°C. There was a significant increase in proline concentration after WRT but no difference was observed after WRT except after sixty days of ripening at 7.2°C and ninety days at 4°C and 7.2°C.
Tryptophan concentrations in strain P196 increased after WRT but no difference was observed except for a significant decrease after sixty days of ripening at 4°C but concentration at 4°C after ninety days was significantly higher. There was a significant increase in amino acid concentration during CRT but there was not a significant difference between WRT and CRT. The highest total concentration for all amino acids 73 during CRT in strain P196 was seen after sixty days of ripening at 7.2°C and the lowest after sixty days at 4°C suggesting temperature may enhance proteolysis during cool ripening. The length of the ripening period may also play a role in promoting proteolysis in strain P196, which was observed in significantly high amino acid concentration totals after ninety days.
Unlike strains P572 and P196, there were thirteen amino acids in strain P873 that showed a significant increase in concentration between make day and WRT with no observed difference during CRT (Table 4.4). Lysine and alanine were the dominant amino acids after make day but glutamic acid, leucine, and lysine were dominant after
WRT. Citrulline, glycine, glutamic acid, and proline showed significant differences after
WRT and during CRT. Citrulline concentration increased after WRT but significantly decreased during CRT. An increase in glutamic acid concentration occurred after WRT and during CRT with a significant increase after ninety days at 4°C. Glycine concentrations increased as temperature and ripening period increased with differences seen after sixty days at 7.2°C and ninety days at all temperatures. The same trend was observed with proline concentrations with increased differences after sixty days at 7.2°C and ninety days at 4°C and 7.2°C. There was a significant increase in total amino acid concentrations for strain P873 from make day to WRT and CRT with differences seen during CRT. An increase was seen after sixty days at 7.2°C and after ninety days at all temperatures. These findings also suggest an increase in temperature and prolonged ripening enhances proteolysis.
After comparing differences in amino acid concentrations for each strain, amino acid concentrations were compared between strains. Differences were only observed 74 after ninety days at 4°C and 7.2° between all strains. Eight amino acids (alanine,
glutamine, isoleucine, methionine, phenylalanine, threonine, tyrosine, and valine) were
significantly higher at 4°C in strain P873 whereas only lysine was significantly higher at
this temperature in strains P572 and P196. At 7.2°C, eleven amino acids (alanine,
arginine, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, and valine) were significantly higher in strains P196 and P873 while asparagine was higher only in strain P873. Total amino concentrations were higher in strains P196 and P873 after ninety days at 7.2°C.
Proline and glutamic acid were the amino acids of interest because of their ability
to serve as compatible solutes for P. freudenreichii when exposed to low temperatures.
Proline concentration increased from make day and during the ripening period for all P.
freudenreichii strains with an observed difference between make day, warm room, and cool ripening. There was a difference in glutamic acid concentration in all strains from
make day to warm room ripening but no difference was seen throughout the cooler
ripening period. Although not an amino acid of interest, the trend in citrulline
concentration deserved attention. Its concentration, by all strains, decreased to levels below the detection limit during CRT suggesting that this compound is further metabolized.
Evaluation of Eyes/Splits. Cheeses were sliced after sixty and ninety days of storage for evaluation of eyes for splits. No difference was seen after sixty days of ripening but a significant increase was seen for each strain after ninety days at 7.2°C
(Table 4.5). Cheeses manufactured with strain P572 and ripened for ninety days at 4 and
7.2°C had significantly more eyes suggesting an extended ripening period and increased 75 temperature play a role in enhancing eye development. A difference was seen after sixty days at 4°C among all strains and cheese manufactured with strain P196 possessed the most eyes at this time point.
Slices were also analyzed for splits using the same program and splits were compared for each strain at each timepoint. Splits increased with an increase in temperature. The number of splits was higher with each strain after sixty and ninety days at 7.2°C (Table 4.5). A significant increase was seen in strain P572 when ripened at
7.2°C with the number of splits increasing as ripening increased. For strain P196 there were more splits seen after sixty days at 7.2°C but a decrease in splits after ninety days at the same temperature.
Splits were also compared among strains. After ninety days at 0°C the most splits were seen in cheese manufactured with strain P873 and the fewest with strain P196
(Table 4.6). Cheese manufactured with strain P873 had the highest number of splits after sixty days of ripening at 4°C and 7.2°C indicating temperature does have an effect on split formation. A significant difference was seen among all strains after ninety days at
7.2°C. Splits increased from strain to strain during this ripening period. Cheese manufactured with strain P873 had 0.717% splits, strain P572 had 0.483%, and strain
P196 had 0.250%. These findings suggest not only do temperature and the length of ripening have an effect on split formation but the strain used in manufacture does as well.
Figure 4.1 provides images of some visible eyes and some visible splits observed among the cheeses.
76 4.4 Discussion
Ripened cheeses are purposely exposed to warm temperatures or held for long periods at colder temperatures to permit bacteria and enzymes to transform fresh curd to cheese (Kosikowski and Mistry, 1997). Protein, fat, moisture, salt, and pH are important
Swiss cheese components. In addition to temperature, they contribute to the microbiological and chemical changes that occur during the ripening process. These changes help in flavor and texture development. The microbiological changes that occur involve the death of starter culture bacteria and possible growth of nonstarter bacteria from nutrients provided by autolysis of the starter bacteria. The chemical changes that occur include degradation of carbohydrates, proteins, and lipids which provide a more pliable body and more flavor. The degradation of proteins or proteolysis contributes to textural changes of the cheese matrix due to breakdown of the protein network (Sousa et al., 2001).
Proteolytic and peptidolytic enzymes of microbial origin, usually mesophilic and thermophilic starters, are mainly responsible for the subsequent secondary proteolysis, finally leading to the formation of small peptides and free amino acids (Weinrichter et al.,
2004). The final products of proteolysis are free amino acids and their concentration in cheese at any stage of ripening is the net result of the liberation of free amino acids from casein, their degradation to catabolic products and perhaps some synthesis by the cheese microflora (Sousa et al., 2001). Proteolysis has been shown to occur mainly in the warm room (Thierry et al., 1998 and Valence et al., 2000). Amino acid concentrations increased during WRT for all strains suggesting proteolysis does occur in the warm room and increased temperatures contribute to its occurrence. Ji et al. (2004) found that warm
77 room holding time affected free amino acid concentration at the end of ripening, seventy
days after warm room. Glutamic acid concentration in cheese manufactured with strains
P572 and P196 increased during WRT but no difference was seen thereafter. Its
concentration did increase in cheese manufactured with strain P873 after ninety days at
4°C suggesting this strain may have some proteolytic activity. Proline is more abundant
in Swiss-type cheeses with concentrations reported to range from 150 mg to 215 mg per
100 g of cheese (Mitchell, 1981). In the present study, proline concentration increased during CRT for all strains with observed differences at higher temperatures and extended
ripening periods with concentrations similar to previous reports (2500 micrograms per
gram of cheese). There was no difference in the concentration of glutamic acid and
proline during CRT between the strains.
Total amino acid concentrations were higher as temperature and ripening duration
increased for cheese manufactured with P572 and P196. There was no difference in total
amino acid concentrations in cheese made with strain P873 after WRT and during CRT.
All observed increases were at 4°C and 7.2°C or extended ripening suggesting these
factors may contribute to further proteolysis after the warm room ripening. The increase
in free amino acid concentrations is likely due to increased activity of proteases and peptideases at these temperatures. Sources of these enzymes include rennet, native milk
enzymes, such as plasmin, and starter and nonstarter LAB. Amino acid concentrations
can depend on the peptidase activity by LAB and non-starter LAB factors that were not
explored in this study (Ardo and Petterson, 1988, Peterson et al., 1990, Ji et al., 2004).
LAB posses a very comprehensive proteinase/peptidase system capable of hydrolysing
oligopeptides to small peptides and amino acids (Sousa et al., 2001). While PAB are
78 weakly proteolytic, they do contain various peptidases that may contribute to proteolysis in the cheese upon lysis. Therefore, starter and nonstarter PAB may also contribute to proteolysis in the later stages of ripening.
Eyes are an important characteristic associated with Swiss cheese. Eye development depends on time, quantity, intensity and rate of CO2 production as well as
the number and size of the nucleation sites (Polychroniadou, 2001). Carbon dioxide
produced by PAB results in this eye formation, which occurs during WRT, and growth of
PAB is encouraged at this time because of the pliable cheese matrix. Eye formation
should take place at a slow, uniform rate. If the rate of gas production is too rapid the
casein network is unyielding to increased gas pressure and splits form and if too slow
cheese will become underset with small eyes or no eyes (White et al., 2003). Eyes were
observed in all cheeses after CRT with differences among the ripening period. This study
showed the length of the ripening period influences eye formation with an increase seen
after ninety day while extending the ripening period at the highest temperature resulted in
all cheese having the most eyes formed at that time.
Splits are one of many eye deformities observed after cool ripening once the
cheese is sliced. Their presence leads to the cheese being difficult to slice as well as being
downgraded. Some phenomena that contribute to splits are late production of CO2, fermentation of citrate, and changes in CO2 solubility with changes in temperature (Noël
et al., 1999). Once the cheese enters the cool ripening period its body becomes more rigid and unable to withstand further CO2 production. Splits may result if PAB continue to be metabolically active during this time producing more CO2. Cheese is usually
ripened for sixty days, packaged, and distributed for further processing into slices or
79 chunks, making it difficult to determine when and where eye deformities may occur.
Defects may not become apparent until after distribution to the cutting and slicing facility.
In this study cheeses underwent different cool ripening temperatures and different ripening periods. Splits were observed in all cheeses. Visible splits were higher in cheeses made with the most cold tolerant P. freudenreichii strain (P873). All cheeses held at the highest temperature also had a large number of splits. In 1967, Parks et al. reported the commercial storage temperature for Swiss cheese is 7.2°C but it can be ripened in a range of 2.8 to 7.2°C. Some commercial storage temperatures used today range from 0-7.2°C. Hettinga and Reinbold (1965) found that the degree of proteolysis was not correlated with splitting. The fewest slits were seen among strains P572 and
P196 at the lowest temperature suggesting strain and temperature do have an affect on split occurrence. Careful P. freudenreichii strain selelction and temperature control during distribution and further processing are essential to reducing the occurrence of splits.
Some limitations to this study were the inability to precisely control all storage temperatures due to the commercial environment and the inability to slice the cheese after
WRT to follow the progression of eye formation. Also the confirmation of all starter and potential non-starter bacteria was not performed but it is recommended because their presence may contribute to some of the abovementioned results. It may also be important to evaluate the rate of gas production during both ripening periods. Overall the findings of the this study provide sufficient evidence that the combination of ripening temperature,
80 strain, and length of ripening are factors that should be considered for Swiss cheese manufacture in helping reduce the occurrence of splits.
81 Strain %Fat (FDB)b %Moisture %Protein %Salt(D30) pH
P572d 49.75(0.4) 38.83(0.7) 25.28(0.8) 0.61(0.1) 5.21(0.01)
P196e 49.89(0.5) 38.40(0.2) 24.59(0.2) 0.61(0.1) 5.22(0.04)
P873f 49.87(0.1) 38.43(0.2) 25.04(1.6) 0.39(0.1) 5.21(0.01) a Standard deviation in parenthesis (n = 3). b FDB = fat on a dry basis. c FOC = fat on cheese. d Strain characterized as cold sensitive. e Strain characterized as intermediate. f Strain characterized as cold tolerant.
Table 4.1 Make day and day 30 composition of Swiss cheese manufactured with different P. freudenreichii strains.a
82 Duration and Temperature of Ripening
0 d 30 d 60 d 90 d 0°C 4°C 7.2°C 0°C 4°C 7.2°C ala 210.7 576.9 531.1 263.0 388.6 677.3 551.92 368.72 arg 208.9 b 821.7 a 693.8 a 558.8 a 847.6 a 1605.9 a 1149.3 a 801.4 a,2 asn 17.8 b 518.2 a 609.7 a 420.1 a 560.9 a 1078.7 a 881.2 a 534.4 a,2 asp 65.0 240.2 191.0 148.1 205.2 356.1 213.0 155.6 citrulline 21.8 b 35.1 a 27.1 a,b 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c cys 0.1 14.3 10.5 0.0 0.0 0.0 0.0 0.0 gln 60.6 b 430.5 a 489.6 a 304.5 a 402.7 a 902.5 a 669.6 a,2 435.6 a,2 glu 89.3b 1078.1 a 1213.4 a 738.8 a 735.5 a 1491.6 a 1767.1 a 1105.2 a gly 22.2 b 142.5 a 193.7 a 122.6 a 176.8 a 323.2 a 265.8 a 173.2 a,2 his 63.8 b 291.3 a 308.5 a 192.4 a 229.1 a 508.9 a 445.7 a 273.7 a ile 21.2 b 313.1 a 343.5 a 233.5 a 277.2 a 813.2 a 457.6 a,1 302.2 a,2 leu 57.3 b 1307.3 a 1319.5 a 826.6 a 1083.7 2331.2 a 1250.2 a 1065.8 a a,2 lys 254.5 b 1188.3 a 1070.8 a 805.8 a 1046.0 2192.4 a 1520.5 1000.2 a a,2 a,2 met 11.1 186.2 190.8 127.0 150.9 315.9 194.52 135.62 phe 30.1 b 654.4 a 621.4 a 351.3 a 493.8 a 1071.1 a 667.4 a,2 467.2 a,2 pro 139.7 c 383.3 b 435.4 b 468.1 b 745.0 749.6 a,b 1153.0 a 673.4 a,b a,b ser 21.1 b 176.0 a 157.1 a 127.8 a 177.6 a 379.0 a 264.9 a 168.4 a,2 thr 29.5 b 250.5 a 265.0 a 175.2 a 248.0 a 531.6 a 345.2 a,2 235.1 a,2 trp 6.0 c 54.3 b 44.2 b 0.0 c 29.6 b 133.2 a 54.8 b 34.3 b tyr 32.9 b 325.3 a 250.5 a 163.9 a 210.5 a 645.1 a 323.3 a,2 222.1 a val 73.5 b 818.2 a 870.7 a 557.8 a 715.9 a 1553.2 a 1065.2 729.4 a,2 a,2 Total 1437.1 9805.6 9837.2 6585.1 8724.7 17659.6 13495.9 8881.7 b c a,b a,b a,b b a a *Values with different superscript letters within the same row are significantly different (P<0.05). #Values with different superscript numbers are significantly different from the corresponding value in Tables 4.2 and 4.3. Table 4.2 Amino acid concentrations (ug/g) in cheeses prepared with P. freudenreichii strain P572.
83 Duration and Temperature of Ripening
0 d 30 d 60 d 90 d 0°C 4°C 7.2°C 0°C 4°C 7.2°C ala 216.1 578.9 356.4 270.5 839.5 538.2 562.52 686.91 arg 207.2 b 788.7 a 520.8 a 560.8 a 1344.0 a 1129.7 a 1228.5 a 1481.4 a,1 asn 17.8 b 501.1 a 409.7 a 416.8 a 1076.4 a 717.0 a 944.3 a 1004.2 a asp 57.9 249.6 a 163.4 132.7 254.6 313.9 261.4 293.2 citrulline 22.2 b 32.6 a 19.9 b 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c cys 0.1 9.2 2.5 0.0 0.0 0.0 0.0 0.0 gln 62.5 b 459.6 a 352.6 a 266.6 a 862.2 a 623.0 a 740.2 792.9 a,1 a,1,2 glu 74.0 b 1303.7 a 1008.8 845.2 a 1690.1 a 1269.7 a 1843.7 a 1775.2 a a gly 20.9 b 148.4 a 132.4 a 117.1 a 347.3 a 259.8 a 291.6 a 334.0 a,1 his 60.2 b 326.3 a 223.9 a 166.1 a 548.3 a 388.7 a 492.0 a 492.6 a ile 17.6 b 338.2 a 250.9 a 200.8 a 465.5 a 493.2 a 538.0 549.3 a,1 a,1,2 leu 57.1 b 1260.0 a 902.6 a 827.6 a 2025.9 a 1710.0 a 1084.0 a 1922.4 a,1 lys 240.6 b 1285.3 a 846.0 a 682.5 a 2148.0 a 1583.4 a 1607.9 1839.3 a,2 a,1 met 8.7 191.3 a 132.0 102.4 221.3 215.7 206.52 249.01 phe 25.4 b 624.5 a 421.4 a 322.6 a 936.9 a 747.0 a 719.0 a,2 810.5 a,1 pro 127.5 c 397.9 b 353.9 b 412.0 b 1068.1 a 629.1 b 1438.7 a 1055.6 a ser 19.6 b 175.8 a 129.5 a 119.0 a 270.2 a 277.4 a 285.9 a 324.7 a,2 thr 28.7 b 254.8 a 187.2 a 152.2 a 500.4 a 380.6 a 365.6 a,2 430.1 a,1 trp 5.0 c 46.4 a 28.9 a 0.0 b 60.4 a 76.2 a 54.8 a 57.3 a tyr 28.0 b 291.8 a 178.9 a 158.7 a 342.4 a 342.6 a 298.8 a,2 391.5 a val 66.2 b 828.5 a 604.9 a 503.8 a 1185.3 a 1146.7 a 1189.7 1328.8 a,1,2 a,1 Total 1363.3 10093.5 7226.6 6257.3 16189.7 12842.1 14663.6 15819.1 c a,b b b a a a a,1 *Values with different superscript letters within the same row are significantly different (P<0.05). #Values with different superscript numbers are significantly different from the corresponding value in Tables 4.1 and 4.3. Table 4.3 Amino acid concentrations (ug/g) in cheeses prepared with P. freudenreichii strain P196.
84 Duration and Temperature of Ripening
0 d 30 d 60 d 90 d 0°C 4°C 7.2°C 0°C 4°C 7.2°C ala 233.9 b 651.8 a,b 471.8 276.7 b 809.0 a,b 547.0 a,b 917.5 a,1 643.2 a,b a,b,1 arg 179.5 b 830.6 a 722.9 a 794.6 a 1175.9 a 1143.3 a 1842.2 a 1360.9 a,1 asn 14.7 b 555.4 a 546.7 a 573.9 a 974.3 a 792.0 a 1374.1 a 939.2 a,1 asp 67.9 273.0 225.6 190.5 146.2 219.4 174.5 191.6 citrulline 21.5 a 37.0 a 28.8 a 0.0 b 0.0 b 0.0 b 0.0 b 0.0 b cys 0.3 23.7 2.9 0.0 0.0 0.0 0.0 0.0 gln 59.4 b 484.5 a 475.7 a 401.5 a 801.5 a 720.5 a 1135.7 726.2 a,1 a,1,2 glu 56.6 c 1247.2 b 1190.3 879.3 b 1438.2 b 1372.5 b 2539.9 a 1388.9 b b gly 24.4 c 154.1 b 170.6 b 166.2 b 303.9 a 219.6 a,b 421.5 a 293.5 a,1,2 his 55.1 b 322.5 a 287.0 a 237.3 a 484.0 a 404.2 a 692.8 a 418.5 a ile 17.7 b 360.2 a 336.0 a 323.3 a 562.9 a 719.9 a 773.2 a,1 492.8 a,1,2 leu 59.3 b 1429.0 a 1229.0 1128.0 2055.9 a 1728.6 a 2489.3 a 1788.9 a a a,1,2 lys 227.3 b 1336.0 a 1158.5 986.2 a 1925.9 a 1707.4 a 2450.5 1656.3 a a,1 a,1 met 9.3 205.0 185.0 149.0 273.8 237.4 316.81 211.41,2 phe 27.1 b 726.6 a 616.1 a 458.9 a 904.9 a 839.5 a 1082.5 766.1 a,1 a,1,2 pro 137.5 c 464.5 b 467.1 b 701.4 b 1323.4 a 611.5 b 1326.5 a 1036.3 a ser 21.5 b 201.2 a 185.6 a 190.9 a 319.4 a 311.8 a 457.9 a 303.3 a,1 thr 28.3 b 265.4 a 251.1 a 221.3 a 463.9 a 410.3 a 563.4 a,1 387.5 a,1,2 trp 4.8 b 52.4 a 40.8 a 0.0 b 53.8 a 101.4 a 81.6 a 46.7 a tyr 27.1 b 338.0 a 250.9 a 221.8 a 312.5 a 382.0 a 480.8 a,1 348.7 a val 70.4 b 934.8 a 835.6 a 741.3 a 1308.5 a 1204.6 a 1736.5 1179.4 a,1 a,1,2 Total 1343.5 10892.8 9678.4 8641.9 15637.7 13672.8 20857.2 14179.4 c a,b b b a a a a,1 *Values with different superscript letters within the same row are significantly different (P<0.05). #Values with different superscript numbers are significantly different from the corresponding value in Tables 4.1 and 4.2
Table 4.4 Amino acid concentrations (ug/g) in cheeses prepared with P. freudenreichii strain P873.
85
% Eyes Timepoint (Temp.) P572 P196 P873
Day 60 (0°C) 8.83b 8.32b 7.97b Day 90 (0°C) 10.95b 8.58b 13.62b
Day 60 (4°C) 7.80b,2 14.52b,1 7.20b,2 Day 90 (4°C) 17.68a,b 14.13b 12.88b
Day 60 (7.2°C) 10.33b 10.73b 10.80b Day 90 (7.2°C) 27.28a 35.13a 27.80a * Values within the same column with different letters are significantly different (P<0.05). # Values within the same row with different numbers are significantly different (P<0.05). Table 4.5 Percent of cheese slice surface area covered by eyes at sixty and ninety days of cool ripening at 0, 4, and 7.2°C.
% Splits Timepoint (Temp.) P572 P196 P873
Day 60 (0°C) 0.033c 0.033c 0.200b Day 90 (0°C) 0.050c,2 0.017c,2 0.380b,1
Day 60 (4°C) 0.017c,2 0.067c,1,2 0.300b,1 Day 90 (4°C) 0.000c 0.117c 0.067b
Day 60 (7.2°C) 0.117b,2 0.917a,1 1.350a,1 Day 90 (7.2°C) 0.483a,2 0.250b,3 0.717a,1 * Values within the same column with different letters are significantly different (P<0.05). # Values within the same row with different numbers are significantly different (P<0.05). Table 4.6 Percent of cheese slice surface area covered by splits at sixty and ninety days of at 0, 4, and 7.2°C.
86
A1 B1
A2 B2
A3 B3
Figure 4.1 Digital images of cheeses manufactured with different P. freudenreichii strains ripened at different temperatures. Column A represents visible eyes and column B epresents visible splits. Row 1 represents strain P572, row 2 represents strain P196, and row 3 represents strain P873. 87 CONCLUSIONS
• The dairy propionibacteria differ in their abilities to grow at lower temperatures.
• The maximum growth rates and lag phases for the four strains varied in CDM
with glycine betaine, proline, or glutamate.
• In CDM, strains P728 and P873 were the most tolerant at the lowest temperature
(7.2°C) while strains P843 and P572 were the most sensitive.
• All strains showed improved growth at low temperatures when any of the three
compatible solutes were added to the medium.
• Importation of glycine betaine, proline, and glutamate at low temperatures differs
among P. freudenreichii strains.
• The ability of dairy propionibacteria to transport proline is not affected by
temperature but temperature does affect glycine betaine and glutamate transport.
• Proline was transported most abundantly by all strains at all temperatures but at
various degrees.
• At the lowest temperature (7.2°C) all solutes were transported by all strains.
• Freeze-thaw tolerance is strain dependent in propionibacteria.
• The effect of growing cells in glycine betaine at 28-30°C on subsequent freeze-
thaw stress appears to be strain dependent.
• Glycine betaine was found to exert a better cryoprotective effect on strain P728
when subjected to multiple freeze thaw cycles.
88 • Some P. freudenreichii strains may synthesize cold shock/cold induced proteins
in response to a rapid downshift in temperatures making them ideal for use in
starter cultures.
• Proline concentration increased from make day and during the ripening period for
all P. freudenreichii strains with an observed difference between make day, warm
room, and cool ripening.
• There was a difference in glutamic acid concentration in all strains from make day
to warm room ripening but no difference was seen throughout the cooler ripening
period.
• There was no difference in the concentration of glutamic acid and proline during
CRT between the strains.
• Visible splits were higher in cheeses made with the most cold tolerant P.
freudenreichii strain (P873).
• Temperature and the length of ripening have not only had an effect on split
formation but the strain used in manufacture does as well.
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Appendix A: Graphs displaying cold tolerance of Propionibacterium strains.
98 2.5 2.5 P572 P843 2.0 2.0
1.5 1.5 A600nm A600nm 1.0 1.0
0.5 0.5
0.0 0.0 MMC 024681012 024681012 1GB 100GB Time (Days) Time (Days) 1Pro 100Pro 2.5 2.5 1Glu 100Glu P728 P873 2.0 2.0
1.5 1.5 A600nm
A600nm 1.0 1.0
0.5 0.5
0.0 0.0 024681012 024681012 Time (Days) Time (Days)
Figure A.1 Effects of various concentrations of glycine betaine, proline and glutamate on the growth of P. freudenreichii strains P843, P572, P728, and P873 at
30°C. Strains were grown in chemically defined media containing 1 mM or 100 mM of glycine betaine, proline, or glutamate. Absorbance was read spectrophotmetrically at
600nm.
99 2.0 2.0
P572 P843 1.5 1.5
1.0 1.0 A600nm A600nm
0.5 0.5
0.0 0.0 0 2 4 6 8 10 12 14 16 0246810121416 MMC Time (Days) Time (Days) 1GB 100GB 2.0 2.0 1Pro P728 100Pro P873 1Glu 100Glu 1.5 1.5
1.0 1.0 A600nm A600nm
0.5 0.5
0.0 0.0 0246810121416 0246810121416 Time (Days) Time (Days)
Figure A.2 Effects of various concentrations of glycine betaine, proline and glutamate on the growth of P. freudenreichii strains P843, P572, P728, and P873 at
22°C. Strains were grown in chemically defined media containing 1 mM or 100 mM of glycine betaine, proline, or glutamate. Absorbance was read spectrophotmetrically at
600nm.
100 2.0 2.0
P843 P572
1.5 1.5
1.0 1.0 A600nm A600nm
0.5 0.5
MMC 0.0 0.0 0 20406080100 0 204060801001GB 100GB Time (Days) Time (Days) 1Pro 100Pro 2.0 2.0 1Glu 100Glu P728 P873 1.5 1.5
1.0 1.0 A600nm A600nm
0.5 0.5
0.0 0.0 0 204060801000 20406080100
Time (Days) Time (Days)
Figure A.3 Effects of various concentrations of glycine betaine, proline and glutamate on the growth of P. freudenreichii strains P843, P572, P728, and P873 at
10°C. Strains were grown in chemically defined media containing 1 mM or 100 mM of glycine betaine, proline, or glutamate. Absorbance was read spectrophotmetrically at
600nm.
101 2.0 2.0
P843 P572
1.5 1.5
1.0 1.0 A600nm A600nm
0.5 0.5
0.0 0.0
0 20406080100 0 20406080100 MMC Time (Days) Time (Days) 1 GB 100 GB 1 Pro 2.0 2.0 100 Pro P728 1 Glu P873 100 Glu 1.5 1.5
1.0 1.0 A600nm A600nm
0.5 0.5
0.0 0.0
0 20406080100 0 20406080100
Time (Days) Time (Days)
Figure A.4 Effects of various concentrations of glycine betaine, proline and glutamate on the growth of P. freudenreichii strains P843, P572, P728, and P873 at
7.2°C. Strains were grown in chemically defined media containing 1 mM or 100 mM of glycine betaine, proline, or glutamate. Absorbance was read spectrophotmetrically at
600nm.
102
Appendix B: Bacteria enumeration and digital images of Swiss cheese manufactured with different Propionibacterium strains.
103
PABb strain Lactobacillus species Streptococcus thermophilus
P572c 5.56 x 108 8.34 x 109 P196d 1.10 x 109 5.85 x 109 P873e 1.47 x 109 9.39 x 107 a Cells were enumerated from make day (Day 0) samples. b PAB = propionibacteria. c Strain characterized as cold sensitive. d Strain characterized as intermediate. e Strain characterized as cold tolerant.
Table B.1 Viable cell counts of lactic acid bacteria used to manufacture Swiss cheese in triplicate with different P. freudenreichii strains.a
104
A B C D E F
Figure B.1 Digital images of P. freudenreichii strain P527 (cold-sensitive) at 0, 4, and
7.2°C for 60 days (A, C, and E) and 0, 4, and 7.2°C for 90 days (B, D, and F).
105
A B C D E F
Figure B.2 Digital images of P. freudenreichii strain P196 (intermediate) at 0, 4, and
7.2°C for 60 days (A, C, and E) and 0, 4, and 7.2°C for 90 days (B, D, and F).
106
A B C D E F
Figure B.3 Digital images of P. freudenreichii strain P572 (cold-tolerant) at 0, 4, and
7.2°C for 60 days (A, C, and E) and 0, 4, and 7.2°C for 90 days (B, D, and F).
107