1 Author: Vanevenhoven, Daniel W. A Characterization of the ......Raw milk Gouda cheese was sourced...

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Author: Vanevenhoven, Daniel W. Title: A Characterization of the Rheology of Raw Milk Gouda Cheese

The accompanying research report is submitted to the University of Wisconsin-Stout, Graduate School in partial

completion of the requirements for the

Graduate Degree/ Major: MS Food and Nutritional Sciences

Research Adviser: Naveen Chikthimmah, Ph.D.

Submission Term/Year: Summer, 2012

Number of Pages: 71

Style Manual Used: American Psychological Association, 6th edition

I understand that this research report must be officially approved by the Graduate School and that an electronic copy of the approved version will be made available through the University Library website

I attest that the research report is my original work (that any copyrightable materials have been used with the permission of the original authors), and as such, it is automatically protected by the laws, rules, and regulations of the U.S. Copyright Office.

My research adviser has approved the content and quality of this paper. STUDENT:

NAME Daniel Vanevenhoven DATE: July, 2012

ADVISER: (Committee Chair if MS Plan A or EdS Thesis or Field Project/Problem):

NAME Naveen Chikthimmah, PhD DATE: July, 2012

--------------------------------------------------------------------------------------- ------------------------------------------

This section for MS Plan A Thesis or EdS Thesis/Field Project papers only Committee members (other than your adviser who is listed in the section above) 1. CMTE MEMBER’S NAME: Eun Joo Lee, PhD DATE: July, 2012

2. CMTE MEMBER’S NAME: James Burritt, PhD DATE: July, 2012

3. CMTE MEMBER’S NAME: Hans Zoerb, PhD DATE: July, 2012

----------------------------------------------------------------------------------------------------------------------------- ---- This section to be completed by the Graduate School This final research report has been approved by the Graduate School.

Director, Office of Graduate Studies: DATE:

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Vanevenhoven, Daniel W. A Characterization of the Rheology of Raw Milk Gouda Cheese

Abstract

The objective of this study was to characterize the rheology of raw milk Gouda cheese aged up

to six months. Raw milk Gouda cheese was obtained from a local artisan. Rheological

characterization included the compression test to determine yield stress, yield strain, and 5%

strain secant modulus. The single-edge notched bend test was used to determine the critical

energy release rate. Hardness, cohesiveness, adhesiveness, chewiness, gumminess, and

springiness were calculated using Texture Profile Analysis (TPA). Cheese samples were

analyzed for proteolysis using SDS-PAGE. In the compression test, yield stress and 5% strain

secant modulus significantly increased with aging time (p < 0.05). Yield strain significantly

decreased with aging time (p < 0.05). For the single-edge notched bend test, the critical energy

release rate significantly decreased with aging at 2.3 months (p < 0.01) and then remained

approximately constant for the rest of the aging time. The TPA values for hardness and

chewiness significantly decreased at 2.3 months of aging (p < 0.01). SDS-PAGE studies showed

rapid degradation of the αs1- and αs2- caseins. This study has documented significant changes in

rheology by 2.3 months and the rapid degradation of the αs1- and αs2- caseins in raw milk Gouda

cheese.

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Acknowledgments

I would like to extend my appreciation to several individuals for the contributions they

have made to this research.

I would like to thank Dr. Hans Zoerb for his critical involvement during the inception of

this study, Dr. Naveen Chikthimmah for his guidance through the course of this work, and Drs.

Eun Joo Lee and James Burritt for their advice during this work.

I would like to express my gratitude to Holland’s Family Cheese, LLC for supporting this

research through the provision and donation of raw milk Gouda cheese.

I would like to thank my parents, family, and friends, for the support and encouragement

they have provided me throughout this thesis work.

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Table of Contents

Page

Abstract ........................................................................................................................................... 2

List of Figures ................................................................................................................................. 7

Chapter I: Introduction .................................................................................................................... 8

Statement of the Problem .......................................................................................................... 10

Purpose of the Study ................................................................................................................. 10

Assumptions of the Study ......................................................................................................... 11

Definition of Terms ................................................................................................................... 11

Limitations of the Study ............................................................................................................ 11

Methodology ............................................................................................................................. 12

Chapter II: Literature Review ....................................................................................................... 13

Overview of Cheese .................................................................................................................. 13

Gouda Cheese ........................................................................................................................ 14

Milk Protein............................................................................................................................... 15

Caseins ................................................................................................................................... 15

Whey Proteins........................................................................................................................ 16

Cheesemaking ........................................................................................................................... 16

Raw Milk Cheese ...................................................................................................................... 19

Aging of Cheese ........................................................................................................................ 21

Proteolysis ............................................................................................................................. 21

Rheology ................................................................................................................................... 25

Uniaxial Compression ........................................................................................................... 26

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Texture Profile Analysis ........................................................................................................ 32

Table 1: Texture Profile Analysis Parameters and Definitions ............................................. 35

Bending Test .......................................................................................................................... 37

Chapter III: Methodology ............................................................................................................. 41

Materials .................................................................................................................................... 41

Moisture Content ....................................................................................................................... 41

Proteolysis ................................................................................................................................. 41

Rheology ................................................................................................................................... 43

Uniaxial Compression ........................................................................................................... 43

Texture Profile Analysis ........................................................................................................ 43

Bending Test .......................................................................................................................... 44

Statistical Analysis .................................................................................................................... 45

Chapter IV: Results ....................................................................................................................... 46


Proteolysis ................................................................................................................................. 47

Uniaxial Compression ............................................................................................................... 48

Texture Profile Analysis............................................................................................................ 51

Bending Test ............................................................................................................................. 54

Chapter V: Discussion .................................................................................................................. 55


Proteolysis ................................................................................................................................. 55

Uniaxial Compression ............................................................................................................... 57

Texture Profile Analysis............................................................................................................ 58

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Bending Test ............................................................................................................................. 59

Conclusions ............................................................................................................................... 61

Recommendations ..................................................................................................................... 62

References ..................................................................................................................................... 64

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List of Figures

Figure 1. A Universal Testing Machine (UTM) .......................................................................... 26

Figure 2. A typical stress-strain curve ......................................................................................... 29

Figure 3. A typical Texture Profile Analysis force-time curve for cheese .................................. 34

Figure 4. The Single-Edge Notched Bend (SENB) sample geometry ......................................... 39

Figure 5. Moisture content of raw milk Gouda cheese aged to 6.3 months ................................ 46

Figure 6. SDS-PAGE of protein extracted from raw milk Gouda cheese aged to 6.3 months .... 47

Figure 7. 5% strain secant modulus of raw milk Gouda cheese aged to 6.3 months ................... 48

Figure 8. Yield stress of raw milk Gouda cheese aged to 6.3 months ......................................... 49

Figure 9. Yield strain of raw milk Gouda cheese aged to 6.3 months ......................................... 49

Figure 10. Stress-strain curves for raw milk Gouda cheese aged to 6.3 months ......................... 50

Figure 11. Mean TPA parameters (I) of raw milk Gouda cheese aged to 6.3 months ................ 52

Figure 12. Mean TPA parameters (II) of raw milk Gouda cheese aged to 6.3 months ............... 53

Figure 13. Mean fracture toughness of raw milk Gouda cheese aged to 6.3 months .................. 54

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Chapter I: Introduction

Cheese is a popular product throughout the United States and many other regions of the

world. The United States produced 10.6 billion pounds of cheese in 2011 (National Agricultural

Statistics Service, 2011). Cheese production and consumption in the United States has seen a

steady increase over the last 10 years.

Cheese is a particularly popular product in the Western world. Cheese types have been

developed all over the world to fit the needs of chefs and consumers (Gunasekaran & Ak, 2003).

Cheese can be hard or soft, bland or pungent. Cheesemaking is an essential milk preservation

technique and cheese is an important source of nutrition. Many of the nutrients in milk are found

highly concentrated in cheese (The Dairy Council, 2012). Cheese is a source of high quality

protein, calcium, phosphorus, iodine, vitamin A, and calories.

Cheesemaking begins with the coagulation of the milk proteins. This coagulation results

in the formation of curd. The curds are separated from the remaining milk fraction. The curd is

then salted and shaped. Depending on the cheese type, but particularly in hard cheeses, the curd

may be pressed to increase cohesion. Salting is a necessary step in all cheese for the

development of texture and flavor.

Structurally, cheese is a matrix formed by the milk proteins known as caseins (Luyten,

van Vliet, & Walstra, 1991a). Upon its formation, this matrix traps milk fat globules dispersed

throughout the milk. Cheese can be considered a hydrated protein matrix, with interspersed filler

fat particles (Luyten, 1988).

Cheese ripening is a complex internal process involving multiple simultaneous

interactions. Ripening is primarily the result of the action of enzymes and bacteria present in the

cheese. Ripening is necessary for the development of the characteristic flavor, color, and texture

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of the different cheese types and ages (Gunasekaran & Ak, 2003). Ripening involves the

breakdown of proteins. This proteolysis contributes to the changing rheology of the cheese. The

level of proteolytic activity varies by cheese type and production methods. Proteolysis is largely

responsible for the flavor and texture development in cheese (Gunasekaran & Ak, 2003).

The complex and varied methods of cheesemaking and the complex nature of the cheese

itself make it difficult to control or modify the properties of cheese (Gunasekaran & Ak, 2003).

The physical properties of cheese are influenced by milk composition, manufacturing methods,

and the maturation environment (Lucey, Johnson, & Horne, 2003).

Many of the mechanical properties of cheese are related to the structure of casein proteins

(Lucey et el., 2003). In the United States, cheese is often used as an ingredient where its

rheological properties are as important as its flavor. Cheese is used as a topping on pizza and

breads, in foods such as lasagna, and on cheeseburgers. In these applications, physical properties

such as the ability to slice, stretch, and melt are of primary importance.

Rheology is the study of deformation and flow of matter (Gunasekaran & Ak, 2003).

Several instruments have been used to study the rheology of cheese. These include the shear-

press, viscometer, consistometer, and many others (Friedman, Whitney, & Szczesniak, 1963).

Several methods have been developed for use with the universal testing machine, including the

bending test, compression test, tension test, and others.

The effects of cheesemaking techniques have been studied anecdotally and controlled for

centuries by experienced cheesemakers. A better understanding of the cause and effect of the

changing protein structure will aid in quality control during cheese manufacture and for judging

maturation. Understanding mechanisms for the development of the rheological properties of

cheese may help develop cheeses with specific properties.

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A type of cheese that is of interest is Gouda cheese. Young Gouda cheese has a mild

flavor; aged Gouda cheese is complex in its sensory profile. Gouda cheese is a semi-hard and

full fat cheese. Although Gouda cheese represents a small share of the total United States cheese

consumption, sales of Gouda cheese have recently increased (Buragas, 2005). Rheology and

flavor has been shown to be a factor in the overall acceptance of Gouda cheese (Yates & Drake,

2007). Proteolysis is an important factor in the development of texture and flavor in cheese.

Few rheological and proteolytic studies exist on raw milk Gouda cheese. This study will

therefore investigate the relationships between age, rheology, and proteolysis in raw milk Gouda

cheese.

Statement of the Problem

The rheology of cheese is an important quality parameter. The rheology of cheese affects

how it is consumed and its overall consumer acceptance. It is important to quantify rheology so

that it may be used as a measure for producers and processors in maintaining product

consistency. Gouda cheese is increasing in popularity in the United States. The rheology of raw

milk Gouda cheese has not been well studied.

Purpose of the Study

The objective of this study was to characterize the rheology and the extent of proteolysis

in raw milk Gouda cheese up to 6 months of age. This study was an original look at the

changing rheology and degree of proteolysis in raw milk Gouda cheese during aging. Aging

time is an important parameter influencing the quality of cheese. For artisanal cheese producers,

determining the effect of aging time on cheese rheology is important for quality and process

optimization. Quantifying the rheology of raw milk Gouda cheese will provide an understanding

for manufacturers to aid in determining maturation and quality. Proteolysis has been shown to

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affect the rheology of cheese and is useful in understanding the changing rheology of aging

cheese.

Assumptions of the Study

Raw milk Gouda cheese was sourced from an artisanal Gouda cheese producer who

maintained consistency in process. It was assumed that the Gouda cheese samples were made

according to style. It was also assumed that raw milk Gouda cheese made during different times

of the year did not vary considerably.

Definition of Terms

Gouda cheese. Gouda cheese must have at least 46% milkfat by weight of total solids

and not more than 45% moisture (21 C.F.R. § 133.142, 2012). The cheese is semi-hard. The

flavor is buttery and slightly sweet with a firm texture (Wisconsin Milk Marketing Board, 2012).

Viscoelastic. A property of materials that exhibit both viscous flow and elastic

deformation.

Rheology. The study of deformation and flow of matter (Gunasekaran & Ak, 2003).

Often used as an equivalent term to texture, which in food applications is used to describe the

flow, deformation, and disintegration of a sample under force (Tunick, 2000). Rheology is

primarily concerned with the study of viscoelastic materials.

Proteolysis. The breakdown of proteins. In cheese it is primarily the result of enzymatic

and bacteriological action.

Limitations of the Study

Pasteurized milk Gouda cheese was not used as a control for the comparison of raw milk

Gouda cheese. The purpose of this study was to characterize the rheology of raw milk Gouda

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cheese. A comparison with the rheology of pasteurized milk Gouda cheese would provide useful

information on the effect of pasteurization on rheology but was beyond the scope of this study.

Methodology

Raw milk Gouda cheese was obtained from a local artisanal Gouda cheese producer in

Central Wisconsin. Cheese rheology was evaluated using an Instron Universal Testing Machine.

Rheological characterization methods included the compression test, the single-edge notched

bend test, and Texture Profile Analysis (TPA). Cheese samples were analyzed for proteolysis

using SDS-PAGE.

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Chapter II: Literature Review

This chapter will begin with an overview of cheesemaking. The properties of milk

protein and raw milk will follow. The effects of aging on cheese will be discussed. This chapter

will conclude with a discussion of cheese rheology.

Overview of Cheese

Cheese is a popular product in the United States and throughout the world. The United

States produced 10.6 billion pounds of cheese 2011 (National Agricultural Statistics Service,

2011). Cheese production and consumption in the United States has seen a steady increase over

the last 10 years. Production is dominated by Cheddar and Mozzarella cheese, which account for

two-thirds of the total United States cheese production.

Cheese styles have been developed all over the world and vary considerably in their

rheology. It can be difficult to categorize the many styles of cheese. Where rheology is the

primary interest, a classification based on firmness and ripening method may be most relevant

(Gunasekaran & Ak, 2003). Cheese can first be divided by manufacturing method (acid or

enzyme coagulated). Enzyme coagulated cheese can be further divided by ripening method

(bacteria or mold). Lastly, cheese can be categorized by firmness (soft, semisoft, hard, or very

hard).

Cheese can be made from raw (unpasteurized) or pasteurized milk. Pasteurized milk is

commonly used in cheesemaking. In the United States, raw milk cheese must be “cured for a

period of 60 days at a temperature not less than 35°F” (7 C.F.R. § 58.439, 2012).

In the first stage in cheesemaking, the milk proteins are coagulated to form a semisolid

gel. The gel is cut to allow the whey to be drained from the curds. The curds are then pressed

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into a definite shape and salted. Finally, many cheeses are ripened for a period of time to

develop flavor and texture.

Gouda Cheese. The United States has increased its total cheese production over the last

five years to 10.6 billion pounds in 2011 (National Agricultural Statistics Service, 2011). Gouda

cheese is small part of the total United States production with about 2 million pounds produced

annually (Beck, 2012). However, Gouda cheese represents a growing segment of the cheese

market and has a large potential for growth (Buragas, 2005).

There is not a required manufacturing method for Gouda cheese in the United States.

However, a basic procedure has been defined (21 C.F.R. § 133.142, 2012). The milk is warmed

and acidified by the action of a lactic acid bacterial culture. Rennet or other enzymes are used to

coagulate the mixture. After coagulation, the mass is cut, stirred, and heated. The mixture is

further stirred and heated, diluted with water or brine, and salted to facilitate the removal of

whey. The whey is then drained and the curds pressed. The pressed curds are removed and

salted and cured.

Gouda cheese must have at least 46% milkfat by weight of total solids and not more than

45% moisture by weight (21 C.F.R. § 133.142, 2012). Gouda cheese is a semi-hard, full-fat

cheese (Bertola, Califano, Bevilacqua, & Zaritzky, 2000).

Cheese is a source of high quality protein, calcium, phosphorus, iodine, and vitamin A

(The Dairy Council, 2012). On a 100 g basis, Gouda cheese has 700 mg calcium, 546 mg

phosphorus, 563 IU vitamin A, and 0.33 mg riboflavin (Nutrient Data Library, 2012).

In Gouda cheese, small holes, known as “eyes”, form within the cheese (Luyten et al.,

1991a). The eyes are caused by the production of gas by bacteria. As the gas pressure increases,

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round eyes are formed. In poor quality Gouda cheese, gas production may cause the cheese to

fracture and form slits instead of eyes.

Young Gouda cheese has a mild flavor (Bertola et al., 2000). The cheese is buttery and

slightly sweet with a creamy texture (Wisconsin Milk Marketing Board, 2012). As it ages,

Gouda cheese develops a complex flavor and a firmer texture.

Milk Protein

Milk proteins are classified as either caseins or whey proteins.

Caseins. The caseins are the main protein group in milk. Casein proteins make up

approximately 80% of the total protein content of milk. The caseins, together with calcium and

phosphate called colloidal calcium phosphate (CCP), form colloidally dispersed aggregates in

milk. These aggregates are known as casein micelles (Lucey et al., 2003). The micelle scatters

light, imparting on milk its whiteness. Their primary function is to provide nutrition for the

young offspring. The micelle is 50-300 nm in diameter (Choi, Horne, & Lucey, 2011). The

molecular weight of CCP has been measured at about 7,000 g/mol. The casein micelle is stable

even at boiling temperatures (Holt, 1992). However, the addition of rennet to the milk or

adjusting the milk to pH 4.6 readily coagulates the caseins.

The caseins are the milk phosphoproteins that precipitate at pH 4.6 and 20°C (Farrell et

al., 2004). The caseins are grouped into families called αs1-, αs2-, β-, and κ-casein, and are found

in proportions of approximately 3:1:3:1, respectively.

The precise structure of the casein micelle is unknown. Several models have been

proposed, including micelles comprised of smaller submicelles. The most recent and generally

accepted theory proposes a dual-binding model for the casein micelle (Horne, 1998). It consists

of a single primary structure involving interactions between the caseins and CCP. The Horne

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model proposes a dual binding of the caseins. The proposed bonding is between two

hydrophobic casein regions and between a hydrophilic casein region and CCP. The κ-casein acts

as an outside layer. The hydrophobic region of κ-casein faces inwards and the hydrophilic

region extends outward into the aqueous phase. New imagery of the casein micelle supports the

Horne model through the observation of evenly dispersed CCP and the absence of a micellar

substructure (Trejo, Dokland, Jurat-Fuentes, & Harte, 2011).

Whey Proteins. The whey is the liquid portion of milk that contains all the initial

components of milk that remain after the cheesemaking process. Whey contains the water

soluble components of milk including lactose, minerals, and water soluble vitamins.

The term whey proteins refer to the water soluble milk proteins that remain after

precipitation of the caseins at pH 4.6 and 20°C (Farrell et al., 2004). Whey proteins make up

approximately 20% of the total protein in milk. The whey proteins include β-lactoglobulin, α-

lactalbumin, bovine serum albumin, immunoglobulins, and lactoferrin. The whey also includes

some peptides cleaved from casein during the initial stage of cheesemaking.

Whey protein has an isoelectric point at pH 5.2 and can be denatured by heating (Singh,

Roberts, Munro, & Teo, 1996). When heat denatured, the whey proteins aggregate or interact

with the casein micelle. This coating of micelle with denatured whey proteins affects the

structure formation of acidified milk gels (Singh et al., 1996; Vasbinder, van de Velde, & de

Kruif, 2004).

Cheesemaking

Although the exact execution of the cheesemaking process varies between the many

cheese varieties, many cheesemaking processes have several commonalities. Before

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cheesemaking begins, milk is often pasteurized. Some cheeses are still made from raw

milk, especially in Europe (Fox, Guinee, Cogan, & McSweeney, 2000). However, many cheeses

today are made from pasteurized milk.

Pasteurization of the milk affects the rheological and flavor development of cheese

(Olson et al., 2011; Tunick, Van Hekken, Call, Molina-Corral, & Gardea, 2007). Pasteurization

destroys harmful bacteria that are present in the milk (Gunasekaran & Ak, 2003). Pasteurization

also destroys the lactic-acid bacteria and inactivates native enzymes. This has consequences

during the cheesemaking process as well as during cheese ripening. Traditionally, the lactic-acid

bacteria in milk lower the pH to an appropriate level for cheesemaking. However, if the milk is

pasteurized lactic-acid bacteria are added to the milk (starter culture) or the milk is directly

acidified by the addition of an acid (Gunasekaran & Ak, 2003). The starter bacteria added

during cheesemaking remain active during ripening, contributing to the flavor and texture

development. It is therefore desirable to use a starter culture when making cheese to be aged.

The diverse microflora and the enzymes of raw milk are also active during the ripening of raw

milk cheeses.

Prior to cheese making, milk is often standardized (Gunasekaran & Ak, 2003).

Standardization typically involves a normalizing of the fat content. Cream or skim milk may be

added as needed. Standardization is done to produce a consistent product regardless of possible

fluctuations in milk from several suppliers.

The first step in cheesemaking is the coagulation of the milk proteins known as caseins

(Gunasekaran & Ak, 2003). Casein can be coagulated through enzymatic action, acidification to

a pH of 4.6, or by heating and acidifying to a pH of 5.2. Enzymatic coagulation through the use

of rennet is the most common method (Lucey et al., 2003). Rennet contains two proteolytic

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enzymes, chymosin and bovine pepsin (Sousa, Ardo, & McSweeney, 2001). Chymosin and

pepsin contribute approximately 90% and 10% of the milk clotting activity of rennet,

respectively.

The first phase of enzymatic coagulation is the cleaving of the hydrophilic outside layer

of the casein micelle. This destabilizes the casein micelles and leads to its aggregation. The

network that forms entraps the fat globules. The formation of this gel shows a great deal of

dependence on the pH, Ca concentration, protein content, and temperature of the milk (Guinee,

Feeney, Auty, & Fox, 2002; Gunasekaran & Ak, 2003; Lucey, 2002). Eventually, the

aggregation forms a cohesive gel throughout the entire volume of milk. The firmness of the gel

is generally believed to be related to the number and strength of the bonds between the casein

micelles (Gunasekaran & Ak, 2003).

The gel matrix has a natural tendency to contract, leading to the expulsion of the liquid

phase (Gunasekaran & Ak, 2003). This is known as syneresis. The expulsed liquid is the whey.

The whey contains water soluble components including lactose, whey proteins, casein peptides

formed during coagulation, and salts. This process continues naturally over several hours. To

speed the removal of whey, the gel is cut into small cubes. This reduces the distance a given

volume of whey must travel to be expelled from the casein matrix. Cutting of the gel is a critical

process that allows the cheesemaker to control the moisture content of cheese (Lucey, 2002).

Cutting the gel early will allow more whey to be removed, resulting in cheese with lower

moisture content (Lucey et al., 2003). A late cutting results in a cheese with higher moisture.

The dependence of moisture content on cutting time is probably the result of more developed

bonding between the caseins.

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Correct moisture content is important in the manufacture of different cheese types. The

moisture content of cheese has a strong effect on its rheological properties (Luyten, 1988).

Moisture content also affects the microbial and enzymatic activity in cheese (Fox et al., 2000).

This activity in turn affects the ripening, stability, and quality of the cheese. The removal of

whey, which contains the lactose, removes a substrate for potential microbial activity, thereby

minimizing unwanted microbial action (Scott, Robinson, & Wilbey, 1998).

The curds are then drained of whey and typically salted and formed into a shape

(Gunasekaran & Ak, 2003). Salt affects the functionality and microbial properties of the cheese

(Fox et al., 2000; Paulson, McMahon, & Oberg, 1998). Hard and semi-hard cheeses (e.g.,

Gouda) are shaped by pressing (Gunasekaran & Ak, 2003). Pressing removes whey and

expedites the fusion of curds into a cohesive mass (Lucey et al., 2003).

Cheese ripening occurs after manufacturing and is essential for the development of the

characteristic flavor, texture, and appearance of different cheese types (Gunasekaran & Ak,

2003). During ripening, cheese undergoes proteolysis, lypolysis, and glycolysis which result in

structural and sensory changes. Ripening is primarily the result of microbial and enzymatic

action. Ripening will be discussed in more detail in a later section. A number of ripening

conditions exist to produce specific cheese varieties. The manufacturing and ripening

procedures of cheesemaking present a complex set of conditions that must be tightly controlled

during production.

Raw Milk Cheese

Pasteurization is a heat treatment process designed to reduce the harmful microorganisms

in milk for safety and quality reasons (Gunasekaran & Ak, 2003). Pasteurization has been

shown to reduce native bacterial populations in milk by > 90% (Grappin & Beuvier, 1997).

20

Pasteurization also destroys lactic-acid bacteria and inactivates native enzymes in milk (Sousa et

al., 2001). The diverse microflora and enzymes of raw milk are active during cheese ripening.

Pasteurization affects the rheological and flavor development of cheese (Olson et al., 2011;

Tunick et al., 2007).

During cheese ripening, proteolysis is accomplished through the action of a variety of

bacteria and enzymes native and non-native to the milk (Sousa et al., 2001). This proteolysis

contributes to the changing rheology of the cheese. The level of proteolytic activity varies by

cheese type and production methods. Proteolysis is largely responsible for the flavor and texture

development in cheese (Gunasekaran & Ak, 2003).

Proteolytic agents in cheese come from several sources; these sources include milk which

contains plasmin, coagulant such as rennet, starter culture, secondary starter, and nonstarter

microorganisms (Fox, Singh, & McSweeney, 1994). Pasteurization affects the activity of many

of these enzymes (Grappin, Rank, & Olson, 1985; Sherwood, 1936).

Raw milk cheese has been shown to have significantly more bacteria than pasteurized

milk cheese (Tunick et al., 2007). Those researchers attributed the differences in the rheological

properties of raw and pasteurized milk cheeses to higher bacterial levels. A similar conclusion

was reached by Van Hekken, Tunick, Tomasula, Molina Corral, and Gardea (2007). Olson et al.

(2011) also observed differences in the microorganism levels of raw and pasteurized milk

cheeses. It was concluded that pasteurization affected the functional properties of the cheese

studied. The proteolysis of β-casein was also observed to be greater in raw milk cheese (Olson et

al., 2011). This was hypothesized to be the result of different enzymatic activity in the raw milk

cheese.

21

Pasteurization has been shown to affect the extent of proteolysis in Cheddar cheese

during aging (Lau, Barbano, & Rasmussen, 1991). Two explanations were given for the

influence of pasteurization on proteolysis. Firstly, whey proteins denatured during pasteurization

may interact with casein and interfere with the action of proteinases. Secondly, pasteurization

may destroy native proteolytic agents.

Aging of Cheese

Cheese ripening is an essential part of the cheese making process for nearly all cheeses.

Ripening is needed for the development of characteristic rheological and flavor properties of the

various cheese types (Gunasekaran & Ak, 2003). Ripening involves proteolysis, glycolysis, and

lipolysis (Lucey et al., 2003). Proteolysis has been cited as the most important biochemical

reaction in cheese ripening (Fox, Lucey, & Cogan, 1990). Proteolysis is a major factor affecting

cheese rheology (Lawrence, Creamer, & Gilles, 1987). In cheese, proteolysis is the breakdown

of intact caseins and peptides into smaller peptides and free amino acids.

During traditional cheese aging, moisture is lost over the aging period. Today some

cheeses are packaged and aged in plastic wrap. These cheeses don’t typically undergo the same

degree of moisture loss, if any, as observed by Pollard, Sherkat, Seuret, and Halmos (2003).

Moisture has been shown to affect the rheology of cheese (Everard et al., 2006; McMahon,

Paulson, & Oberg, 2005; Rinaldi, Chiavaro, & Massini, 2010).

Proteolysis. Proteolytic agents in cheese come from several sources; these sources

include milk which contains plasmin, coagulant such as rennet, starter culture, secondary starter,

and nonstarter microorganisms (Fox et al., 1994). Pasteurization affects the activity of many of

these enzymes (Grappin et al., 1985).

22

The first proteolytic action during cheesemaking usually occurs by the action of rennet.

Rennet cleaves the outer hydrophilic κ-casein segment of the casein micelle at the Phe105-

Met106 bond (Gunasekaran & Ak, 2003). This proteolysis results in water soluble peptides and

the remaining κ-casein fraction, known as para-κ-casein, inside the micelle. The resulting

destabilization of the micelle leads to the coagulation of the caseins in the early stages of

cheesemaking.

The primary purpose of the starter culture is to lower pH through the production of lactic

acid (Sousa et al., 2001). Opportunistic nonstarter microorganisms increase in population as the

starter culture eventually declines. Secondary starters are used in the production of specific

cheese styles and are not used to produce lactic acid.

One model of relating proteolysis to rheology assumes that each casein is part of the

cheese structure. Once a casein is cleaved, it can no longer contribute to the structure of the

cheese (Lucey et al., 2003). Using this model, the degree of proteolysis may be used as a

predictor of rheology.

Polyacrylamide gel eletrophorsis (PAGE) is commonly used to determine the level of

proteolysis in cheese (Basch, Farrell, Walsh, Konstance, & Kumosinski, 1989; Hesari, Ehsani,

Mosavi, & McSweeney, 2007; Hynes, Delacroix-Buchet, Meinardi, & Zalazar, 1999). The two

typical types are SDS-PAGE and Urea PAGE. PAGE makes use of differences in the electrical

charge of the proteins as well as differences in their size. Protein separation can be achieved by

applying an electrical force across a gel. Separation will occur according to the charge and size

of the protein. In SDS-PAGE, SDS binds with the proteins, coating them with a uniform charge

to weight ratio. Therefore in SDS-PAGE, proteins only migrate in the gel according to their size.

However, when using SDS-PAGE in the separation of cheese protein, the caseins do not migrate

23

according to size (Basch, Douglas, Procino, Holsinger, & Farrell, 1985). It is likely that such

abnormalities are the result of inconsistent binding of SDS to casein. In this case, isolated

caseins can be used as markers to identify the proteins in cheese since molecular weight markers

cannot be used effectively.

Plasmin is the primary native proteolytic agent in milk (Lucey et al., 2003). Plasmin

works on all the caseins and primarily on αs2- and β-casein. Plasmin is not inactivated by

pasteurization. Milk coagulants are added to milk during the cheese making process. These

enzymes retain varing levels of activity (0-15%) (Sousa et al., 2001). Residual chymosin acts on

αs1-casein early during ripening by cleaving it at the Phe23-Phe24 bond. The resulting fragments

are αs1-casein (f1-23) and αs1-casein (f24-199). The αs1-casein (f1-23) quickly hydrolyzes while

αs1-casein (f24-199) degrades steadily and may be used as proteolytic indicator (Tunick et al.,

2007). However, αs1-casein (f24-199) comigrates with β-casein in the SDS-PAGE system

(Basch et al., 1989). It is important to realize this when anaylzing β-casein degradation by using

SDS-PAGE.

The αs1-caseins break down more rapidly than the β-caseins. In Cheddar cheese, Basch et

al. (1989) found the half life of αs1- and β-casein to be 2 and 37 weeks, respectively. In a study

on raw milk and pasteurized milk cheese, pasteurized milk cheese β-casein remained intact while

raw milk cheese β-casein degraded over the 16 week ripening period (Tunick et al., 2007). This

increase in proteolysis may be attributed to plasmin and enzymes not inactivated by

pasteurization.

During cheese making, rennet cleaves κ-casein into para-κ-casein and other peptides

(Basch et al., 1989). The para-κ-casein remains generally unchanged throughout the cheese

ripening period (Nath & Ledford, 1973).

24

Proteolysis has been used as an indicator of cheese age (Fallico et al., 2004). Ripening

time has been shown to be the most significant factor in the proteolysis of cheese protein

(Costabel, Pauletti, & Hynes, 2007). Besides age, proteolysis is also affected by pH, moisture

content, and salt content (Gunasekaran & Ak, 2003). The pH affects the amount of enzyme that

is removed with the whey during straining of the cheese. Other studies have concluded pH has

little affect on proteolysis (Hynes et al., 1999).

The coagulating enzymes are generally thought to have less impact on the proteolysis of

cooked cheese due to enzyme denaturation (Sousa et al., 2001). Hynes, Candioti, Zalazar, and

McSweeney (2004) investigated rennet activity in hard cooked cheese. Rennet activity was

detectable at the tested cooking temperaures. The αs1-casein (f24-199), a peptide associated with

rennet activity was also able to be measured. This suggests renaturation or incomplete

denaturation of the rennet enzymes.

In SDS-PAGE, several proteins that migrate slower than para-κ-casein and faster than β-

casein have been observed that are not easily identified (Basch et al., 1989). These bands are

probably peptides from the proteolysis of the αs1- and αs2-caseins.

Pasteurization has been shown to affect protyeolysis (Tunick et al., 2007). The αs1 was

found to degrade faster in raw milk cheese than in pasteurized milk cheese. This increased level

of proteolysis may be due to active enzymes not subjected to pasteurization. Raw milk cheese

also has higher and more diverse populations of microflora than pasteurized milk cheese (Van

Hekken et al., 2007). These bacteria affect the degree of proteolysis in cheese.

The αs1-casein has been correlated with hardness as determined by Texture Profile

Anaylsis (TPA). Hardness may decrease over time due to a breakdown of the cheese structure.

25

Fracture toughness has been correlated with the degradation of the α-caseins (Charalambides,

Williams, & Chakrabarti, 1995).

Rheology

Rheology is the study of deformation and flow of matter (Reiner, 1964). Stated another

way, rheology is a measure of the material properties that control deformation and flow behavior

when subject to external forces (Gunasekaran & Ak, 2003). Deformation occurs when an

external force is applied to a purely elastic material. Flow occurs when an external force is

applied to a purely viscous material, such as a liquid. Restated, solids deform and liquids flow.

Most real materials exhibit properties of elastic and viscous materials and both deform and flow.

Such materials are called viscoelastic. Rheology is primarily concerned with these materials.

Cheese, like most foods, exhibits viscoelastic behavior.

Rheology relates stress, strain, and time (Gunasekaran & Ak, 2003). Stress is related to

the force applied to a sample. Strain is related to the reaction and change in shape of that force.

Both stress and strain account for the size of the sample, allowing for the calculation of material

properties independent of sample size. As a viscoelastic material, the timescale of a rheological

experiment is important (Lucey et al., 2003). The timescale of an experiment is the duration that

a stress is applied to the sample. A longer timescale provides more time for the material to flow

viscously and results in less elastic deformation.

The rheology of cheese has been shown to be a critical measure of the acceptability of

cheese (Yates & Drake, 2007). The rheological properties of cheese can be quantified using

fundamental instrumental methods that are independent of the machine used. This allows for the

comparison of results from different researchers. Fundamental methods in rheology include the

26

compression, tension, bending, torsion, and relaxation tests. Some fundamental methods are

describe is more detail in the following sections.

Uniaxial Compression. Uniaxial compression is one the most popular methods for

testing the rheology of cheese (Gunasekaran & Ak, 2003). Uniaxial compression is typically

conducted on a type of instrument known as a Universal Testing Machine (UTM). A UTM

consists of upper and lower loading points and a load cell. The loading points can be fitted with

flat plates to conduct the compression test (Figure 1). A number of other tests can be performed

on a UTM using various attachments including tension, bending, and shear. The UTM allows

for precise and accurate control and measurement of deformation and force. The ease of

execution is a possible reason for the popularity of the compression test (Luyten, van Vliet, &

Walstra, 1992).

Figure 1. A Universal Testing Machine (UTM).

In a uniaxial compression test, a sample is placed between the two parallel plates and is

compressed by the plates while closing at constant speed. A cylindrical sample is the most

commonly used specimen shape (Gunasekaran & Ak, 2003). Cubic shapes can be used but are

27

not preferred due to non-uniform deformation during compression, resulting in stress

concentrations in the sharp edges of the cube. Cylindrical samples are typically cut using a cork

borer and wire cutter. A true cylindrical shape is essential for testing accuracy. Some

recommendations for cutting cheese are to perform cutting at refrigeration temperature, to

perform cutting as slowly as possible, and to lubricate the cutting edges and borer surfaces with

oil (Gunasekaran & Ak, 2003).

The dimensions of the sample require some consideration. Samples should be large

compared to the fat globules and curd particles in the cheese (Walstra & van Vliet, 1982).

Testing inaccuracy increases with decreasing sample size. Cylindrical samples with a diameter

around 20 mm are commonly used. If the length, L, is much greater than the diameter, D, the

sample may buckle during compression. In the opposite situation, frictional effects at the

sample-plate surface may become unacceptably large. Luyten (1988) found a ratio of 1.3 to

2.0 to be acceptable. A ratio less than 1.0 and greater than 2.5 is not acceptable.

During compression, a barreling or bulging of the sides of the cylindrical specimen may

occur. This indicates that friction is affecting the sample. The surfaces between the sample and

plates can sometimes be lightly lubricated, typically with oil, to minimize the effect of friction

(Charalambides et al., 1995). Minimization of non-uniform deformation is important when

making the constant volume assumption. Assuming constant volume of the specimen during

compression is useful when calculating true stress and will be discussed further below.

The rheology of cheese is greatly affected by temperature and therefore must be

controlled during testing (Lucey et al., 2003). Temperature affects the bonding behavior

between the caseins. More significantly, temperature affects the properties of the fat within the

casein matrix, thereby affecting the properties of whole cheese (Luyten, 1988). It is also

28

important to maintain the sample and the UTM plates at the same temperature to minimize

temperature gradients during testing (Gunasekaran & Ak, 2003).

During testing, the UTM records force-time data. These data are converted into stress

and strain values, which are in turn used to calculate rheological properties such as Young’s

modulus and the yield point. The yield point is often taken as the point of maximum stress on

the stress-strain curve, seen as a peak where fracture quickly reduces stress (Gunasekaran & Ak,

2003). It is important to note that maximum stress may not correspond to the point of fracture

initiation (Luyten, 1988). However, during testing the fracture point is difficult to observe

visually. A further complication is that the yielding may first occur within the compression

sample and may not be observable. Therefore, for reasons for convenience and consistency, the

yield point is often taken as the point of maximum stress on the stress-strain curve. It is possible

that the stress-strain curve may not show a definite peak. In this case, the minimum slope of the

stress-strain curve has been used (Guinee & O'Kennedy, 2009; Lee, Imoto, & Rha, 1978). A

typical stress-strain curve for cheese is shown in Figure 2.

Young’s modulus is the slope of the stress-strain curve during the initial period of linear

elastic deformation. Calculation of Young’s modulus for cheese is difficult or impossible for

two reasons. As a viscoelastic material, cheese does not exhibit straight, linear elastic

deformation; the initial part of the stress-strain graph is instead curved (Figure 2). Secondly, due

to the difficulty in producing perfectly cylindrical samples with parallel faces, the UTM may

register small stress until the full sample comes under full compression. For these reasons the

strain secant modulus is sometimes used (Charalambides et al., 1995). The strain secant

modulus is the slope of a line from a stress at a specific strain through the origin, for example,

the 5% strain secant modulus.

29

Figure 2. A typical stress-strain curve.

Strain is a measure of the degree of deformation. In the compression test, a cylindrical

sample is compressed to a shorter height. This change in height is quantified by the strain value.

The engineering or Cauchy strain, ε, is calculated

(1)

where h is the current sample height, and H is the original height. Strain can have a

positive or negative value depending on the direction of the strain (compression or tension).

When the h is less than H, as it is during compression, strain will be negative.

The engineering strain is useful only when measuring small strains, typically up to 1%

(Gunasekaran & Ak, 2003). This is because the engineering strain does not account for the

changing height of the sample during compression. The true strain uses the current height rather

than the initial height of the sample to calculate strain. Using some mathematical manipulations,

the true strain can be expressed

(2)

Stre

ss

Strain

30

This is known as the true or Hencky strain. Compressive true strain will have a negative

value as in engineering strain. A simple validation of true strain at large deformation can be

made by comparing equations (1) and (2). If the engineering strain has a value of 1, the current

height must be 0, which is impossible. On the other hand, a true strain value of 1 would indicate

a current height , which is meaningful.

When measuring deformation up to about 25%, both measures of strain are

approximately equal (Peleg, 1984). Foods are often tested at deformations greater than 25%.

The true strain is the most commonly used measure of deformation in food testing (Gunasekaran

& Ak, 2003).

The stress, σ, is defined as the force per unit area and can be written

(3)

where F is the force in the direction of testing, and A is the area the force is applied to.

Engineering stress uses the initial area in calculating stress. A more accurate method of

representing stress is to use the true stress. Rewriting equation (3) in terms of the current area

and assuming a cylindrical sample yields

(4)

where P represents force, and R the original radius. This calculation of true stress

assumes a constant volume deformation. This is a reasonable assumption for cheese (Luyten,

1988). The true stress is commonly used in compression testing (Charalambides et al., 1995).

Choosing a set of uniaxial compression testing conditions is not straightforward

(Charalambides et al., 1995). At a given displacement rate, the applied strain rate will be higher

for a shorter specimen. As a viscoelastic material, cheese will appear stiffer at higher strain

31

rates. Additionally, simply decreasing the displacement rate for shorter specimens will affect the

friction at the contact surfaces.

A standard testing condition does not exist for cheese. It would seem almost all possible

combinations have been used to suit the particular needs of researchers. However, a certain

range of conditions seem to be typical (Gunasekaran & Ak, 2003). Typical crosshead speeds are

between 50 and 100 mm/min. Sample diameter and length are often about 20 mm with a L/D

ratio close to 1. Samples seem to be tested at a variety of temperatures between refrigeration and

room temperature. A standardization of testing conditions would greatly enhance the ability to

compare results from different laboratories.

The effect of strain rate has been studied (Ak & Gunasekaran, 1992; Luyten et al.,

1991a). At lower strain rates cheese dissipates more energy as viscous flow, lowering the stress

needed to achieve a given deformation. However, at higher strain rates there are more frictional

effects within the protein matrix, increasing the energy lost and lowering the stress needed to

achieve a given deformation. The results of the studies indicate that increased strain rates

increase the stress necessary for deformation. However, due to these two counteracting

mechanisms, each cheese needs to be tested individually.

The compression test has been used to calculate Young’s modulus, yield strain, and yield

stress of Gouda cheese (Bertola et al., 2000; Luyten, 1988). A comparison of the modulus and

yield point of Gouda cheese calculated from different tests has been done using compression,

tension and bending tests (Luyten etal., 1992; Luyten et al., 1991a). The different tests used

produced similar results. It was concluded that the compression test can be used to determine the

rheological properties.

32

Charalambides et al. (1995) used the compression test to quantify the material properties

of Cheddar cheese aged to 6 months. The modulus and yield stress followed a pattern of

significant increase during aging. It was noted by that these two values showed an

approximately inverse relationship with the β-casein. An increase in modulus was also observed

by Rinaldi et al. (2010) with increasing age. This increase was attributed to decreasing moisture

content.

Often the goal of rheological studies is to eliminate the need for a trained sensory panel

by making correlations between sensory and rheological properties (Bourne, 1982). Texture and

flavor have been shown to be major factors in consumer acceptance of Gouda cheese (Yates &

Drake, 2007). Correlations have been made between the yield stress and yield strain and the

sensory perception of Cheddar cheese (Everard et al., 2006).

Texture Profile Analysis. Texture is a complex property. The only way to truly

quantify texture is through a trained human sensory panel. However, due to the cost and time of

setting up a trained panel, there is a clear advantage to quantifying texture using mechanical

methods. Procedures have been developed to maximize the correlations between instrumental

analysis and the human perception.

There are many different instruments that can be used to quantify texture (Friedman et

al., 1963). However many are only able to quantify a single texture parameter and not useful in

describing the complex texture profile of food. Texture Profile Analysis (TPA) was first

developed in early 1960s. TPA compresses a food sample in two consecutive compressions.

An early predecessor of TPA was the denture tenderometer designed at the Food

Technology Laboratory at Massachusetts Institute of Technology (Proctor, Davison, & Brody,

1955). This was an imitative test which attempted to imitate mastication. To mimic the action

33

of the human mouth, the tenderometer had actuating jaws with dentures and cheeks to hold the

sample. An important modification was made by Friedman et al. (1963). This group replaced

the jaws with a plunger and plate. This device was called the texturometer.

TPA was further developed by Bourne (1968) who adapted the existing procedure to an

Instron universal testing machine. This replaced hinged compression with the uniaxial

compression of the Instron. The result was that TPA essentially became a compression test with

two consecutive compressions. This “two-bite” procedure is used to mimic mastication.

Differences in the response of food to each compression are used to calculate the TPA

parameters.

A typical TPA testing curve for cheese is shown in Figure 3. Several texture properties

can be calculated from the TPA curve such as hardness, cohesiveness, adhesiveness, springiness,

gumminess, and chewiness. A summary of these terms and their method of calculation is given

in Table 1. Many of these terms were defined by Friedman et al. (1963) for use with the

texturometer. When calculating the area under the force-time curve for the first and second

bites, Bourne (1968) modified the calculation by only including work during compression of the

samples, excluding the work done during withdrawal of the plate. It should be noted that time is

converted to distance for the calculation of the TPA parameters. This is easily done when the

crosshead rate is known.

34

Figure 3. A typical Texture Profile Analysis force-time curve for cheese (time is converted to

distance for calculation of TPA parameters). A1 and A2 represent the positive area under the first

and second compression curves, respectively. A3 represents the negative area under the curve

during the first withdrawal. d1 and d2 represent the crosshead travel distance during the first and

second compression, respectively. P1 represents the force at maximum compression during the

first compression.

35

Table 1

Texture Profile Analysis Parameters and Definitions

TPA Parameter Units Calculation Method

Definition

Hardness

(N)

Force at P1

Peak force during the first compression

Cohesiveness (--) A2/A1 The ratio of the positive work of the first compression to the positive work during the second compression

Adhesiveness

(J)

A3

The negative force area for the first withdrawal, representing the work to pull the plunger away from the sample

Springiness

(m)

d2

The height that the sample recovers by the second compression, originally called elasticity

Gumminess

(N)

Hardness Cohesiveness

Energy needed to disintegrate the sample until it is ready for swallowing

Chewiness

(J)

Hardness Cohesiveness Springiness

Energy needed to chew the sample until it is ready for swallowing

(Bourne, 1968; Friedman et al., 1963; Szczesniak, 1963)

TPA has allowed for the breakdown of texture into components by mechanical methods

(Bourne, 1978). TPA is the primary method of quantifying texture and has been used with many

different foods. Several researchers have used TPA to analyze texture development in cheese.

A wide range of test conditions have been used with TPA. Without a standard testing

protocol, comparison of results can be difficult. Bourne and Comstock (1981) studied the effect

of compression ratio on the TPA parameters. Compression ratio is the compressed height of the

sample over the original height of the sample. Using a variety of foods, TPA was conducted

using compression ratios between 50% and 93%. In general, the TPA parameters increased with

an increase in compression ratio. Large increases occurred at a compression ratio of 80% and

36

greater. However the changes in TPA parameters varied so widely that an overall statement

regarding the effect of compression ratio could not be made. It was concluded that each food

needs to be tested individually. Little effect of age on the TPA parameters springiness,

cohesiveness, and resilience was found at a 30% compression ratio (Pollard et al., 2003). The

typical compression ratio is about 70% (Gunasekaran & Ak, 2003).

The effect of compression ratio on cheese has been studied (Imoto, Lee, & Rha, 1979;

Lee et al., 1978). The degree of compression was shown to affect the measured texture

properties. However, it was determined that compression ratio had less affect on the strength of

correlations to sensory evaluation. A range of responses were observed for the cheese used,

demonstrating the need for individual analysis of cheese.

A wide range of compression rates have been used from 30 to 1000 mm/min (Everard et

al., 2006; Pollard et al., 2003; Tunick et al., 2007). A rate of approximately 100 mm/min is

common. The effect of compression rate was studied by Lee et al. (1978). Increasing the

compression rate increased compression force. This is as expected for a viscoelastic material.

Lee et al. (1978) concluded that hardness, springiness, and adhesiveness are the important

textural properties in describing cheese.

Often the goal of rheological studies is to eliminate the need for a trained sensory panel

by making correlations between sensory and rheological properties (Bourne, 1982). The TPA

parameters have been correlated well to the sensory evaluations of cheese (Everard et al., 2006;

Imoto et al., 1979; Lee et al., 1978).

In an analysis of Pecorino of Appennino cheese, cohesiveness values decreased with

aging (Rinaldi et al., 2010). Adhesiveness and springiness did not change during aging.

Hardness and chewiness increased over the aging period. Pecorino of Appennino cheese is a

37

semi-hard cheese made with raw milk. It was concluded that the TPA parameters are useful in

predicting age.

Semi-hard, raw milk Chihuahua cheese, a style similar to Cheddar cheese, has also been

studied used TPA (Tunick et al., 2007). Hardness, chewiness, and cohesiveness decreased over

the aging period. Springiness did not demonstrate a strong trend. Some similarities can be seen

in the TPA of semi-hard raw milk cheeses. However, each cheese must be tested individually to

ensure accurate measurements are taken. Pasteurization was also found to affect the TPA

parameters of Chihuahua cheese (Van Hekken et al., 2007).

Everard et al. (2006) used TPA to study the effect of maturation on Cheddar cheese.

Hardness, springiness, and chewiness decreased with maturation. This was determined to be the

result of proteolysis. Everard et al. (2006) also determined that hardness, springiness, and

chewiness increased with decreasing moisture content. Increased moisture allows for more

flexibility of the protein matrix. In the aging of Gouda cheese, proteolytic activity and moisture

loss occur simultaneously and exert an opposite influence on rheology (Luyten, 1988).

Bending Test. The fracture properties of cheese are important in evaluating cheese

(Luyten, van Vliet, & Walstra, 1991b). Fracture of cheese is desirable during chewing. The

fracture properties affect the overall acceptability of cheese. Therefore, quantifying fracture

characteristics is important for cheese quality.

Fracture mechanics considers the presence of defects in determining theoretical material

strength (Anderson, 1991). Fracture mechanics considers both the applied stress and defect size

when calculating fracture toughness. Fracture toughness is independent of testing conditions.

Fracture toughness is a measure of the energy per unit area necessary to form a new crack

surface (Williams, 1984).

38

The energy criterion states that propagation results when the energy available for crack

formation is greater than the resistance energy of the material. The energy release rate, G, is

defined as the energy per unit of crack area of a linear elastic material (Anderson, 1991)

(5)

where σ is the applied stress, a is half the crack length, and E is Young’s modulus. The

critical energy release rate, Gc, is the energy release rate necessary for new crack formation and

is a measure of fracture toughness. The critical energy release rate relates stress and crack size.

Near a defect, the local stress is higher and therefore the maximum stress will occur at an

existing crack (Luyten et al., 1991b). The stress intensity of a material relates the stress at the

crack tip. The stress intensity factor, K, is used to determine conditions around the crack tip in a

linear elastic material and is given as

√ (6)

where Y is a dimensionless testing condition constant (Gunasekaran & Ak, 2003). The

stress intensity that results in failure is the critical stress intensity factor, Kc. Therefore, Kc is also

a measure of fracture toughness.

In specific testing configurations, it is possible to define K of a crack. Typical testing

configurations include Single-Edge Notched Tension (SENT), Single-Edge Notched Bend

(SENB), and the Double-Edge Notched Tension (DENT). An example of the SENB

configuration is shown in Figure 4.

39

Figure 4. The Single-Edge Notched Bend (SENB) sample geometry. L denotes the length, S the

span, W the width, B the breadth, P the load, and a the notch length.

Using the SENB testing configuration, Kc can be defined

√ ⁄ (7)

where P is the applied force, B is the breadth, w is the width, and ⁄ is a calibration

factor based on the testing geometry (Williams, 1984).

The SENB test is a convenient method to determine Gc when testing cheese (Kamyab,

Chakrabarti, & Williams, 1998). Using this testing configuration, Gc can be calculated by

integration of the load-displacement curve, that is by using the energy from test initiation to

fracture. In the SENB testing configuration Gc is defined

⁄ (8)

where U is the area under the load-displacement curve until fracture (ASTM, 2007). The

recommended geometry dimensions are: , and ⁄ (ASTM,

2007). The deep notches proposed are used to create brittle fracture.

Equation (8) assumes linear elastic behavior. Strictly speaking, this condition is not met.

Cheese is a viscoelastic material, and therefore the fracture properties cannot be theoretically

40

calculated using the above equations. However the above methods can be assumed to be

reasonably accurate for the testing of cheese (Charalambides et al., 1995).

The effect of strain rate on fracture stress was studied by Luyten et al. (1991b). As

expected for a viscoelastic material, shorter stain times allow for less viscous dissipation of

energy, and therefore result in higher fracture strain. Higher strain rates also increased fracture

stress. Strain rate had a greater effect on young cheese, while mature cheese was relatively

unaffected. This is likely due to the higher degree of viscous flow in young cheese.

Using tension tests, young Gouda cheese was found to be more notch sensitive than more

mature cheese (Luyten et al., 1991b). This means that the length of the notch, used to create a

stress concentration, had a more pronounced effect on young Gouda cheese. Young cheese did

not immediately fail once fractured; whereas mature cheese, once fractured, experienced faster

crack propagation. This property of cheese is perceived as brittleness in sensory evaluations.

Luyten et al. (1991b) estimated the fracture toughness in Gouda cheese using the SENT

test. Fracture toughness was found to decrease over the 10 month aging period. Fracture

toughness was also estimated by cutting cheese with wire. These results agreed well with other

tests of fracture toughness.

The SENB test has been used to study the affect of age on Gc of Cheddar cheese

(Charalambides et al., 1995). The Gc of mild Cheddar cheese was found to generally decrease

with increasing age. The Gc was visually compared to the α-casein in the cheese and a

relationship was suggested. The SENB, SENT, and wire cutting test have also been used to

determine Gc (Kamyab et al., 1998).

41

Chapter III: Methodology

The objective of this study was to describe the effect of aging time on the rheological

properties and the extent of proteolysis in raw milk Gouda cheese of varying aging times (0.5,

2.3, 4.3, and 6.3 months). This chapter will present the methodology used to assess rheology,

proteolysis, and moisture content of raw milk Gouda cheese.

Materials

Raw milk Gouda cheese samples were obtained from Holland’s Family Cheese, LLC

(Thorp, WI). Four ages of Gouda cheese were used, aged approximately 0.5, 2.3, 4.3, and 6.3

months. Cheese was taken directly from the aging cellar and wrapped in plastic wrap to

minimize moisture loss during transport to UW-Stout laboratory facilities. At UW-Stout the

Gouda cheese was stored at refrigeration temperatures. During testing, care was taken to keep

the Gouda cheese in plastic wrap as much as possible.

Moisture Content

Moisture content was determined based on the method of the International Organization

for Standardization (ISO 5534:2004(E)). Weigh boats were pre-dried for 12 hr at 102°C.

Approximately 3 g Gouda cheese samples were dried in a draft oven. Drying occurred at 102°C.

The mass was recorded in grams to four decimal places. The mass of the samples were checked

hourly until a reduction of 0.002 g per hour was recorded.

Proteolysis

Proteolysis was observed by SDS-PAGE. The protein in the Gouda cheese samples was

separated by adding 20 mL of 0.5 M sodium citrate to 10 g of cheese sample and ground to

homogeneity using a mortar and pestle. 90 mL of water was added to the mixture. After a short

rest of several minutes, visibly separated fat was removed. The mixture was adjusted to pH 4.6

42

by dropwise addition of 1.0 M HCl. It was then centrifuged at approximately 1600 g for 10 min.

The insoluble protein pellet was prepared for SDS-PAGE.

Preparation of the protein samples for SDS-PAGE was based on the method of Pardo and

Natalucci (2002). A milk protein sample was prepared for use as a reference for the

identification of the cheese proteins (Basch et al. 1989). Milk protein isolate was used as a

reference protein (Milk Protein Isolate – 9060BM I8871, Kerry Ingredients, Tralee, Ireland).

0.25 g of protein was combined with 25 mL Tris-HCl buffer (pH 8.0) and heated in a boiling

bath for 20 min. The solution was immediately gravity filtered through Whatman 40 filter paper.

The solution was diluted with an equal volume of double-concentrated Laemmli sample buffer

(125 mM Tris-HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 0.4% bromophenol blue, 20%

glycerol) (Sigma-Aldrich, St. Louis, MO). The solution was heated in a boiling bath for 5 min.

The solution was stored at -17°C until use in SDS-PAGE.

SDS-PAGE was performed using precast 4 to 20% gradient polyacrylamide gels (Thermo

Scientific, Rockford, IL). The running buffer, Tris-HEPES-SDS (100 mM Tris, 100 mM

HEPES, 3 mM SDS, 8.0 pH) (Thermo Scientific, Rockford, IL) was kept at refrigeration

temperature until use. The four cheese protein samples and the milk protein isolate sample were

injected at 4 μl and 8 μl volumes. Samples were injected using a 10 μl Hamilton syringe. SDS-

PAGE was run at 30 V for 20 min, 80 V for the next 30 min, and 90 V for the remainder of the

run.

The gel was placed in a staining and fixing solution (25% methanol, 10% acetic acid,

0.1% Coomassie Brilliant Blue R-250) for 1 hr. The gel was then destained with washes of

destain solution (25% methanol, 10% acetic acid).

43

Rheology

Rheological tests were conducted on an Instron universal testing machine (Model 3342,

Instron, Norwood, MA) with a 500 N load cell. All cheese samples were allowed to equilibrate

at refrigeration temperature. The samples were cut immediately after being removed from

refrigeration. After cutting, the samples were sealed in plastic wrap, and equilibrated at room

temperature (21 - 23°C). Rheological values were the mean of six trials.

Uniaxial Compression. Samples had dimensions of length, 31.8 mm, and diameter, 25.4

mm. Samples therefore had a length to diameter aspect ratio of 1.25. Samples were cut using a

25.4 mm lubricated cork borer and wire. Samples were compressed between 45 mm platens.

The platens were lightly coated with oil to minimize barreling. The test was initiated at a preload

of 0.03 N. The crosshead speed was 100 mm min-1.

The 5% strain secant modulus was calculated (Charalambides et al., 1995). Yield stress

and yield strain were calculated at the yield point. The yield point was determined using a 2%

strain offset. A typical stress-strain curve is shown in Figure 2. True stress, σ, and true strain, ε

were calculated by

(9)

(10)

where P represents the force, h the current height, R the original radius, and H the

original height.

Texture Profile Analysis. Texture Profile Analysis (TPA) was conducted using

cylinders with dimensions of length, 31.8 mm, and diameter, 25.4 mm. Samples therefore had a

length to diameter aspect ratio of 1.25. Samples were compressed between 45 mm platens.

44

Samples were cut using a 25.4 mm lubricated cork borer and wire. Samples were compressed

twice to 70% of the original height with a 3 sec hold between compressions. The test was

initiated at a preload of 0.03 N. The crosshead speed was 100 mm min-1. The TPA parameters

hardness, cohesiveness, adhesiveness, chewiness, gumminess, and springiness were calculated as

described in Table 1.

Bending Test. The bending test used was the single-edge notched bend test (SENB).

The method was based on the work of Charalambides et al. (1995). Samples were cut using a

wire. Samples had a length, L, span, S, width, W, and breadth, B, of 79, 64, 16, and 8 mm

respectively (Figure 4). A knife was pressed into the sample to create a notch length, a, of 2-3

mm. The test was initiated at a preload of 0.01 N. The crosshead speed was 60 mm min-1.

The critical energy release rate, Gc, was calculated

⁄ (11)

where U is the energy under the load-displacement curve from test initiation to crack

initiation and ⁄ is a calibration factor based on the testing geometry (ASTM, 2007). The

appropriate calibration factor from Anderson (1991) is

⁄

√

[ ] (12)

where

(13)

As stated in equation (11), U represents the area under the load-displacement curve up to

crack initiation. Although difficult to measure, crack initiation was observed to occur near the

point of maximum load. Therefore for consistency, crack initiation was taken as the point of

maximum load.

45

Statistical Analysis

Data were analyzed for significant effects using one-way ANOVA. Pairwise

comparisons of means were analyzed using the two-sample t-test. Compression test comparisons

were described as significant when . All other comparisons were described as

significant when .

46

Chapter IV: Results

Moisture Content

An ANOVA showed a significant effect of age on moisture content .

Moisture content decreased steadily from 40.4% to 34.4% during the aging period (Figure 5).

Figure 5. Moisture content of raw milk Gouda cheese aged to 6.3 months. Standard errors are

marked with vertical bars. Means which significantly differ between ages are marked with

different letters ( ).

a

b

c

d

32%

33%

34%

35%

36%

37%

38%

39%

40%

41%

42%

0 1 2 3 4 5 6 7 8

Mo

istu

re C

on

ten

t

Age (months)

47

Proteolysis

The results of the SDS-PAGE are shown in Figure 6. Breakdown of the α-caseins was

nearly complete by 2.3 months. The β-casein appeared to breakdown steadily until 4.3 months.

A constant amount of β-casein remained from 4.3 to 6.3 months. The products of β-casein

breakdown, the γ-caseins, increased with age. The para-κ-casein did not appear to be affected by

cheese age. Several bands migrated slower than para-κ-casein and faster that γ1-casein. These

bands were not easily identified.

Figure 6. SDS-PAGE of protein extracted from raw milk Gouda cheese aged to 6.3 months.

Lane 1, milk protein isolate; Lanes 2-5, cheese protein aged to 0.5, 2.3, 4.3, 6.3 months; Lanes 6-

10 are in the same order as lanes 1-5 with twice the protein volume. Lf = lactoferrin, BA =

bovine serum albumin, IgG = immunoglobulin heavy chain, Lg = lactoglobulin, La =

lactalbumin, CN = casein.

1 2 3 4 5 6 7 8 9 10

α-La

β-Lg

Lf BA IgG

para-κ-CN γ2, γ2-CN

γ1-CN

β-CN

κ-CN

αs1-CN

αs2-CN

48

Uniaxial Compression

The uniaxial compression test was used to determine the 5% strain secant modulus, the

yield stress, and the yield strain (Figures 7-9). An ANOVA showed age had a significant effect

on the measured properties . The 5% strain secant modulus increased over the aging

period. A significant increase was only observed between 2.3 and 4.3 month Gouda cheese

. Values of the 5% strain secant modulus ranged from 132 to 315 kPa. The yield

stress increased significantly between 2.3 and 4.3 months , from 13 to 23 kPa. The

yield strain initially decreased and then stabilized. The yield strain had a maximum value of 0.13

at 0.5 months and a minimum value of 0.10 at 4.3 months. Stress-strain curves for Gouda cheese

are shown in Figure 10.

Figure 7. 5% strain secant modulus of raw milk Gouda cheese aged to 6.3 months. Standard

errors are marked with vertical bars. Means which significantly differ between ages are marked

with different letters ( ).

a a

b b

0

50

100

150

200

250

300

350

400

0 2 4 6 8

5%

Str

ain

Se

can

t M

od

ulu

s (k

Pa)

Age (months)

49

Figure 8. Yield stress of raw milk Gouda cheese aged to 6.3 months. Standard errors are



Figure 9. Yield strain of raw milk Gouda cheese aged to 6.3 months. Standard errors are



a a

b b

0

5

10

15

20

25

30

35

0 2 4 6 8

Yie

ld S

tre

ss (

kPa)

Age (months)

a

b c

bc

0.06

0.07

0.08

0.09

0.1

0.11

0.12

0.13

0.14

0 2 4 6 8

Yie

ld S

trai

n

Age (months)

50

Figure 10. Stress-strain curves for raw milk Gouda cheese aged to (●) 0.5 mo, (▲) 2.3 mo, (■) 4.3

mo, (♦) 6.3 mo.

0

10

20

30

40

50

60

0.0 0.2 0.4 0.6 0.8 1.0

Stre

ss (

kPa)

Strain

6.3

4.3

2.3

0.5

51

Texture Profile Analysis

The Texture Profile Analysis (TPA) parameters hardness, springiness, cohesiveness,

adhesiveness, gumminess, and chewiness were calculated (Figures 11, 12). An ANOVA showed

age had a significant effect on the measured parameters except adhesiveness .

Hardness significantly decreased at 2.3 months, then remained approximately stable .

Springiness followed a general decreasing trend. At 6.3 months springiness appeared to

increase, but the increase was not significant. Cohesiveness followed a pattern similar to

springiness. Adhesiveness did not appear to trend throughout the aging period and did not show

any significant changes. Gumminess and chewiness followed similar patterns. Both parameters

significantly decreased at 2.3 and 4.3 months . The decrease was largest at the

beginning of the aging period. Between 4.3 and 6.3 months, those parameters appeared to

stabilize.

All of the TPA parameters decreased over the aging period, except adhesiveness. Most

of the parameters exhibited a decreasing trend between 0.5 and 4.3 months and a stabilization

between 4.3 and 6.3 months.

52

Figure 11. Mean TPA parameters (I) of raw milk Gouda cheese aged to 6.3 months. Standard



a

b b b

0

10

20

30

40

50

60

70

80

0 2 4 6 8

Har

dn

ess

(N

)

Age (months)

a

b

c c

0

2

4

6

8

10

12

14

0 2 4 6 8

Spri

ngi

ne

ss (

mm

)

Age (months)

a

b

c c

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 2 4 6 8

Co

he

sive

ne

ss (

--)

Age (months)

53

Figure 12. Mean TPA parameters (II) of raw milk Gouda cheese aged to 6.3 months. Standard



a a

a

a

-12

-10

-8

-6

-4

-2

0

0 2 4 6 8

Ad

he

sive

ne

ss (

mJ)

Age (months)

a

b

c c

0

5

10

15

20

25

30

35

0 2 4 6 8

Gu

mm

ine

ss (

N)

Age (months)

a

b

c c

0

50

100

150

200

250

300

350

400

0 2 4 6 8

Ch

ew

ine

ss (

mJ)

Age (months)

54

Bending Test

An ANOVA showed a significant effect of age on fracture toughness, Gc

(Figure 13). Young cheese aged to 0.5 months had a Gc of 7.4 J m-2. Gc significantly decreased

to approximately 4.3 J m-2 at 2.3 months and remained approximately constant for

the remainder of the aging period.

Figure 13. Mean fracture toughness of raw milk Gouda cheese aged to 6.3 months. Standard



a

b b b

0

1

2

3

4

5

6

7

8

9

0 1 2 3 4 5 6 7 8

Gc

(J/m

2)

Age (months)

55

Chapter V: Discussion

Moisture Content

Moisture content significantly decreased during the aging period . Moisture

decreased from 40.4% to 34.4% for 0.5 and 6.3 month old cheese, respectively (Figure 5).

In this study, moisture content was not controlled. During aging moisture content

decreased concomitantly with an increase in proteolysis. Therefore it is difficult to attribute

changing rheological properties to either moisture or proteolysis. However, in other studies

moisture has been shown to affect the rheology of cheese (Everard et al., 2006; Luyten, 1988;

McMahon et al., 2005).

Luyten (1988) reported that decreasing moisture content corresponded to an increase in

Young’s modulus. A similar pattern was noticed in this study with the 5% strain secant modulus

increasing as the moisture content decreased. Cheese has been described as a matrix of protein

and water with fat acting as a filler (Luyten, 1988). The increase in the modulus may then

possibly be due to the relative increase in protein, which provides structure. The increase may

also be related to less freedom of movement for the protein particles as moisture decreases. An

increase in modulus was also observed by Rinaldi et al. (2010). The increase in modulus was

attributed to decreasing moisture content.

Proteolysis

The protein profile of cheese in this study appeared to correlate well with the findings of

Basch et al. (1989). In the current study, the αs1- and αs2-caseins were almost completely

degraded by 2.3 months of age. The effect of raw and pasteurized milk on the proteolysis and

rheology of cheese has been previously studied (Tunick et al., 2007). Tunick et al. (2007) found

the αs1-casein to degrade faster in raw milk cheese than in pasteurized milk cheese. Greater

56

proteolysis of the αs1-casein may be due to the proteases in milk (plasmin) and other enzymes not

inactivated during pasteurization. Raw milk cheese has also been shown to have more diverse

and greater bacterial population than pasteurized milk cheese (Van Hekken et al., 2007). These

bacteria may be responsible for differences in the level of proteoysis in raw and pasteurized milk

cheese.

It has been suggested that the degradation of the α-caseins may be related to fracture

toughness (Charalambides et al., 1995). The results of the current study show an early reduction

in the α-caseins and a similar pattern was observed in the fracture toughness. The αs1-caseins

have been well correlated with the Texture Profile Anaylsis (TPA) value of hardness (Tunick et

al., 2007). The TPA hardness value in this study appeared to agree well with those results. In

this study, hardness decreased with the apparent proteolysis of αs1-casein and then remained

constant. The trend of hardness is very similar to the trend of fracture toughness. There may be

a possible relationship between these two measures of rheology.

The β-casein degraded slower than the α-casein. This is in agreement with other studies

(Basch et al., 1989). The products of β-casein proteolysis are the γ-caseins. As β-casein

decreased, the γ-caseins can be seen to increase (Figure 6). These bands have previously been

observed by Basch et al. (1989). Tunick et al. (2007) found β-casein to remain relatively

unchanged in pasteurized milk cheese over a 16 week period, however, the β-casein was found to

breakdown in raw milk cheese. The results of the current study indicated that the β-casein

underwent proteolysis in raw milk cheese over a 6 month period. These results are in agreement

with Tunick et al. (2007). This increase in proteolytic activity in raw milk cheese may be

attributed to enzymes not inactivated by pasteurization (Tunick et al., 2007).

57

Of interest are several bands that migrated slower than para-κ-casein and faster that β-

casein and were not easily identified (Figure 6). These bands were also observed by Basch et al.

(1989) in an analysis of Cheddar cheese. It is probable that the bands in this range are peptides

from the proteolysis of the αs1- and αs2-caseins.

During cheese making, rennet cleaves κ-casein into para-κ-casein and other peptides

(Basch et al., 1989). The para-κ-casein remains generally unchanged throughout the cheese

ripening period (Grappin et al., 1985; Nath & Ledford, 1973). A unchanging pattern was

observed in this study.

Uniaxial Compression

The uniaxial compression test was used to determine the 5% strain secant modulus, the

yield stress, and the yield strain (Figures 7-9). An ANOVA showed age had a significant effect

on the measured properties . A significant increase in the 5% strain secant modulus

was only observed between 2.3 and 4.3 months . Values of the 5% strain secant

modulus ranged from 132 to 315 kPa. The yield stress increased significantly between 2.3 and

4.3 months , from 13 to 23 kPa. The yield strain initially decreased and then

stabilized. The yield strain had a maximum value of 0.13 at 0.5 months and a minimum value of

0.10 at 4.3 months.

The results of the compression test in this study agree well with the results of Luyten

(1988), who used the compression test to calculate the Young’s modulus, yield strain, and yield

stress of Gouda cheese. The yield stress and the modulus increased and the yield strain

decreased with age, as was the case in this study. It was noticed that trends in the material

properties seemed to develop at a slower pace than in this study. This may be due to our use of

58

raw milk, which has been shown to affect maturation rate (Olson et al., 2011; Tunick et al.,

2007).

Charalambides et al. (1995) used the compression test to quantify the material properties

of Cheddar cheese aged to 6 months. The modulus and yield stress significantly increased

during aging, as was observed in this study. The values in the mentioned study were much

higher than the values in this study. This may be due to the type of cheese used (Sharp Cheddar)

and also the testing temperature (4°C opposed to 22°C). Charalambides et al. (1995) noted that

the modulus and yield stress showed an approximately inverse relationship with the quantity of

β-casein. An increase in modulus was also observed by Rinaldi et al. (2010) with increasing age

and was attributed to decreasing moisture content.

Texture Profile Analysis


adhesiveness, gumminess, and chewiness were calculated (Figures 11, 12). All of the TPA

parameters, except adhesiveness, significantly decreased over the aging period .

Most of the parameters exhibited a decreasing trend between 0.5 and 4.3 months and a

stabilization between 4.3 and 6.3 months.

An analysis of Pecorino of Appennino cheese produced many results similar to those in

this study (Rinaldi et al., 2010). Pecorino of Appennino is a semi-hard cheese made with raw

milk. Cohesiveness values were similar in both trend and value. Adhesiveness did not change

during aging. Springiness did not show any trend, contrary to the decreasing trend observed in

this study. Hardness and chewiness increased over the aging period. No mention is made about

the degree of proteolysis or moisture content over the aging period. This information would be a

starting point for further discussion on the rheology of that cheese. Nevertheless, this

59

comparison suggests possible textural similarities within the semi-hard raw milk cheese type.

Rinaldi et al. (2010) concluded textural properties were useful in predicting cheese age.

Semi-hard, raw milk Chihuahua cheese, a style similar to Cheddar cheese, has been

studied used TPA (Tunick et al., 2007). The TPA hardness was similar in trend and value to the

results of this study. Chewiness and cohesiveness decreased over the aging period. Springiness

did not exhibit a strong trend. By comparing the results of semi-hard, raw milk cheese studies, it

would seem there are similarities across some of the TPA parameters. However the differences

demonstrate the need for the individual analysis of each cheese variety for accurate results.

TPA has been used to study the maturation of Cheddar cheese (Everard et al., 2006).

Hardness, springiness, and chewiness decreased with maturation, as was observed in this study.

This was determined to be the result of proteolysis. Everard et al. (2006) also determined that

hardness, springiness, and chewiness increased with decreasing moisture content. Higher

moisture content allows for more flexibility of the protein matrix. Over the aging period,

individual samples were wrapped in plastic, which prevented moisture loss. Therefore, moisture

content does not have much effect on the changing rheology of wrapped cheese. However, in

traditionally aged cheese, cheese may lose moisture during aging, as was the case in this study.

These two mechanisms work against one another in that proteolysis decreases hardness while

moisture loss increases hardness.

Bending Test

An ANOVA showed that age had a significant effect on fracture toughness, Gc

) (Figure 13). Young cheese aged to 0.5 months had a Gc of 7.4 J m-2. Gc decreased

significantly to 4.3 J m-2 for 2.3 month cheese and remained approximately constant for the

remainder of the aging period. These results agree well with Luyten et al. (1991b) who

60

determined the fracture energy of Gouda cheese using tension testing. They found fracture

energy to be approximately 7.5, 6.0, 5.0, and 2.0 J m-2, for 0.5, 1, 2, and 10 month cheese,

respectively. Luyten et al. (1991b) also estimated fracture energy by using the wire cutting test.

These results agreed well with other tests of fracture energy. Kamyab et al. (1998) determined

Gc using the SENB test and found values of about 3 J m-2.

The effect of aging on Cheddar cheese has been studied (Charalambides et al., 1995).

Charalambides et al. (1995) used the SENB test on 37, 89, 155, and 182 day old mild Cheddar

and determined Gc values of 41, 29, 17, and 30 J m-2, respectively. A similar trend was observed

in this study. Charalambides et al. (1995) visually compared the Gc to the α-casein content of the

cheese, which appeared to correlate well. Gc and the α-casein decreased with age suggesting a

relationship between Gc and the α-casein in cheese. In the current study, the α-casein was visible

at 0.5 months and then appeared to be completely degraded by 2.3 months. The Gc significantly

decreased from 0.5 months to 2.3 months and then remained constant. This observation supports

a possible relationship between Gc and the α-casein.

The practical meaning of lower fracture toughness is that a cheese will fracture with less

force. A young cheese that has a greater resistance to fracture must be completed bitten or cut

for fracture to occur compared to an aged cheese that requires less deformation for fracture. This

is perceived as the brittleness of a cheese during mastication. Mature cheese is therefore more

brittle. In this study, the TPA parameter hardness followed a pattern similar to fracture

toughness. A TPA value for brittleness was not able to be calculated; however hardness may be

an equivalent indicator for brittleness in this case.

61

Conclusions

The rheology of raw milk Gouda cheese was successfully characterized up to 6 months of

age. Aging time had a significant effect on the compression properties. The results of the

compression test in this study agree well with previous studies. It was noticed that trends in the

mechanical properties developed at a faster pace than in other studies. This may be due to our

use of raw milk which has been shown to affect maturation rate.


adhesiveness, gumminess, and chewiness were calculated. All of the TPA parameters except

adhesiveness significantly decreased over the aging period. Those affected TPA parameters

exhibited a decreasing trend between 0.5 and 4.3 months and a stabilizing trend between 4.3 and

6.3 months.

Aging time had a significant effect on the fracture toughness, Gc. Initially, Gc decreased,

and then remained approximately constant for the remainder of the aging period.

Proteolysis was observed through SDS-PAGE. The αs1- and αs2-caseins were almost

completely degraded by 2.3 months of aging time. This appeared to be more rapid than in

studies using pasteurized milk.

This study was an original look at the changing rheology and degree of proteolysis in raw

milk Gouda cheese during aging. For artisanal cheese producers, determining the effect of aging

time on cheese rheology is important for quality and process optimization. Quantifying the

rheology of raw milk Gouda cheese will aid in determining maturation and quality.

62

Recommendations

Future studies can be undertaken with the aim of better understanding the rheology of

raw milk Gouda cheese, specifically on the effect of proteolysis and moisture content during

aging and on the relationship between consumer acceptability and rheology.

1. The extent of proteolysis can be quantified through the use of PAGE and densitometry.

This would allow for the statistical comparison of the degree of proteolysis and the

rheology of cheese. This is necessary to study the effect of proteolysis on the rheology of

cheese.

2. The effect of moisture content was not directly addressed in this study. Moisture content

is recognized as a major factor affecting cheese rheology. Moisture content may also

affect maturation events, such as proteolysis. Therefore further study of the effect of

moisture content on the rheology and proteolysis in raw milk Gouda cheese would be

beneficial. This may be accomplished by controlling initial moisture content and

moisture loss during aging.

3. There is a need to study seasonal effects on raw milk Gouda cheese. Seasonal variations

in cattle feed and in weather may affect the microbiology, proteolysis, and rheology of

the final cheese product.

4. Consumer acceptability can be studied through sensory evaluations. Correlations

between consumer acceptably and the instrumental texture analysis of raw milk Gouda

cheese would benefit manufactures by reducing the dependence on sensory evaluations.

These correlations would allow manufactures to use instrumental texture analysis as an

indicator of cheese maturation and quality.

63

5. This study addressed the rheology of raw milk Gouda cheese up to 6 months of age.

Characterization of older cheese is necessary to determine the rheology of that cheese and

if the trends observed in this study continue with age.

64

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