EUROPEAN M.SC. DEGREE IN FOOD SCIENCE, TECHNOLOGY AND

NUTRITION

THESIS

AUTHOR

MARIA GABRIELA ARAUJO MIÑO

TITLE

Influence of standardization, rennet type, curd wash level and cook

temperature on the composition, microbiology, functionality, flavour

and ripening of novel Swiss-type cheeses

June 2012

Dublin Institute of Technology

Teagasc Food Research Centre Moorepark

II

DECLARATION

I hereby certify that the material which is submitted in this thesis towards award of the European

M.Sc. degree in Food Science, Technology and Nutrition is entirely my own work and has not

been submitted for any academic assessment other than part-fulfillment of the award named

above.

Signature of candidate:…………………………………..

Date:………………………

III

ACKNOWLEDGEMENTS

I would like to thank The National Secretary of Higher education, Science, Innovation and

technology in Ecuador (SENESCYT) for providing me the funding which enabled me to carry out

this project.

I would like to thank the Irish Dairy Board and Teagasc for providing me the opportunity to become

part of their team.

This dissertation would not have been possible without the guidance and the help of several

individuals who in one way or another contributed and extended their valuable assistance in the

preparation and completion of this study.

First and foremost, I would like to acknowledge the advice and guidance of my placement

supervisor Dr. Diarmuid Sheehan Research Official of the Food Chemistry and Technology

department in Teagasc Food Research Centre Moorepark (TFRCM).

I would also like to express my thanks and gratitude to my laboratory supervisor Dr. Nuria Costa,

who has supported me throughout this thesis with her patience, knowledge, encouragement

friendship, helpful advice, and valuable guidance.

I would like to extend my sincere appreciation to Mairead Stack for putting time on her own

schedule to read this thesis and for the opportunity she gave me to perform my intership in the Irish

Dairy Board and Teagasc.

I thank all Teagasc staff and especially Joanne Hayes, Paula O’Connor, Siobhán Ryan and Anne

Marie McAuliffe for their technical support during the course of this study.

To my friends Julia Adriana, Cristina, Daniel, Maria, thanks for your help, friendship and for the

many laughs shared.

It gives me immense pleasure to thank all my family members, especially my parents, Ana and

Bolivar, my brothers, Emilio and Vanessa, my aunt, Patricia, and my uncle, Fernando for supporting

and encouraging me to pursue this degree.

I am deeply grateful with Carlos for his patience, support, tolerance, love and friendship.

IV

ABBREVIATIONS

BSI British Standard Institution

Ca Calcium

CFU Colony Forming Units

FAA Free amino acids

FDM Fat in Dry Matter

FDM Fat in dry matter

g grams

GLM General linear model

HPLC High Performance Liquid Chromatography

IDF International Dairy Federation

kPa Kilopascal

L Litres

LAB Lactic Acid Bacteria

Lb Lactobacillus

LBS Lactobacillus selective medium

LM17 Lactose medium

MCA Milk clotting activity

MNFS Moisture in the non fat substance

N Newton

NSLAB Non -starter lactic acid bacteria

P Probability value

PAB Propionic acid bacteria

PCA Principal component analysis

pH 4.6 SN pH 4.6-soluble nitrogen

pH 4.6 SN% TN pH 4.6-soluble nitrogen expressed as a % of total nitrogen

S/M Salt in moisture

SLA Sodium lactate agar

TCA Trichloroacetic acid

TN Total nitrogen

YGC Yeast extract agar

V

TABLE OF CONTENTS

Declaration ........................................................................................................................................ II

Acknowledgements .......................................................................................................................... III

Abbreviations ................................................................................................................................... IV

Table of contents ............................................................................................................................... V

Introduction ....................................................................................................................................... 1

CHAPTER 1. Literature Review ...................................................................................................... 2

1.1. Introduction ............................................................................................................................ 3

1.2. Rennet ................................................................................................................................... 4

1.3. Rennet substitutes ................................................................................................................. 5

1.3.1. Microbial rennet: Rhizomucor miehei ............................................................................. 6

1.3.2. Recombinant Calf Chymosin .......................................................................................... 6

1.3.2.1. Chymosin B produced from Aspergillus niger var. awamori containing calf

prochymosin B gene. ........................................................................................................... 7

1.4. Technical aspects of rennet substitutes ................................................................................. 8

1.5. Starter Bacteria ...................................................................................................................... 9

1.6. Secondary flora .................................................................................................................... 10

1.6.1. Propionic bacteria ......................................................................................................... 10

1.6.2. Yeast as a cheese adjunct ........................................................................................... 11

1.7. Effect of reducing lactose concentration in cheese curd on proteolysis ............................... 12

CHAPTER 2. Characterisation of the manufacture, composition and ripening of Novel Swiss-

type cheese produced using different rennet-types, variations in milk standarization and

with or without curd wash...................................................................................... ....................16

Abstract ........................................................................................................................................... 17

2.1. Introduction .......................................................................................................................... 18

2.2. OBJETIVES OF THE STUDY .............................................................................................. 20

2.3. Materials and methods ......................................................................................................... 20

2.3.1. Milk analysis ................................................................................................................. 20

2.3.2. Strains .......................................................................................................................... 20

2.3.3. Coagulant type ............................................................................................................. 21

2.3.4. Cheese manufacture .................................................................................................... 22

2.3.5. Gross Composition ....................................................................................................... 22

2.3.5.1. Cheese sampling .................................................................................................. 22

VI

2.3.5.2. Composition .......................................................................................................... 23

2.3.6. Assessment of Proteolysis in Cheese. ......................................................................... 23

2.3.6.1. Primary proteolysis ................................................................................................ 23

2.3.6.2. Secondary proteolysis ........................................................................................... 24

2.3.7. Functionality ................................................................................................................. 24

2.3.7.1. Texture .................................................................................................................. 24

2.3.7.2. Flowability of the heated cheese .......................................................................... 25

2.3.8. Flavour ......................................................................................................................... 25

2.3.8.1. Assessment of short chain volatile fatty acids ....................................................... 25

2.3.8.2. Volatiles profile ...................................................................................................... 25

2.3.9. Statistical analysis ........................................................................................................ 26

2.4. Results ................................................................................................................................. 27

2.4.1. Raw milk composition ................................................................................................... 27

2.4.2. Composition of standardized milk ................................................................................. 27

2.4.3. Gross composition ........................................................................................................ 27

2.4.3.1. Lactates and sugars .............................................................................................. 28

2.4.4. Proteolysis .................................................................................................................... 29

2.4.5. Functionality ................................................................................................................. 32

2.4.5.1. Texture profile analysis (TPA) ............................................................................... 32

2.4.5.2. Flowability of the heated cheese ........................................................................... 34

2.4.6. Flavour ......................................................................................................................... 34

2.4.6.1. Acetic, propionic and butyric acid levels during ripening ...................................... 34

2.4.6.2. Volatiles................................................................................................................. 35

2.5. Discussion ........................................................................................................................... 42

2.6. Conclusion ........................................................................................................................... 46

2.7. References .......................................................................................................................... 47

CHAPTER 3. Influence of cooking temperature during cheese manufacture on the composition, microbiology, proteolysis, functionality and flavour of novel Swiss-type cheese made with yeast adjunct...........................................................................................57 Abstract ........................................................................................................................................... 58

3.1. Introduction .......................................................................................................................... 59

3.2. Objective of study ................................................................................................................ 60

3.3. Materials and Methods ......................................................................................................... 60

3.3.1. Starters Strains ............................................................................................................. 60

3.3.2. Cheese Manufacture .................................................................................................... 60

VII

3.3.3. Enumeration of starter bacteria, non starter bacteria, propionic acid bacteria and yeast

............................................................................................................................................... 62

3.3.4. Cheese Analysis ........................................................................................................... 62

3.3.4.1. Cheese sampling .................................................................................................. 62

3.3.4.2. Gross composition ................................................................................................ 62

3.3.5. Assessment of Proteolysis in Cheese .......................................................................... 63

3.3.5.1. Primary proteolysis ................................................................................................ 63

3.3.5.2. Secondary proteolysis ........................................................................................... 63

3.3.6. Functionality ................................................................................................................. 63

3.3.6.1. Texture .................................................................................................................. 63

3.3.7. Flavour ......................................................................................................................... 64

3.3.7.1. Assessment of short chain volatile fatty acids ....................................................... 64

3.3.7.2. Volatiles profile ...................................................................................................... 64

3.3.8. Statistical analysis ........................................................................................................ 65

3.4. Results ................................................................................................................................. 65

3.4.1. Gross Composition ....................................................................................................... 65

3.4.2. Lactates and sugars ..................................................................................................... 66

3.4.3. Viability of starter bacteria: Streptococcus thermophilus and Lactobacillus helveticus

during cheese ripening. .......................................................................................................... 67

3.4.4. Viability of Lactobacillus (Lb. helveticus and NSLAB) during cheese ripening .............. 68

3.4.5. Viability of adjunct culture: yeast and propionic bacteria .............................................. 69

3.4.6. Proteolysis .................................................................................................................... 70

3.4.7. Functionality ................................................................................................................. 73

3.4.7.1. Texture profile analysis (TPA) ............................................................................... 73

3.4.8. Flavour ......................................................................................................................... 74

3.4.8.1 Acetic, propionic and butyric levels during ripening ................................................ 74

3.4.9. Volatiles ........................................................................................................................ 75

3.5. Discussion ........................................................................................................................... 80

3.6. Conclusion ........................................................................................................................... 85

CHAPTER 4 .................................................................................................................................... 91

4. General Discussion ....................................................................... Error! Bookmark not defined.

4.1. References ............................................................................................................................... 86

1

INTRODUCTION

Cheese is a key product for the Irish dairy industry, with six of the major Irish dairy companies

involved in its production including: Glanbia, Kerry Group, Dairygold, Carbery Foods, Wexford

Creamery, and Tipperary Co-op. Irish National cheese output is steadily increasing and production

is over 170,000 tonnes per annum (Beresford, 2011). Cheese markets are expanding globally but

in particular potential opportunities exist in markets such as the UK, Europe and the USA. It is

estimated that cheese consumption in Europe will increase by 300,000 tonnes per annum during

the period 2010 to 2020, thus offering Ireland a unique opportunity to increase its market share

(Beresford, 2011).

There has been an overdependence on Cheddar output in Ireland. Over 80 per cent of the cheese

currently produced in Ireland falls into the Cheddar category, however there is a need to expand

the product portfolio and many different approaches have been investigated to add value

(Beresford, 2011).

Wilkinson et al. (1997) stated the ability to diversify the range of cheese produced in modern high-

volume industrial plants may proceed via two routes: (1) production of soft, semi-soft, mould, smear

ripened and other speciality cheeses, but which requires major capital investment in specialised

curd manufacture or ripening facilities; or (2) production of diverse, innovative and novel cheese

types in existing Cheddar or Emmental-type cheese plants with a resulting broader product

portfolio.

The production of novel cheese types on existing equipment involves the manipulation of process

and ripening variables, e.g., starter type, cook temperature, rennet type, draining pH; to generate

unique flavours and textures.

The objectives of this study were to evaluate the effects of process variables on cheese

parameters, specifically 1) the combined effect of rennet type, milk composition and curd washing

level on the composition, functionality, proteolysis and flavour of novel Swiss-type cheese and 2)

the influence of cooking temperature on the composition, microbiology, proteolysis and flavour of

novel Swiss-type cheese made with yeast as an adjunct culture.

2

CHAPTER 1

Literature review

3

1. Literature review

1.1. Introduction

Swiss cheese is a generic name in North America for several related varieties of cheese which

resemble Swiss Emmental. Some types of Swiss cheese have a distinctive appearance, as the

blocks of the cheese are riddled with holes known as "eyes". Swiss cheese without eyes is known

as "blind" (Fox et al., 2000).

Three types of bacteria are used in the production of Emmental cheese: Streptococcus salivarius

subspecies thermophilus, Lactobacillus (Lactobacillus helveticus or Lactobacillus delbrueckii

subspecies bulgaricus), and Propionibacterium (Propionibacterium freudenreichii subspecies

shermani) (Fox et al., 2000).

Swiss-type cheeses are characterized by large eyes produced by P. freudenreichii subsp.

shermanii, which metabolizes lactate to propionate, acetate, and CO2 (Fox et al., 2000).

Propionibacterium do not grow in the milk during cheese making but grow in the cheese during

maturation, when it is transferred to a hot room (20°C-22°C). The curd of these cheeses is quite

rubbery and is able to trap the CO2 (which migrates through the curd until it reaches a fissure or

weakness, at which place an eye develops). The texture of these cheeses is influenced by a high

cook temperature (~ 55°C), which inactivates most of the coagulant, and a high pH at draining,

which leads to a high concentration of calcium in the curd Mullan (2005).

Swiss cheese can be classified in the follow categories (Mullan, 2005):

Extra-Hard: Sbrinz

Hard: Emmentaler (or Emmenthaler or Emmental), Gruyère/Greyerzer, Sapsago, Vacherin

Fribourgeois

Semi-Hard: Bündner Bergkäse, Mutschli, Raclette cheese, Tête de Moine, Tilsiter, Semi-

Soft: Vacherin Mont d’Or

Soft: Gala

4

1.2. Rennet

Production of cheese curd is essentially a concentration process in which the milk fat and casein

are concentrated about tenfold while the whey proteins, lactose and soluble salts are removed in

the whey (Fox, 1997). The acid coagulated and acid/heat-coagulated cheeses are normally

consumed fresh but the vast majority of rennet-coagulated cheeses are ripened (matured) for a

period ranging from three weeks to more than two years, during which numerous microbiological,

biochemical, chemical and physical changes occur, resulting in characteristic flavour, aroma and

texture (Fox, 1997).

Coagulation is achieved by the addition of rennet, or rennet-like enzymes, a range of which are

available, from both animal and microbial sources (Ramet, 2000). The rennet-induced coagulation

of milk has been studied extensively and can be divided into 2 main stages, namely the primary

(enzymatic) and secondary (aggregation) stages of coagulation (Fox et al., 2000). The primary

involves hydrolysis of Κ-casein at the Phe105-Met106 bond to give a hydrophilic macro peptide

(residues 106-169), called the casein macro peptide, which diffuses into the whey, and para-Κ-

casein, which remains attached to the casein micelle. As a result, the casein micelles become

destabilized and there is a reduction in the electrostatic repulsion between them, allowing them to

move closer to each other. The secondary (non-enzymatic) stage of coagulation involves

flocculation or aggregation of the rennet altered micelles to give a three-dimensional gel network

known as coagulum. The primary and secondary phases of milk clotting generally overlap, as

aggregation of micelles begins while enzymatic hydrolysis is still ongoing (Ruettimann and Ladish,

1987; Brule et al., 2000; Lucey, 2003; Crabbe, 2004; Dejmek and Walstra, 2004; Horne and Banks,

2004).

When the gel is at the required firmness, usually after a period of 45 min, it is cut into small pieces

using horizontal wired knives (Scott et al., 1998). This action increases the curd surface area many

times and results in expulsion of whey (synergetic) from the curd to give mixture of curds and whey

(Kosikowski and Mistry, 1997). After cutting, the curd particles are soft with a very open coat

surrounding them. In order to prevent the loss of fat and other milk components, the curds are

allowed to stand in the whey (for 10 min) during which time they ‘heal’ and the curd coat becomes

more membrane-like (Scott et al., 1998).

5

1.3. Rennet substitutes

Owing to the increasing world production of cheese (roughly 2-3% per annum over the past 30

years) and the reduced supply of calf veils (due to a decrease in calf numbers and a tendency to

slaughter calves at an older age), the supply of calf rennet has been inadequate for many years

(Fox et al., 2000). This has led to an increase in the price of veal rennet and to a search for rennet

substitutes. Despite the availability of numerous potentially useful milk coagulants, only six rennet

substitutes (all aspartyl proteinases) have been found to be more or less acceptable for cheese

production: bovine, porcine, and chicken pepsins, acid proteinases from Rhizomucor miehei,

Rhizomucor pusillus, and Cryphonectria parasitica (Rhizomucor and Cryphonectria were

previously known as Mucor and Endothia, respectively) (Fox et al., 2000).

Chicken pepsin is the least suitable of the commercial rennet, owing to its low MCA (milk clotting

activity) and proteolytic activity ratio, chicken pepsin promotes extensive degradation of both α- and

β-caseins in Cheddar cheese, leading to the development of flavour defects (e.g., bitterness) and

textural defects (soft body and greasiness) during maturation. Bovine pepsin is probably the most

satisfactory. Its proteolytic specificity is similar to that of calf chymosin, and it gives generally

satisfactory results with respect to cheese yield and quality (Fox and McSweeney, 1997).

The proteolytic specificity of the three commonly used fungal rennets (Rhizomucor miehei, R.

pusillus and Cryphonectria parasitica) is considerably different from that of calf chymosin, but they

have given generally satisfactory results when used in the manufacture of most cheese varieties.

However, the proteolytic activity of all the rennet substitutes is higher than that of calf chymosin,

resulting in higher levels of protein in the cheese whey and lower cheese yields (Fox and

McSweeney, 1997).

The MCA varies among commercial rennet and it is dependent on factors such as temperature, pH

and CaCl2 content. For instance the MCA of commercial rennets (calf rennet, R. miehei, R. pusillus,

and C. parasitica) increases with temperature in the range 28°C-36°C. The MCA of porcine pepsin,

calf rennet, and bovine pepsin at pH 6.6 increases with temperature up to 44°C, 45°C and 52°C,

respectively. The fungal enzymes (R. miehei, R. pusillus, and C. parasitica) lose activity at 47°C

and 57°C, respectively. The MCA of the pepsins, especially porcine pepsin, is more pH dependent

than that of chymosin, while that of the fungal rennets is less sensitive in the pH region 6.2-6.8

6

The coagulation of milk by C. parasitica proteinase is also less sensitive to added Ca2+ than

coagulation by calf rennet, but coagulation by Rhizomucor proteinases is more sensitive (Fox and

McSweeney, 1997).

1.3.1. Microbial rennet: Rhizomucor miehei

Microbial rennet has higher proteolytic activities than chymosin (Guinee and Wilkinson, 1992;

Harboe and Budz, 1999) and thus their use is dependent on the level of proteolysis desired, on the

degree of heat stability of the enzyme and on the temperatures used during the cook manufacture.

Proteolytic enzymes are produced by submerged fermentation on a vegetable substrate with a

selected strain of the fungus Rhizomucor miehei which is kept under contained conditions,

concentrates and purified to avoid contamination with unpleasant by-products of the mold growth.

The typical enzymatic activities in Rhizomucor miehei have significant influence on the yield and

the flavour and texture development of cheeses as compared to calf- and fermentation produced

chymosin. However the flavour and taste of cheeses produced with microbial rennets tend towards

some bitterness, especially after longer maturation periods (Samson A, 2004). These so-called

"microbial rennets" are suitable for vegetarians, provided no animal-based alimentation was used

during the production. R. miehei cleaves the bond Phe105-Met106 of Κ-casein (Drohse and Foltmann,

1989).

1.3.2. Recombinant Calf Chymosin

Several rennet substitutes, including bovine pepsin (from adult cows), fungal proteinases and other

proteolytic enzymes, have shown to have a much greater level of non-specific proteolytic activity,

and in some cases higher thermo stability that causes more degradation of milk proteins to

peptides, leading to a reduction in yield and poor flavour development in some types of cheese

(Fox et al,. 2000). Consequently, there have been numerous attempts to produce chymosin in

micro-organisms.

The gene for prochymosin has been cloned in E. coli, Saccharomyces cerevisiae, Kluyveromyces

marxianus var. lactis, Aspergillus nidulans, Aspergillus niger, and Tricoderma reesei (Foltmann,

1993, Pitts et al., 1992).

Recombinant chymosins have been approved for commercial use in many, but not all, countries.

The cheese making properties of recombinant chymosin have been assessed on many cheese

varieties, always with very satisfactory results (Fox and Stepaniak, 1993). Furthermore, varieties of

7

cheese made with recombinant chymosin have been evaluated in comparison to cheese produced

using the natural enzyme. No significant differences could be detected between them, regarding

recovery of milk solids, rate of proteolysis during ripening, as well as in the characteristics of the

final cheese products (Green et al., 1985; Kawaguchi et al., 1987; Hicks et al., 1988; Bines et al.,

1989; Ward and Kodama, 1991).

1.3.2.1. Chymosin B produced from Aspergillus niger var. awamori containing calf prochymosin B gene.

Production

The bovine prochymosin B gene is cloned in Aspergillus niger var. awamori. The producer

organism is subjected to fermentation process where it grows in several stages to build up the

inoculums for large-scale production. After inoculation the cells are grown aerobically under the

proper conditions of pH, temperature, nutrient composition, etc. When chymosin reaches a desired

level in the fermentation broth, the fermentation is stopped and the fungal cells are inactivated and

separated from the liquid. Chymosin is then recovered from the broth by one of two methods:(1)

the broth is filtered, followed by chromatographic purification and concentration of chymosin; or (2)

the chromatographic step is preceded by extraction of chymosin from the fermentation broth

(Pfizer, 1988).

Properties

Recombinant chymosin from Aspergillus niger var. awamori cleaves a single bond in Κ- casein.

The recombinant chymosin B has been extensively characterized and it has shown to be enzymatic

and immunologically identical to calf chymosin B. The recombinant enzyme differed from calf

chymosin B only in the degree of glycosylation, but is otherwise biochemically identical.

The chymosin B preparation has been tested for other enzyme activities that could be present

(peptidase, lipase, amylase, aspergillopepsin, etc.). In all cases, the activities were either not

detected or detected at low levels. Results from additional studies indicated the absence of

mycotoxins, of antimicrobial activity, of residues of polyethylene glycol and of producing organisms

(Pfizer, 1988).

In a sub chronic (90-d) feeding study in rats, no adverse effects were noted at levels up to 10 mg of

chymosin preparation kg/d. Negative results were also obtained in a series of standard

mutagenicity and clastogenicity tests (Van Eekeen et al.,1988)

8

1.4. Technical aspects of rennet substitutes

In addition to fulfilling the criteria lay down by legislative agencies regarding purity, safety, and

absence of antibiotics (IDF, 1990), rennet substitutes must possess the following characteristics

(Guinee and Wilkinson, 1992):

A high MCA : proteolytic ratio, as for example with calf rennet, prevents excessive nonspecific

proteolysis during manufacture and hence protects against a weak gel structure, high losses of

protein and fat in the whey, and reduced yields of cheese solids. Moreover, it avoids excessive

proteolysis during maturation and thus ensures the correct balance of peptides of different

molecular weights and hence desirable flavour, body, and functional characteristics in the ripened

cheese. Excessive proteolysis, especially of β-casein, is associated with the development of a bitter

flavour.

An MCA that is not very pH dependent in the region 6.5-6.9. A sharp decrease in MCA

combined with increasing pH may lead to slow gelation and a low curd tension at cutting,

especially if the milk pH at setting is high (e.g., 6.7-6.8, as may occur in late lactation) or

when the casein concentration is low (e.g., < 2.4%, w/w). These conditions are conducive

to low recovery of fat and reduced cheese yield and can occur in large factories, where the

duration of milk ripening is short (especially with the use of direct vat starters) and

production steps (including cutting) are generally carried out according to a fixed time

schedule. The addition of CaCl2 or acidulants (e.g., gluconic acid-5-lactone) may overcome

the latter problems.

Thermo stability comparable to that of calf rennet at the pH values and temperature used

during cheesemaking. This can markedly influence the level of residual rennet in high-cook

cheeses such as Emmental, Romano, Provolone, and low-moisture Mozzarella and hence

the level of proteolysis, texture, and functionality of the cheese during maturation.

The ability to impart desired flavour, body, and texture characteristics to the finished

cheese.

9

1.5. Starter Bacteria

The primary function of starter bacteria is to produce acid during the fermentation process. Starter

bacteria are either added deliberately at the beginning of manufacture or may be natural

contaminants of the milk, as is the case in many artisanal cheese varieties made from raw milk

(Beresford et. at., 2002). The starter bacteria grow rapidly in cheese milk and curd during

manufacture, reaching 108- 109 cfu/g, but subsequently decrease to approximately 1% of maximum

numbers within 1 month of ripening due to the low curd pH, depletion of lactose and the high salt

concentration in the curd. The death and lysis of the starter cells are important as

intracellular proteolytic enzymes are released into the cheese matrix where they degrade

oligopeptides (casein derived peptides produced by the coagulant and milk proteinase) to smaller

peptides and amino acids (Lane and Fox, 1996). Their enzymes are involved in the conversion of

proteins into amino acids and fatty acids from which flavour compounds are produced (Cogan and

Beresford, 2002).

The starter bacteria are members of the genera Lactococcus, Lactobacillus, Streptococcus,

Leuconostoc, and Enterococcus spp. (Cogan and Beresford, 2002). Also, Streptococcus

thermophilus, Lactobacillus delbrueckii and Lactobacillus helveticus are regarded as starter

bacteria (Beresford and Williams, 2004).

Either mesophilic or thermophilic starter cultures are used, depending on the cheese being

manufactured; mesophilic cultures are used in the production of Cheddar, Gouda, Edam, Blue and

Camembert, while thermophilic cultures are used for high temperature (50–55°C) cooked hard

cheeses such as Emmental (Swiss-type), Gruyere, Parmesan and Grana.

Production of acid in the early stages of the manufacture of Swiss-type cheeses depends

substantially on the activity of Streptococcus thermophilus, while the Lactobacillus spp. e.g.,

Lactobacillus helveticus, are responsible for continuing the acidification process in the cheese

press post drainage (Martley, 1983).

Manufacturing parameters such as curd cooking temperature, the subsequent holding time at the

cooking temperature before drainage of the curd and the degree of sensitivity of Streptococcus

thermophilus and Lactobacillus to cooking temperatures used in cheese manufacture also affect

the starter growth, activity and acidification profile (Martley, 1983).

10

Martley (1983) reported that the temperature at which most rapid acid production for St.

thermophilus occurred varied from 39.3 to 46.1°C depending on strain with a mean value of 42.7°C

for 36 strains examined. Similarly, the most rapid acid production for 38 strains of Lactobacilli

occurred in the range of 41.8-46.6°C with a mean temperature for maximum acid production by 27

Lb. helveticus strains of 44°C (Giraffa et al., 1993).

1.6. Secondary flora

The microorganisms involved in the secondary flora include propionic acid bacteria, coryneform

bacteria, yeasts and molds. In addition to these, cheeses contain adventitious nonstarter lactic acid

bacteria (NSLAB) that originate from the milk or the environment. This adventitious microflora is

composed mainly of mesophilic lactobacilli and, to a lesser extent, pediococci (Fox et al., 2000)

1.6.1. Propionic bacteria

Propionic acid bacteria (PAB; Propionibacterium freudenreichii) are used in the manufacture of

certain Swiss-type cheeses to achieve their characteristic nutty sweet flavour and eyes (Fröhlich-

Wyder and Bachmann, 2004). Nutty flavour in cheese has been positively correlated with

concentration of propionic acid (Vangtal and Hammond, 1986; Wilkinson and Sheehan, 2002) and

with levels of calcium and magnesium propionate (Warmke et al., 1996).

Propionic acid bacteria in the cheese milk survive the relatively high cooking temperature, ~54°C

used in the manufacture of Swiss-type cheeses and their growth is stimulated by increasing the

ripening temperature to 18-22 °C.

PAB grow at low oxygen concentrations (anaerobic to aerotolerant) (Fröhlich-Wyder and

Bachmann, 2004) and their growth is inhibited by high salt in moisture (S/M) levels, to an extend

dependant on strain type. Thierre et al. (2005) attributed the lower PAB numbers in experimental

Raclette (6% S/M) than in Emmental (1% S/M) to a combination of lower ripening temperature and

higher S/M contents. PAB grow well in brine salted cheeses where slow migration of salt inwards

allows PAB counts to increase during the hot room step of ripening; however Fernández-Esplá and

Fox (1998) reported and initial increase followed by a decrease of 1 to 2 log cycles in PAB counts

during ripening in a dry-salted Cheddar type containing PAB adjuncts and ripened at 7°C.

PAB grow optimally between pH 6 and 7 with a pH maximum for growth at 8.5 and minimum at 4.6

(Langsrud and Reinbold, 1973). Curd washing enables regulation of acid production and thus pH

during cheese manufacture and ripening (Czulak et al., 1969). Water may be added during cheese

11

manufacture to achieve a higher pH after lactic fermentation (5.20-5.30) to accelerate PAB

fermentation (Fröhlich-Wyder and Bachmann, 2004).

The optimal growth temperature for PAB is 30°C, but growth also occurs between 7 and 45°C as

reviewed by Langsrud and Reinbold (1973). Most cheeses with a PAB fermentation undergo a hot

room ripening step (20-24°C) (Fröhlich-Wyder and Bachmann, 2004) including Emmental, Iowa-

style Swiss cheese, Jarlsberg (Reinbold, 1972) while others including Gruyère and Bergkäse are

held at lower temperatures (12°C to 15.5°C) throughout ripening (Reinbold, 1972).

Previous studies by Sheehan et al., (2007) reported that the washed-curd, dry-salted cheeses

ripened for 182 d at 12°C were similar to those of Swiss-type control cheeses ripened with a hot

room temperature. The approach described allows manufacturing Swiss-type cheese using

techniques associated with Cheddar type manufacture.

1.6.2. Yeast as a cheese adjunct

Specific surveys have shown that the most frequently occurring species in cheeses are

Debaryomyces hansenii, Yarrowia lipolytica, Pichia membranifaciens, Pichia fermentans,

Kluyveromyces lactis, Saccharomyces cerevisiae, and Geotrichum candidum (Fleet and Mian,

1987).

Yeasts are widely dispersed in the dairy environment and appear as natural contaminants in raw

milk, air, dairy implements, surfaces, equipments, brine and smear water (Mounier et al., 2006).

They can either cause spoilage or affect desirable biochemical changes (Seiler and Busse, 1990;

Eliskases-Lechner, 1998). Yeasts are involved in the ripening process of cheese and partake in

microbial interactions, contribute to texture changes and the biosynthesis of aromatic compounds

like volatile acids and carbonyl compounds (Fleet and Mian, 1987; Roostita and Fleet, 1996; Rossi

et al., 1998; Welthagen and Viljoen, 1999). Due to features such as high proteolytic and lipolytic

activities, some yeast species play an important role in the formation of aroma precursors such as

amino acids, fatty acids and esters (Lenoir, 1984).

Yeasts can inhibit undesired microorganisms (Kaminarides and Laskos, 1992) and excrete growth

factors like B-vitamins, pantothenic acid, niacin, riboflavin and biotin (Purko et al., 1951; Lenoir,

1984; Jakobsen and Narvhus, 1996). The main contribution of yeasts to the cheese maturation

12

process is the utilization of lactic acid which in turn increases the pH and therefore favoring

bacterial growth and initiating the second stage of cheese ripening (Fleet, 1990).

Kluveromyces lactis is well known for its ability to assimilate lactose, glucose, and mixtures of

glucose and galactose. The yeast produces a β-galactosidase that hydrolyzes lactose, allowing

utilization of the sugars by the yeast (Spencer et al., 2002). Kluveromyces lactis has enzymatic

activities like amino peptidase activity (Kagkli et al., 2006), and it’s able to produce esters (Arfi et

al., 2004), that are associated with the formation of fruity flavours such as alcohols (isoamyl

alcohol, isobutyl alcohol and 2- phenyethanol), aldehydes (2-phenyacetaldehyde), esters

(ethylacetate and 2-phenyacetate) as well as monoterpenes (Martin et al., 2002). These

compounds play a major role in the final development of the cheese flavour and aroma (Law,

2001).

The possibility of using Kluveromyces lactis as starter cultures for production of a semi-hard French

cheese “Cantalet” was proposed by De Frietas et al., (2009). Cheese milk was inoculated with a

cocktail of yeast species containing Kluyveromyces lactis, Yarrowia lipolytica, and Pichia

fermentans to promote flavour development. In the study only Kluyveromyces lactis survived

throughout the ripening period, whereas Pichia fermentans and Yarrowia lipolytica died off after 3d

and 45d, respectively. However, the highest cook temperatures used during the manufacture of

Cantalet cheese reached 31°C, which differs to the cook temperatures (~50°C) which are

commonly applied to the manufacture of a Swiss-type cheese. The viability of yeast subjected to

high cook temperatures could be affected, and hence its ability to enhance flavour during cheese

ripening, and to our knowledge this approach has not been fully investigated.

1.7. Effect of reducing lactose concentration in cheese curd on proteolysis

The fermentation of lactose by lactic acid bacteria to lactic acid (mainly the L+ isomer) is an

essential feature of the manufacture of most cheese varieties (Fox et al., 1996). The average

content of lactose in bovine milk is 4.6%, ~98% of which is lost in the whey as lactose or lactic acid.

The remaining 2% is fermented by starter bacteria during the manufacture of cheese curd or the

early stages of ripening. As a consequence of the production of lactic acid, the pH of the curd is

reduced, affecting the retention and activity of coagulant during ripening, curd tension, syneresis,

and mineral content of the curd and the growth of non-starter lactic acid bacteria (NSLAB) (Fox et

al., 1990). Removing a portion of whey and replacing it by warm water is a common practice in the

manufacture of Gouda and Edam cheese and enables the cheese manufacturer to regulate acid

13

production. Gouda and Edam cheeses are submerged in brine (NaCl) and salt penetrates slowly

during brining and the subsequent storage period. Consequently, ample time remains for the virtual

complete fermentation of lactose to lactic acid by the starter culture before the salt concentration in

the centre of the cheese becomes inhibitory (although inhibition may occur in the surface layer).

Hence, the pH in brine-salted cheese is determined by the degree of curd washing which

determines the level of lactose, and ultimately the level of lactic acid.

By contrast, in dry-salted cheeses such as Cheddar, salt is added directly to the curd (milled chips,

typically 6-10 g) at a desired target pH (e.g., 5.3-5.4 for Cheddar). Owing to the larger surface area

of the dry-salted curd, salt reaches all parts rapidly, becoming inhibitory to starter cultures to a

degree dependent on salt sensitivity. This inhibits the fermentation of lactose (to lactic acid) by the

starter culture prior to the completion of its fermentation (Broadbent et al., 2003; Sheehan et al.,

2008). Hence, it is important that the fermentation proceeds close to the target pH prior to salting,

otherwise the pH may not decrease adequately and the cheese may not ripen correctly.

Cheddar cheese is not a washed curd variety; hence, seasonal variation in the concentration of

lactose in milk, which can range from ~4.0 to 4.8% (w/w) (O’Brien et al., 1999), is expected to

influence the composition and quality of the cheese. Nevertheless, relatively few studies have

investigated the potential relationship between the lactose content of milk, and the levels of residual

lactose and lactate on Cheddar cheese quality. Huffman and Kristoffersen (1984) investigated the

effect of altering the lactose content of Cheddar cheese, either by adding lactose to the curd whey

mixture (high lactose, HL) or by curd washing (replacing whey with simulated milk ultrafiltrate; low

lactose, LL).They found that the residual lactose contents of the control (CL), HL and LL cheeses at

1 day were 0.27, 0.41 and 0.06% (w/w), respectively. Lower levels of lactose at day one led to

lower levels of lactate and higher pH values in the mature 9 month-old cheese; flavour developed

more slowly in the LL cheeses, which were described as being less sharp.

Shakeel-Ur-Rehman et al., 2004) also prepared HL and LL cheeses, with lactose levels of 2.3 and

0.25% (w/w) at day one, by fortifying the milk to 8.4% (w/w) lactose with lactose powder (HL) or by

washing of the curds from control milk (LL; lactose concentration not given), respectively. Although

the pH of the HL cheese decreased significantly during maturation from 5.3 at 1 d to 4.8 at 180d,

that of the LL cheese remained relatively constant at 5.3-5.4. Modification of the lactose content

did not affect the gross composition of the CL, HL, and LL cheese; minor differences were found in

the levels of primary proteolysis, and secondary proteolysis between CL and LL cheeses. Only HL

14

cheese had higher levels of total free amino acids and on grading, was found to have a harsh

coarse flavour and a crumbly body. Based on these studies, curd washing do not affect gross

composition or proteolysis, but it does increase the pH of the resultant cheese.

15

OBJECTIVES OF THE STUDY The overall objective of this study was to identify suitable technologies that can be applied to

produce novel Swiss-type cheeses on existing cheddar plants. The factors investigated include:

The combined effect of milk standardization, rennet type, and curd wash level.

Application of two different cook temperatures during manufacture (48°C and 53°C).

16

CHAPTER 2

Characterisation of the manufacture, composition and ripening of Novel Swiss-type cheese

produced using different rennet-types, variations in milk standardization and with or without

curd wash

17

Abstract

The combined effect of rennet type, milk composition and curd wash on the composition,

proteolysis, functionality and flavour of novel Swiss-type cheeses was evaluated. Two different

coagulants were used (recombinant chymosin produced by the Aspergillum Niger var. awarmori

and the acid proteinase obtained from the Rhizomucor miehei), two wash levels (0 and 30%) and

two milk standarisation levels (0.99:1 and 0.90:1, protein-to-fat) (S1 and S2 treatments,

respectively).

The combined effect of rennet-type, milk composition and curd washing affected primary and

secondary secondary proteolysis, functionality (texture, flowability) and flavour (short chain fatty

acid and volatiles compounds), but otherwise had little impact on cheese composition. Differences

in fat and FDM were mainly attributed to differences in composition of standardized milk, while

differences in pH, total lactates and sugar metabolism were mainly attributed to the application of a

curd washing step. Curd washing reduced lactose content in the resultant cheese and thus

increased the pH due to less lactose being converted into lactic acid.

The treatment affected the levels of primary and secondary proteolysis, with the cheese containing

the Rhizomucur miehei proteinase, no wash and higher fat content, giving significantly higher levels

of proteolysis. The latter cheese was also significantly softer and had higher flowability on heating

at 180° and 280°C. While both cheeses produced flavour compounds that are typical of a Swiss-

type cheese, there were significant differences between the two cheeses for certain compounds

such as propionic acid, acetic acid and ethanol being higher in S1 cheese and diacetyl and

pentanoic acid being higher in S2 cheese.

The higher heat-stability of the Rhizomucur miehei proteinase, resulted in the higher levels of

primary proteolysis observed, which along with the higher fat content of the cheese resulted in the

softening of the texture and increase in flowability.

The results of this study show that the use of different rennet types, wash levels and milk

standardisation levels can be a useful tool to diversify the flavour and functionality of cheeses,

especially for those cheeses cooked at high temperature (50-55oC) where chymosin is most heat

labile.

Key words: Proteolysis, proteinase, functionality, Swiss-type cheese.

18

2.1. Introduction

The flavour and texture of the cheese are greatly affected by the type of coagulant used (Broome et

al., 2006). Glycolysis, lipolysis and proteolysis are three primary events that occur during cheese

ripening. Among these events, proteolysis is the most complex process and is catalysed by

enzymes originating from coagulants: chymosin, pepsin or fungal acid proteinase, starter bacteria,

non-starter bacteria and secondary starter bacteria (Fox and McSweeney, 1996). During cheese

ripening, coagulants are responsible for hydrolysing the caseins to large and intermediate-sized

peptides. These peptides are broken down into smaller peptides and/or amino acids by enzymes

from starter and non-starter bacteria (Grappin et al., 1985; Vicente et al., 2001; Hayaloglu et al.,

2005). However, during the manufacture of Swiss-type cheeses, the curd is subjected to high cook

temperatures (50°C-53°C), leading to partial inactivation of the coagulant (Fox et al., 2000), which

could affect cheese proteolysis, functionality and flavour.

The functional properties of a cheese are mainly affected by the hydrolysis of proteins; the pH and

composition of the cheese. Usually, a higher level of fat and a higher level of moisture lead to a

softer cheese that also has higher flowability, while cheeses with a lower pH tend to be harder (Fox

et al., 2000).

Removing a portion of whey and replacing it by warm water is practised in the manufacture of

Gouda and Edam cheese and enables the cheese manufacturer to regulate acid production. Curd

washing does not generally affect gross composition or proteolysis, but it does increase the pH of

the resultant cheese (Shakeel-Ur-Rehman et al., 2004), hence affecting its functionality.

Various other approaches have been employed to improve the functionality development in

cheeses. These include the use of coagulant of different proteolytic activity (Yun, Barbano and

Kindstedt, 1993; Yun, Kiely, Kindsteadt, and Barbano, 1993). Yun et al., (1993) found that the use

of Endothia parasitica proteinase as coagulant increased proteolysis and heat-induced flowability of

low moisture part-skim Mozarella. However, Endothia parasitica (now known as Cryphonectria

parasitica) is thermolabile (Harboe and Budz, 1999) and thus it is partially inactivated during the

manufacture of high-cook cheeses.

Sheehan, O’Sullivan and Guinee (2004) reported that, based on their ability to coagulate milk at

54°C, the heat stability of coagulants decreased in the order: Rhizomucor miehei > Rhizomucor

pusillus >chymosin. Those authors also reported that use of Rhyzomucor miehei resulted in

significantly higher levels of pH 4.6 soluble nitrogen in reduced-fat low-moisture Mozzarella

cheeses in comparison to cheeses made with Rhizomucor pusillus proteinase or chymosin.

19

The use of rennets which are more thermostable, along with a higher pH of the cheese and

increasing the fat content would appear as a possible strategy to enhance proteolysis and

functionality of cheeses (Guinee and Wilkinson, 1992), while possibly having an effect on the

flavour of the cheese.

20

2.2. OBJECTIVES

The objective of this study was to determine the combined effects of milk standardization, rennet

type, and curd wash level on the composition, proteolysis, functionality and flavour of a novel

Swiss-type cheese.

2.3. Materials and methods

2.3.1. Milk analysis

Protein, fat and lactose content were measured in both raw and standardised milk using the

Milkoscan 605 (A/S N, Foss Electric, Denmark). Casein was analysed using sodium acetate-acetic

acid precipitation (IDF, 1964a) and non-protein N (NPN) by solubility in 12% (w/v) tri-chloroacetic

acid (IDF, 2001) and analysed by Kjeldahl.

2.3.2. Strains

The strains used in the cheesemaking trials are outlined in Table 2.1 Cheese milk was inoculated

with commercial DVS starters containing Streptococcus thermophilus, Lactococcus lactis,

Lactobacillus helveticus and Propionibacterium freudenreichii.

21

Table 2.1. Details and differences between the cheese manufacture of novel Swiss-type cheese

treatment one (S1) and novel Swiss-type cheese treatment two (S2).

Treatment Cheese code

S1 S2

Milk standardization P:F 0.99: 1 0.90: 1

Milk volume 454 kg 454 kg

Starter cultures (w/w) 0.0044% L. lactis

0.0044% S. thermophilus

0.0055% Lb. helveticus

0.007% P. freudenreichii.

0.0044% L. lactis

0.0044% S. thermophilus

0.0055% Lb. helveticus

0.007% P. freudenreichii.

Rennet Recombinant rennet Microbial rennet

Curd Formation Firm Firm

Wash 30% 0%

Cook 1 °C per 1.5 min 1 °C per 1.5 min

Max scald 53 °C 53 ° C

Drain pH 6.15-6.30 6.15-6.30

Curd handling Cheddaring Cheddaring

Salting method Dry salting 1.4%-1.6% Dry salting 1.4%-1.6%

Mellow time 15 min 15 min

Cheese size 24 kg moulds 24 kg moulds

Mill pH 5.35 5.35

Ripening regime 15 °C 30 d

8 °C up to 6 months.

15 °C 30 d

8 °C up to 6 months.

2.3.3. Coagulant type

Two different coagulants, which according to the supplier’s information had different thermal

stabilities, were evaluated: 1) recombinant rennet produced by the fungus Aspergillus niger var.

awamori and 2) microbial rennet produced by submerged fermentation on a vegetable substrate

with a strain of the fungus Rhizomucor miehei.

22

2.3.4. Cheese manufacture

Mid-lactation milk was obtained from a local herd, standardized to a protein to fat ratio of 0:99:1

(S1) and 0.90:1 (S2) stored overnight at 6°C. Standardisation was done by either removal of fat

(cream) or the addition of casein (skim milk). Cheese milk was pasteurized in a heat exchanger at

72°C for 15 seconds and then pumped into cylindrical, jacketed, stainless steel vats to a total

weight of 454 Kg with automated variable-speed cutting and stirring equipment (APV Schweiz AG,

Worb, Switzerland). Starter blend was added to the cheese milk. After 1 h pre-ripening period,

either recombinant rennet (S1) or microbial rennet (S2), diluted 1:5 with de-ionized water, was

added at a level of 18 ml per 100 L of milk for the recombinant rennet or at level of 1.8 ml per 100 L

for the microbial rennet.

A coagulation period of 50 min was allowed to obtain a firm curd prior to a cut programme of 5 min.

After a 10 min healing period, the curd/whey mixture was stirred and drained at 30% of initial weight

for S1 and at 0% drain for S2. The amount whey drained in treatment S1 was recovered with 30%

of warm water, and curds in both treatments were cooked by steam injection into the jacket of the

vat. Curds were cooked at a rate of 1°C per 1.5 min from 32°C to 53°C maximum scald. At pH

6.15-6.30 the whey was drained and the curds were cheddared, milled at pH 5.35, salted at a level

of 1.5%, mellowed for 15 min and moulded in 24 kg moulds. The moulds were pressed in a vertical

press at 3 kPa for 30 min and pressed overnight on a horizontal press at 265 kPa. Cheeses were

vacuum packed next d and stored at 15°C for 30 d and at 8°C thereafter.

2.3.5. Gross Composition

2.3.5.1. Cheese sampling

Cheeses were sampled at various times throughout ripening: at 1 d for cheese composition, at

120 d, 150 d, 180 d for pH 4.6 soluble nitrogen, individual free amino acids, and short chain fatty

acids analysis; at 120 d and 180 d for texture and flowability and at 120 d for volatiles compounds.

At each sampling time, a 7 to 6 cm slab of cheese was cut from the exterior face of the block; the

outer layer (1-2 cm) of the slab was discarded and the remainder was used for analysis.

23

2.3.5.2. Composition

Cheese samples were grated to yield particles of <1 mm, using a food processor. Cheese was

analyzed at 1 d for the follow parameters:

Moisture. - Determined by drying a sample of constant weigh in oven at 100 °C. IDF

method 4A, 1982.

Salt. - By titration with silver nitrate. IDF method 88A, 1988.

Fat content. - Using the Rose-Gottlieb method. IDFM 1D, 1996.

Cheese pH. – pH was determined on slurry prepared from 20 g cheese and 12 g de-

ionized water, according to the British Standards Institution , 1975.

Calcium content. - Quantified by atomic absorption spectrophotometry. IDB method 154,

1992.

Ash content. - Determined gravimetrically by heating a sample in a furnace at or below

550 °C until completely ashed. IDB method 27, 1964.

Protein. – Determined by measuring the Nitrogen content of cheese by the macro-block

digestion. IDF method 20B, 1993.

Lactates were measured using the Megazyme kit (D-/L-Lactic Acid Kit, Megazyme

International Ireland Ltd., Bray, Ireland).

Sugars (lactose and galactose) were analyzed by HPLC as described by the method of

Zeppa et al. (2001).

2.3.6. Assessment of Proteolysis in Cheese.

2.3.6.1. Primary proteolysis

The combined effect of milk standardization, rennet type and curd wash level on primary

proteolysis, was measured by monitoring levels of pH 4.6 soluble nitrogen (SN), at 120 d, 150 d

and 180 d. The amount of nitrogen soluble in water at pH 4.6, expressed as a percentage of total

nitrogen (TN) in cheese was measured using the method of Kuchroo and Fox (1982). A sample of

grated cheese (60 g) was placed in a stomacher bag to which distilled water at 55°C (120 g) was

added and the contents blended into a Stomacher (Lab-Blender 400; Seward Medical, London) for

5 min at room temperature. The resulting homogenate was incubated in a water bath at 55°C and

allowed to stand for 1 hour. The contents of the stomacher bag were centrifuged at 2500 rfc for 20

min at 4°C. (Mistral 3000 centrifuge, Block Scientific Inc, Germany). After centrifuging the

supernatant was filtered through glass wool and the filtrate was adjusted to pH 4.6 using 10% HCl

24

and centrifuged at the same conditions as before. The supernatant was filtered though glass wood

and the pH 4.6 soluble extract was analysed by a macro-block digestion method (IDF, 1993) to

determine the content of water soluble nitrogen.

2.3.6.2. Secondary proteolysis

Free amino acids contents were determined in pH 4.6 SN extracts on cheeses of 120 d, 150 d and

180 d of ripening. Samples were deproteinised by mixing equal volumes of pH 4.6 SN and

trichloroacetic acid (240 g/L). Free amino acids were separated using ion-exchange

chromatography with post-column ninhydrin and visible colorimetric detection as described by

Fenelon et al. (2002). Samples were analysed in duplicate.

2.3.7. Functionality

2.3.7.1. Texture

Cheese samples (25 mm3 cubes) were cut from the slab of cheese (Cheese Blocker; Boos

Kaasgreedschap, Bodengraven, Netherlands) and stored at 4°C overnight before analysis. Six

cheeses cubes were analyzed by compression on a TA-HDi Texture Profile analyzer (model TA-

HDI, Stable Micro Systems, Godalming, UK) with a 5 mm compression plate and a 100 kg load cell

at room temperature. Each sample was subjected to 2 consecutive compressions at a speed of 1

mm/s, each to 30% of original sample height, as described in Rynne et al., (2004). Texture profile

analysis parameters were calculated. Hardness (N) was measured as the force at maximum

compression on the first bite. Fracture stress (kPa) was measured as the force per unit area at the

point of fracture on the first bite, fracture strain (dimensionless) was measured as the strain

corresponding to the minimum slope on the force-displacement curve. Cohesiveness

(dimensionless) was calculated as the ratio of the area of the second bite to that of the first bite.

Springiness (dimensionless) was calculated as the ratio of distance of the second bite (peak) to the

distance of the first bite (peak), and chewiness (N) was calculated as the product of harness x

cohesiveness x springiness. Adhesiveness (N * S) was calculated as the negative area after the

first bite in the texture profile curve as described in Van Vliet (1991), Bourne (1978) and expressed

in absolute values.

25

2.3.7.2. Flowability of the heated cheese

Flowability was measured by (i) the Schereiber method modified by Guinee et al. (2002), defined as

the percentage increase in the diameter of a disc of cheese (45 mm diameter, 6.5 mm thick)

melting at 280°C for 4 min, and (ii) by Olson/Price method as modified by Rynne et al. (2004); in

which a 15 g cylindrical cheese sample (diameter, 22 mm; height, 35 mm) was placed in the centre

of a 100 ml graduated glass tube, one end of which was closed and the other fitted with a rubber

bung. The tube was placed in a horizontal position, on a stainless steel tray, in an electric fan oven

at 180 °C for 7.5 min. The tray was then removed and allowed to cool to room temperature (~ 20

min), and the percentage flow was defined as the percentage increase in the length of the cylinder

of cheese.

2.3.8. Flavour

2.3.8.1. Assessment of short chain volatile fatty acids

Acetate, propionate and n-butyrate (C2:0, C3:0, C4:0) contents were determined in cheeses after

120 d, 150 d, and 180 d of ripening. Short chain volatile fatty acids (SCVFA) were obtained as

described by Kilcawley et al. (2001). Five grams of grated cheese were placed in a distillation tube

with 5 ml of 10% sulphuric acid, 0.5 ml of valeric acid at a concentration of 5 mg/ml and 10 ml of

distilled water. The solution was distilled in the distillation unit (2100 Kjeltec, Foss Tecator) and 100

ml of distillate were collected, filtered (0.2 µm filter) and injected onto the HPLC (Water Alliance

system 2695). Individual fatty acids were quantified by relating the area of each peak to the area of

the peaks of the fatty acids used in the internal standard. The final concentration of individual FFA

was expressed as mg of individual short chain fatty acid per kg of cheese. Analyses were

performed in triplicate and results averaged.

2.3.8.2. Volatiles profile

The volatile profiles of the headspace of each sample was analysed by solid phase micro-extraction

(SPME) gas chromatography mass spectrometry (GCMS).

For volatile analysis, 5 g of sample was added to a 20 ml amber screw capped SPME vial and

equilibrated at 40°C for 5 min with pulsed agitation of 4 s at 400 rpm. Sample introduction was

accomplished using a CTC Analytics CombiPal Autosampler. A single DVD/Carboxen/PDMS 1 cm

fiber was used for all analysis. The SPME fiber was exposed to the headspace above the samples

26

for 25 min at depth of 1 cm with pulsed agitation of 4s at 350 rpm. The fiber was retracted and

injected into the GC inlet at 250°C and desorbed for 2 min. Injections were made on a Varian 450

GC with a Perkin Elmer Elite DMS (60 m x 0.25 mm ID x 0.25 DF µm) column. The detector used

was a Varian 320 triple quad mass spectrometer. Individual compounds were identified using mass

spectral comparisons to the NIST 2005 mass spectral library. Individual compounds were assigned

quantification and qualifier ions to ensure that only the individual compounds were identified and

quantified, especially in the case of co-eluting or semi-co-eluting samples. Compounds were

quantified by calculating the area under the peak of each compound and are expressed in arbitrary

units. An autotune of the GCMS was carried out immediately prior to analysis to confirm that the

GCMS was operating under optimal conditions. Each sample was analysed in duplicate.

2.3.9. Statistical analysis

All statistical analysis was carried out using SAS (version 9.1.3, SAS Institute, Cary, NC). Analysis

of variance was carried out on data using the general linear model procedure of SAS (SAS

Institute). The Tukey honestly significant difference test was used to determine the significance of

difference between the means. The level of significance was determined at P < 0.05.

For variables analysed at several times during ripening, analysis of variance for the split-plot design

was carried out on data using the mixed procedure of SAS (SAS Institute).

Statistically significant differences (P < 0.05) between different treatment levels were determined by

using Tukey honestly significant difference.

Principal component analysis (PCA) of the individual amino acids, short chain fatty acids, texture

parameters, and volatiles compounds were performed by using the statistical software The

Unscrambler (v 9.7, CAMO, Norway).

27

2.4. Results

2.4.1. Raw milk composition

Milk used for the manufacture of S1 cheeses contained on average: 4.20% of fat, 3.55% of protein

and 4.63% of lactose, while milk used for manufacture of S2 cheeses contained: 4.28% of fat,

3.63% of protein and 4.627% of lactose.

2.4.2. Composition of standardized milk

The composition of the standardized milk used for this study can be observed in table 2.2

Table 2.2. Composition of standardized milk used for the manufacture of the novel Swiss-type cheeses.

Composition* Treatment

S1 SD** S2 SD**

Fat % 3.61 ± 0.17 4.02 ± 0.19

Protein % 3.56 ± 0.16 3.61 ± 0.15

Lactose % 4.67 ± 0.09 4.63 ± 0.03

Protein: Fat %

0.99 ± 0.01 0.90 ± 0.00

Casein: Fat %

0.73 ± 0.01 0.67 ± 0.02

Protein: Lactose %

0.76 ± 0.05 0.78 ± 0.03

S1 = Standardized milk for the manufacture of a novel Swiss-type cheese with recombinant rennet, with curd washing step and 0.99:1 of protein: fat ratio S2 = Standardized milk for the manufacture of a novel Swiss-type cheese with microbial rennet, without curd washing step and 0.90:1 of protein: fat ratio. *Values presented are the means of 3 replicates **Standard deviation of 3 replicates

2.4.3. Gross composition

The mean composition of the S1 and S2 cheeses are given in Table 2.3 and are typical for a Swiss-

type cheese (Fox et al., 2000; Sheehan et al., 2007).

28

Table 2.3. Gross composition of the novel Swiss-type cheeses.

Composition* Treatment

S1 SD** S2 SD**

Moisture (%w/w) 37.93a ± 0.65 38.2a ± 0.61

Fat (%w/w) 28.41a ± 0.42 29.7b ± 0.41

Protein (%w/w) 28.34a ± 0.29 26.6a ± 1.39

Salt (%w/w) 1.31a ± 0.23 0.97a ± 0.22

Ash (% w/w) 3.77a ± 0.15 3.53a ± 0.42

Ca (mg/100g) 833a ± 22 866a ± 42.66

Ca: Protein ratio (mg/g)

29.39a ± 0.59 32.6a ± 2.84

P/F 0.99a ± 0.01 0.90a ± 0.06

pH 5.58a ± 0.08 5.21b ± 0.07 1MNFS (%w/w) 52.98a ± 0.89 54.35a ± 0.62 2FDM (%w/w) 45.77a ± 0.75 48.09b ± 0.36 3S/M (w/w) 3.45a ± 0.65 2.94a ± 0.60

a, b Values within a row not sharing a common superscript, differ significantly ( P < 0.05) S1 = cheese produced with recombinant rennet, with curd washing step and std. milk 0.99:1 of protein: fat ratio S2 = cheese produced with microbial rennet, without curd washing step and std. milk 0.90:1 of protein: fat ratio. * Values presented are the means of 3 replicates ** Standard deviation of 3 replicates ¹MNFS= Moisture in the non-fat substance ² FDM= Fat in dry matter ³ S/M= Salt in moisture

2.4.3.1. Lactates and sugars

The mean levels of lactates and sugars are shown in Table 2.4. Significant differences (P < 0.05)

were found in levels of L-lactate, total lactate, galactose, and protein: lactose ratio.

29

Table 2.4. Lactate and sugar contents of the novel Swiss-type cheeses.

Composition* Treatment

S1 SD** S2 SD**

d-lactate g/100g cheese

0.147a ± 0.078 0.215a ± 0.035

l-lactate g/100g cheese

0.549a ± 0.064 1.139b ± 0.073

Total lactate g/100g cheese

0.696a ± 0.058 1.354b ± 0.092

Protein: lactate

40.91a ± 3.272 19.693b ± 1.269

Lactose g/100g cheese

0.006a ± 0.01 0.0006a ± 0.001

Galactose g/100g cheese

0.347a ± 0.12 0.165b ± 0.131

a, b Values within a row not sharing a common superscript, differ significantly (P < 0.05) S1 = cheese produced with recombinant rennet, with curd washing step and 0.99:1 of protein: fat ratio S2 = cheese produced with microbial rennet, without curd washing step and 0.90:1 of protein: fat ratio. * Values presented are the means of 3 replicates ** Standard deviation of 3 replicates

2.4.4. Proteolysis

Primary proteolysis, as measured by levels of pH 4.6 SN and expressed as a percentage of total

nitrogen (Table 2.5), increased significantly (P < 0.05) in all cheeses during the ripening period.

The mean levels of pH 4.6 SN were significantly affected (P < 0.05) by the combined effect of

coagulant type; milk composition and curd wash level.

The levels of total free amino acids (secondary proteolysis) are shown in Table 2.5 and Figure 2.1.

30

Table 2.5. Levels of pH 4.6 soluble nitrogen and total free amino acids during ripening of novel

Swiss-type cheeses

Ripening time (d)

Treatment

S1 S2

pH 4.6 SN (% of total N)

120 16.62 a,A 25.24 b,A

150 18.99 a,B 27.86 b,B

180 21.37 a,C 30.99 b,C

Total free AA (mg/kg of cheese)

120 13395 a,A 23945 b,A

150 18126 a,B 27964 b,B

180 19772 a,B 29061 b,B

a, b Values within a row not sharing a common superscript, differ significantly (P < 0.05) A,B Values within a column not sharing a common superscript, differ significantly (P < 0.05). S1 = cheese produced with recombinant rennet, with curd washing step and std. milk 0.99:1 of protein: fat ratio S2 = cheese produced with microbial rennet, without curd washing step and std. milk 0.90:1 of protein: fat ratio

15.00

20.00

25.00

30.00

35.00

120 140 160 180 200

pH

4.6

-SN

/ %

TN

Time (days)

Primary proteolysis pH4.6-SN (% of total N)

S1

S2

Fig 2.1. The combined effects of S1 = cheese produced with recombinant rennet, with curd washing step and std. milk 0.99:1 of protein: fat ratio and S2 = cheese produced with microbial rennet, without curd washing step and std milk. 0.90:1 of protein: fat ratio, on levels of pH 4.6 soluble nitrogen, expressed as % of total N, during ripening. Values presented are the means of tree replicates.

31

0

10000

20000

30000

40000

120 140 160 180 200

FA

A (

mg

/K

g c

he

ese

)

Time (days)

Secondary proteolysis (as determined by the levels of total FAA) over time

S1

S2

Fig 2.2. The combined effect of S1 = cheese produced with recombinant rennet, with curd washing step and std. milk 0.99:1 of protein: fat ratio and S2 = cheese produced with microbial rennet, without curd washing step and std. milk 0.90:1 of protein: fat ratio, on levels of total free amino acids in pH 4.6 soluble nitrogen extract over ripening. Values presented are the means of tree replicates.

Secondary Proteolysis Individual free amino acids

0

1000

2000

3000

4000

5000

Asp

Thre Se

r

Glu

Gly

Ala

Cys Val

Met Ile Leu

Tyr

Ph

e

His

Lys

Arg

Pro

FAA

mg/

kg o

f ch

ee

se

Free AA120 d of ripenig

S1

S2

0

1000

2000

3000

4000

5000

Asp

Thre Se

r

Glu

Gly

Ala

Cys Val

Met Ile Leu

Tyr

Ph

e

His

Lys

Arg

Pro

FAA

mg/

kg o

f ch

ee

se

Free AA150 d of ripenig

S1

S2

32

0

1000

2000

3000

4000

5000

Asp

Thre Se

r

Glu

Gly

Ala

Cys Val

Met Ile Leu

Tyr

Ph

e

His

Lys

Arg

Pro

FAA

mg/

kg o

f ch

ee

se

Free AA 180 d of ripening

S1

S2

Fig 2.3. The combined effect of S1 = cheese produced with recombinant rennet, with curd washing step and std. milk 0.99:1 of protein: fat ratio and S2 = cheese produced with microbial rennet, without curd washing step and std. milk 0.90:1 of protein: fat ratio, on levels of individual free amino acids in pH 4.6 soluble nitrogen extract from cheeses at 120 d, 150 d and 180 d of ripening. Values presented are the means of tree replicates.

2.4.5. Functionality

2.4.5.1. Texture profile analysis (TPA)

Figure 2.4. shows the texture parameters (hardness, springiness, chewiness, fracture stress,

gumminess and cohesiveness) of the cheeses over ripening.

Hardness, defined as the high resistance to deformation by applied stress, was significantly (P <

0.001) lower in S2 cheeses. Fracture stress, defined as the force at which a cheese crumbles,

cracks, or shatters when deformed, it is the result of a high degree of hardness and a low degree of

adhesiveness (Fox et al., 2000). Fracture stress was significantly lower (P < 0.001) in S2 cheeses.

Gumminess determined as the energy required for disintegrating a piece of cheese to a state ready

for swallowing, was significantly (P < 0.001) lower in S2 cheeses. Gumminess is correlated with

hardness (Fox et al., 2000).

Chewiness, the length of time or the number of chews required to masticate a cheese to a state

ready for swallowing, was significantly lower (P < 0.05) in S2 cheeses.

Springiness (elasticity), defined as the tendency to recover from large deformation (strain) after

removal of deforming stress, was significantly (P < 0.05) higher in S2 cheese. Springiness

increases upon elevation of fat levels (Fox et al., 2000).

No significant differences were found in the parameter of cohesiveness, indicating that the extent to

which cheeses were deformed before they ruptured was the same for both treatments.

33

0

50

100

150

200

250

300

350

120 180

N

Time (days)

Hardness

S1

S2

05

101520253035

120 180

kP

a

Time (days)

Fracture Stress

S1

S2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

120 180

-

Time (days)

Springiness

S1

S2

0.00

0.05

0.10

0.15

0.20

0.25

120 180

-

Time (days)

Cohesiveness

S1

S2

0

10

20

30

40

50

60

120 180

N

Time (days)

Gumminess

S1

S2

020406080

100120140

120 180

N

Time (days)

Chewiness

S1

S2

Fig 2.4. Evolution of texture parameters over ripening of novel Swiss-type cheeses S1= cheese produced with recombinant rennet, with curd washing step and std. milk 0.99:1 of protein: fat ratio S2 = cheese produced with microbial rennet, without curd washing step and std milk. 0.90:1 of protein: fat ratio.

34

2.4.5.2. Flowability of the heated cheese

The ability of the melted cheese to flow during ripening is shown in Figure 2.5. Flow levels of S2

cheese were significantly higher (P < 0.01) than that of the S1 cheese at all ripening times and

measured by the 2 methods (Schreiber and Olson). Flowability significantly (P < 0.001) increased

with ripening time as measured by the Schreiber method for both cheeses, while this increase was

not significant as measured by the Olson test

0

20

40

60

80

120 180

Flo

w %

Time (days)

Flowability Schreiber Test

S1

S2

0

100

200

300

400

120 180

% F

low

Time (days)

FlowabilityOlson Test

S1

S2

Fig. 2.5. Evolution of the flowability as measured by the Schreiber method and the Olson method, during ripening of novel Swiss-type cheeses. S1= cheese produced with recombinant rennet, with curd washing step and std. milk 0.99:1 of protein: fat ratio S2 = cheese produced with microbial rennet, without curd washing step and std. milk 0.90:1 of protein: fat ratio.

2.4.6. Flavour

2.4.6.1. Acetic, propionic and butyric acid levels during ripening

Figure 2.6 shows the mean levels of acetic, propionic and butyric acid over ripening..

35

S1 cheeses had significantly higher (P < 0.05) levels of acetic acid (3000 mg/kg) than S2 cheeses

(1500 mg/kg) (Fig. 2.6). Levels of acetate did not change significantly over ripening for either

cheese.

Levels of propionate were significantly higher (P < 0.001), at 180 d of ripening in S1 cheeses

(4200mg/kg) than S2 cheeses (800mg/kg) (Fig. 2.6) Levels of propionate increase significantly in

S2 and numerically in S1.

Levels of butyrate were not affected. Butyrate increased during ripening but the increment was

numerical rather than statistical (Fig. 2.6)

0

1000

2000

3000

4000

120 150 180

mg

/kg

ch

eese

Time (days)

Acetic Acid

S1S2

0100020003000400050006000

120 150 180

mg

/kg

ch

eese

Time (days)

Propionic Acid

S1

S2

0

200

400

600

800

120 150 180

mg

/kg

ch

eese

Time (days)

Butyric Acid

S1S2

Fig 2.6. Evolution of acetic, propionic and butyric acid during ripening, S1= cheese produced with recombinant rennet, with curd washing step and std. milk 0.99:1 of protein: fat ratio and S2 = cheese produced with microbial rennet, without curd washing step and std. milk 0.90:1 of protein: fat ratio.

2.4.6.2. Volatiles

36

Relevant chromatograms show that cheese samples S1 trial 1, 2 and 3 were highly similar to each

other (Fig. 2.7), as were cheese samples S2 Trial 1, 2 and 3 (Fig. 2.8), confirming good

repeatability. Principal component analysis (PCA) of S1 (Trial 1, 2 and 3) and S2 (Trial 1, 2 and 3)

indicated that these cheeses are quite different from one another (Fig. 2.9), with the cheeses

grouping on opposite sides of the PCA, S2 on the left and S1 on the right hand side. The PCA

explained a total of 72% percent of the total variance.

Lawlor et al, (2002) also grouped volatiles compounds in Swiss-type cheeses using PCA. The PCA

indicated that the main components strongly correlated with Swiss type cheeses (Fig 2.10) were :

ketones: including 2-butanone, 2- pentanone, 2- hexanone; branched methyl ketones: 3-methyl- 2-

pentanone, aldehydes as butanal, heptanal, hexanal octanal,; esters: ethyl butyrate, propyl

butyrate, propyl acetate, 3-methyl butyrate; carboxylic acids: ethyl hexanoate propionic acid, acetic

acid, butyric acid, hexanoic acid; alcohols: 2-pentanol, ethanol, 2-heptanol, 2-butanol, 1 butanol;

benzaldehyde) and dimethyldisulphide. In the Table 2.7 the aroma notes of the main volatiles in a

Swiss-type cheese are described.

Table. 2.6. Important volatile compounds of Swiss-type cheeses.

Volatile Aroma note

2-Butanone sharp, sweet odor reminiscent of butterscotch and

acetone.

2- Pentanone Acetone-like.

2- Heptanone blue cheese, fruity, musty, soapy

2- Hexanone Sharp odor

3-Hydroxy-2-butanone

(Acetoin)

Buttery

2,3-Butanedione (Diacetyl) Buttery

3-Methyl- 2-pentanone -

Octanal green, fatty, soapy, fruity, orange

peel

Butanal pungent

Pentanal pungent, almond-like

Heptanal Fatty, oily, green

37

Ethyl butyrate bubble, gum, fruity

Ethyl hexanoate fruity

Propyl butyrate pineaple

Propyl acetate fruity, pear

3-methyl butyrate fruity odor, pineapple.

Ethyl hexanoate fruity

Propionic acid pungent

Methyl hexanoate pineapple

Acetic acid vinegar

Butyric acid Unpleasant smell, sweetish aftertaste (similar to

ether)

Hexanoic acid Goat-like

1-butanol floral, fragrant, fruity, sweet

2-butanol alcoholic

2-pentanol sweet, alcoholic, fruity, nutty

2-heptanol

3 methyl-1butanol fruity, alcohol, solvent-like, grainy

Benzaldehyde Almond like

Α-pinene pine

Dimethylsulphide cabbage, sulfurous

* The LRI and Odour Database” at www.odour.org.uk (maintained by Dr. R. Mottram, Flavour Research Group, School of Food Biosciences, Univ. of Reading).

F10 Fig. 2.7. GC-MS chromatograms of the headspace volatiles for S1 cheeses at 120d of ripening. S1= cheese produced with recombinant rennet, with curd washing step and std. milk 0.99:1 of protein: fat ratio.

S1T1 4mth

S1T2 4mth

S1T3 4mth

Fig. 2.8. GC-MS chromatograms of the headspace volatiles for S2 cheeses at 120d of ripening. S2 = cheese produced with microbial rennet, without curd washing step and std. milk 0.90:1 of protein: fat ratio.

Fig. 2.9. Result of principal component analysis of volatile compounds of S1 and S2 cheeses at 120 d ripening. S1= cheese produced with recombinant rennet, with curd washing step and std. milk 0.99:1 of protein: fat ratio and S2 = cheese produced with microbial rennet, without curd washing step and std. milk 0.90:1 of protein: fat ratio.

Fig. 2.10. Result of principal component analysis of volatile compounds, free fatty acid, free amino acid and gross compositional constituents of eight hard-type cheeses (in bold font) showing the first two principal components. FDM=fat in the dry matter, S/M=salt in moisture, MNFS=moisture in the non-fat substance, pH 4.6-SN=pH 4.6-solible nitrogen (Lawlor et al., 2002)

2.5. Discussion

The production of novel cheese types on existing equipment involves the manipulation of

process and ripening variables, e.g., starter, rennet types, cook temperature, draining pH, etc;

with the aim of generating unique flavours and textures.

The combined effect of rennet-type, milk composition and curd wash, on the composition,

ripening parameters, functionality and flavour of novel Swiss-type cheeses was assessed in the

present study.

No significant differences were found in the compositional parameters of moisture, protein, salt,

ash, calcium, calcium: protein, P/F, MNFS and S/M. However, significant differences (P < 0.05)

were found in levels of pH, total lactates, fat content and FDM.

According to Fox and McSweeney (1997), Kosikowski and Mistry (1997) coagulant type does

not affect cheese gross composition. The marked differences in sugar metabolism and pH

values are probably due to the curd washing step which was applied to S1 cheeses. Curd

washing reduces lactose content and lactic acid concentration in the curd, affecting the pH of

the resultant cheese (Jia Hou et al., 2012). For instance, levels of total lactates in S1 cheeses

were significantly lower (P < 0.05), and pH was significantly (P < 0.05) higher due to low

lactose levels and reduced levels of lactic acid. Similar results were found by Huffman and

Kristoffersen, (1984) and Shakeel-ur-Rehman et al. (2004), curd washing did not affect gross

composition or proteolysis, but it did increase the pH of the resultant cheese

S2 cheeses contained significantly higher levels (P < 0.05) of fat and FDM due to the milk used

for those cheeses being standardized to a protein:fat ratio of 0.90:1, while milk for S1 cheeses

was standardized to a protein:fat ratio of 0.99:1.

Levels of primary proteolysis, as measured by pH 4.6 SN, increased significantly (P < 0.05) in

all cheeses during ripening, which is attributable to the continuous degradation of casein to low

molecular weight water-soluble peptides and amino acids by the action of the residual

coagulant and the proteolytic activity of the starter culture (Sallami et al., 2004; Awad et al.,

2005).

Levels of pH 4.6 SN were significantly different between treatments, with S2 cheeses having

significantly (P < 0.05) higher mean levels of primary proteolysis. This result is mainly attributed

to the thermostability of the microbial rennet, which was not inactivated during the high cook

temperatures (53°C) and according to Fox et al. (2000) the enzymes in rennet are the ones

responsible for initial proteolysis and the production of most of the water-soluble or pH 4.6 SN.

The current results show that recombinant chymosin from the Aspergillus Niger var. Awamori

(S1 cheeses) had lower thermal stability than the microbial rennet. These results are in

agreement with those obtained by Hyslop et al. (1979) and Thunell et al. (1979), who stated

that heat stabilities of coagulants decreases in the following order: Rhizomucor miehei

proteinase>Rhizomucor pusillus proteinase>veal rennet/fermentation produced chymosin.

However the thermo-stability of the coagulants (rennets) is also influenced by other parameters

including pH, temperature and time (Thunell et al., 1979; Fox, Guinee, Cogan and McSweeney,

2000).

There were significant differences (P < 0.05) in the levels of secondary proteolysis, as

measured by individual and total free amino acids; with S2 cheeses giving significantly (P <

0.05) higher mean levels of total amino acids. It has previously been reported by Yun et al.,

(1993) that coagulant type did not significantly influence the levels of secondary proteolysis.

The production of small peptides and free amino acids is due primarily to the action of enzymes

from starter bacteria (Fox et al., 2000). The marked differences in secondary proteolysis might

be attributed to the fact that more substrate (e.g. oligopeptides) was produced in S2 cheeses

during primary proteolysis; therefore more peptides were hydrolysed into amino acids.

The levels of amino acids in S2 cheeses (26.000 mg/kg-30.000 mg/kg) were much greater than

those reported, at comparable ages, in full fat Cheddar (Guinee et al., 2000) and Gouda (Fox

and Wallace, 1997), but were similar to those observed by Lawlor et al. (2002) in mature

Swiss-type cheeses. Total levels of free amino acids in S1 cheeses (13.000 mg/kg-20.000

mg/kg) were similar, at comparable ages, to full fat Cheddar (Guinee et al., 2000).The most

abundant amino acids found in S1 and S2 cheeses at 180 d of ripening where leucine, proline,

glutamine, lysine, valine, phenylalanine and threonine, typical of a Swiss type and cheddar

cheese varieties (Fox et al., 2000). Leucine, lysine, glutamine, valine and phenylalanine,

impart neutral flavours, proline and glycine impart sweet flavours and threonine is responsible

for the sweet bitter flavour (Fox et al., 2000).

Texture parameters: fracture stress, gumminess, chewiness and springiness, were significantly

affected by the combined effects of rennet type, milk composition and curd wash level, while

cohesiveness was not significant different between S1 and S2 cheeses.

Hardness, fracture stress and guminess were significantly lower (P < 0.001) in S2 cheeses,

which is mainly attributed to the higher proteolityc activity of the residual Rhizomucor miehei

proteinase, that readily hydrolyzes casein, the principal substrate of the proteinase, reducing

the content of intact casein and producing a softer texture (Fox et al., 2000). The higher fat

content of the S2 cheese probably contributed to the softer texture as well. Guminess is

generally correlated with hardness (Fox et al., 2000), the softer a piece of cheese is, the less

energy will be required to disintegrate it to a state ready for swallowing. Springiness (elasticity)

was significantly higher (P < 0.05) in S2 cheeses. Springiness increased upon elevation of fat,

salt in moisture levels and with maturity (Fox et al., 2000). Since S1 and S2 cheeses had

similar values of salt in moisture but differed in the levels of fat content, the higher elasticity of

S2 cheeses is linked to its high FDM and fat content.

Chewiness is the product of hardness, cohesiveness and springiness (Fox, et al 2000).

Chewiness was significantly higher (P < 0.05) in S1 cheese, because it is firmer than S2, and

hence it requires higher time of mastication to reach a state ready for swallowing.

The magnitude of most texture parameters decreased over ripening, an effect that is attributed

to reduction of intact casein content, owing to its hydrolysis by the proteolytic activity of the

residual chymosin and starter cultures enzymes (Guinee, 2003; Gunasekaran and Mehmet,

2004).

The ability of the melted cheese to flow (flowability) was significantly different in S1 and S2

treatments. Flowability of S2 cheeses was significantly higher (P < 0.01) than that of S1

cheeses at all ripening times and measured by the 2 methods (Schreiber and Olson).

According to Fox et al. (2002), increases in the levels of primary and secondary proteolysis

through the use of a more proteolytic coagulant than chymosin (e.g., fungal proteinase),

reduces the apparent viscosity and increases free oil and flowability.

Increases in flowability levels in S2 cheese are probably caused by various factors: the

increase in primary proteolysis in S2 cheeses (softer and more elastic texture) and the higher

level of fat. Increasing the fat content is also associated with greater flowability (Fox et al.,

2000).

The flow levels of S2 cheeses at 150 d of ripening (73%) are typical of a Swiss-type cheese

(Fox et al., 2000), while the flow levels of S1 cheeses at 150 d of ripening (62%) were similar to

those reported for a Cheddar cheese (Fox et al., 2007). Consistent with previous studies

(Sheehan et al., 2007), flowability significantly (P < 0.001) increased with ripening time (as

measured by the Schreiber method). These increases may be attributed to a number of factors,

inter alia, increases in proteolysis, fat coalescence and water binding capacity of the casein

matrix, which promote heat-induced displacement of adjoining layer of the casein matrix on

heating (Guinee, 2003).

Acetate, propionate and butyrate are important contributors to cheese flavour. However

excessive concentrations cause off-favors (rancidity) (Langsrud and Reinbold, 1973).

Levels of acetate and propionate were significantly different between the two treatments. S1

cheeses had significantly (P < 0.05) higher levels of acetate and propionate. This might be

attributed to the higher pH of S1 cheeses at 1d (5.58) in comparison to that of S2 cheeses at

1d (5.21). pH influences the growth of PAB, with the optimum pH for growth between 6 and 7,

maximum at 8.5 and minimum at 4.6 (Langsrud and Reinbold, 1973). High pH favors the

growth of PAB, which transform lactate to propionate, acetate and CO2 during the warm room

period (Fox et al., 2000). The higher pH of the S2 cheese probably allowed for a greater growth

of the PAB, thus translating into higher levels of acetate and propionate.

Levels of acetate in S1 cheese (3000 mg/kg) were similar to those reported in a Swiss-type

cheese, at comparable ages, which range from 3000-7000 mg/kg (Steffen et al, 1987; Lawlor et

al., 2002), while levels of acetate in S2 cheese (1500 mg/kg) were within the range of those

found in Cheddar varieties (100 mg/g kg to 6560 mg/kg) by Kristoffersen et al., (1959).

Propionate levels in S1 cheeses (4200 mg/kg) were similar, at comparable ages, to those

reported by Sheehan et al. (2008) in a Swiss-type cheese and to those reported for Emmental

cheese (5000 mg/kg) by FrÖhlich-Wyder and Bachman (2004). Meanwhile levels of propionate

in S2 cheeses (800 mg/kg) were similar to those reported by St Gelais et al. (1991) and

McGregor and White (1990) (120-750 mg/kg and 1000 mg/kg) respectively in Cheddar cheese.

There were no significant differences between the two treatments on the levels of butyrate.

Butyrate levels ( 600 mg/kg) at 180 d of ripening were similar to those reported for Emmental

cheese (650 mg/kg) (Ji, Alvarez and Harper, 2004).

PCA analysis allowed grouping of the experimental cheeses with their major volatile

compounds. PCA of S1 (Trial 1, 2 and 3) and S2 (Trial 1, 2 and 3) indicated that the grouping

of these cheese are quite different from one another. The major differences between the S1

and S2 samples were in relation to the concentration of specific compounds. For example, the

main volatiles found in S1 cheeses were propionic acid, 2-methyl-1-butanol, acetic acid, 1

propanol, propyl butanoate, ethyl propanoate, ethyl acetate, ethanol, 2-methyl-1-butanol.

Meanwhile, the main volatiles found in S2 cheeses were: 2-nonane, pentanoic acid,

benzaldehyde, diacetyl, hexanoic acid, octanoic acid, 3-methyl-butanal, 2 methyl propanol, 2

hexanol, pentanal. According to these results, it can be stated that the treatment significantly

affected the flavour profile of the cheeses..

The flavour of Swiss cheese is comprised of various compounds, such as furanones, short to

medium chain acids, esters, diacetyl, and branched-chain aldehydes (Preininger et al., 1994;

Preininger et al., 1996). Lawlor et al, (2002) grouped volatiles compounds in Swiss-type

cheeses using PCA (principal components anaysis). PCA indicated that the main components

strongly correlated with Swiss type cheeses were : ketones: including 2-butanone, 2-

pentanone, 2- hexanone; branched methyl ketones: 3-methyl- 2-pentanone, aldehydes as

butanal, heptanal, hexanal octanal; esters: ethyl butyrate, propyl butyrate, propyl acetate, 3-

methyl butyrate; carboxylic acids: ethyl hexanoate propionic acid, acetic acid, butyric acid,

hexanoic acid, pentanoic acid; alcohols: 2-pentanol, ethanol, 2-heptanol, 2-butanol, 1 butanol;

benzaldehyde) and dimethyldisulphide.

Some of the volatiles compounds found in S1 and S2 cheeses are typical of Swiss cheese

varieties (e.g. diacetyl, 2-nonane, propionic acid, acetic acid, 2-methyl-1butanol, pentanoic

acid, benzaldehydes, pentanal etc).

2.6. Conclusion

The combined effect of milk standardization, rennet-type and curd washing affected primary

and secondary proteolysis, functionality (texture, flowability) and flavour (acetate, propianate

and volatiles compounds), but otherwise had not impact on moisture, protein, salt, calcium,

ca:protein ratio, P/F, S/M and MNFS levels. Differences in fat and FDM were mainly attributed

to differences in composition of standardized milk, while differences in pH, total lactates and

sugar metabolism were mainly attributed to the application of a curd washing step. Curd

washing reduced lactose content in the resultant cheese and thus increased the pH due to less

lactose being converted into lactic acid. The levels of primary proteolysis (amino acids) were

greater in S1 cheeses owing to the thermostability of microbial rennet. The increase in primary

proteolysis along with the higher fat content of S2 cheeses can account for the differences

observed in texture and the increase in flowability on heating at 180° or 280°C. Levels of

acetate and propionate were affected mainly by the differences in cheese pH. High pH in

cheese is likely to favor the growth of PAB, which in turn produce higher levels of acetate and

propionate. Flavour profiles were quite different between S1 and S2 cheeses; however they

both share volatiles compounds that are typical of a Swiss type cheese.

2.7. References

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CHAPTER 3

Influence of cooking temperature during cheese manufacture on the composition, microbiology, proteolysis, functionality and flavour of novel Swiss-type cheese made with yeast adjunct

Abstract

Yeast species are frequently observed in cheeses and make a significant contribution to the

maturation process due to their ability to grow at low temperatures, assimilation/fermentation of

lactose, the assimilation of organic acids, resistance against high salt concentrations, tolerance

of low pH values and low water activities. However, the viability of yeast used as an adjunct

culture in Swiss type cheese subjected to high cook temperatures has not previously been fully

investigated.

Pilot scale novel Swiss-type cheeses were manufactured using Streptococcus thermophilus

and Lactobacillus helveticus as the main starter cultures plus yeast Kluyveromyces lactis and

propionic acid bacteria as cheese adjuncts using two different cook temperatures.

Compositional analysis were analyzed at 1d, while cheese microflora, proteolysis, levels of

short chain fatty acids and texture were monitoring during a ripening period of 120 d. Volatiles

compounds were analyzed at the end of ripening.

Increasing cook temperature from 48 °C to 53°C significantly reduced viable cell counts of

Streptococcus thermophilus and Kluyveromyces lactis, while it increased viable cell counts of

propionic acid bacteria and Lb. helveticus. Lactobacilli including NSLAB were not significantly

affected by the increased temperature. Composition parameters were affected in that cheese

produced from curd cooked to 53°C, which had significantly lower levels of moisture in non fat

substances, significantly higher pH, lower levels of proteolysis, and short chain fatty acids in

comparison to cheese cooked to 48°C. A firmer texture was observed in cheeses cooked to

53ºC in comparison to those cooked at 48°C.

Volatile compounds were affected by the different cook temperatures. However, both

treatments had aromatic compounds with fruity notes typical of yeast fermentation, suggesting

that the K. lactis was able to withstand high cook temperatures.

The study showed how the cook temperature process variable may be used to create novel

cheeses with varied composition, ripening characteristics, texture and flavour.

3.1. Introduction

Manufacture of most cheese varieties involves combining four ingredients: milk, rennet, micro

organisms and salt, which are processed through a number of common steps such as gel

formation, whey expulsion, acid production and salt addition, followed by a period of ripening.

Variations in ingredient, starter cultures and subsequent processing techniques have led to the

evolution and diversification of all cheese varieties.

The manufacture of Swiss-type cheeses generally requires thermophilic starter cultures (e.g.,

Streptococcus thermophilus and Lactobacillus helveticus). The growth of the starter cultures is

limited by the high cooking temperature (52-54°C) used for Swiss type cheeses, but growth

and acidification begins again as soon as the temperature decreases (Fox et al., 2000).

The manufacture process also involves pressing the curd under whey, overnight curd

fermentation, brine salting and ripening at elevated temperature during which the propionic acid

bacteria grow and transform the lactate to propionate, acetate, and CO2, which are responsible

for aroma and eye formation. Often, Swiss cheeses are covered by an orange smear, called

the morge, composed mainly of corynebacteria, micrococci, and yeast, whose function is to

improve aroma (Fox et al., 2000). In artisanal Swiss cheeses, propionic acid bacteria are

natural contaminants of the raw milk, but, in the industrial production of Swiss cheeses they are

normally added deliberately to the milk to give initial counts of about 103 to 104 cfu/ml (Fox et al,

2000).

St. thermophilus and Lb. helveticus are responsible for the production of lactic acid growing

optimally in the range of around 42.7 and 44.0 °C, respectively (Martley,1983). Heating

inoculated milk to 53 °C has been shown to result in a variable slowing of the acidification of

thermophilic lactobacilli due to decreased cellular viability, probably because of thermal stress

(Neviani et al., 1995). Similarly, the cooking curds to a maximum scald of 53 °C delayed but

did not arrest the growth of thermophilic lactobacilli in the core of Grana cheese (52 °C after 6

h; Giraffa et al., 1998). Sheehan et al. (2007) evaluated the effect of high cook temperatures

on starter and non-starter lactic acid bacteria viability, cheese composition and ripening indices.

Cheeses produced from curds cooked to 47°C had significantly higher levels of moisture in

non-fat substances, salt-in moisture, significantly lower pH and levels of butyrate compared to

cheeses produced from curds cooked to 50 or 53°C.

However, there are no studies relating to the survival of yeast in Swiss-type cheese

manufactured with different cooking temperatures and in turn its impact on cheese

composition, flavour, proteolysis and functionality.

3.2. Objective of study

The objectives of the study were to determine the influence of cooking temperature during the

manufacture on the composition, microbiology, proteolysis, functionality and flavour of novel

Swiss-type cheese made with yeast as adjunct culture.

3.3. Materials and Methods

3.3.1. Starters Strains

Streptococcus thermophilus and Lactobacillus helveticus, yeast Kluveromyces lactis and

Propionibacterium spp. freudenreichii were obtained as DVS and stored at -80°C until cheese

manufacture (Table 3.1).

3.3.2. Cheese Manufacture

Swiss-type cheeses (S3 and S4, Table 3.1) were manufacture at pilot scale in two vats of 500

L. Raw milk was obtained from a local dairy company, standardized to a casein to fat ratio of

0.80, held overnight at 6°C, pasteurized at 72°C for 15 s, and pumped into cylindrical,

jacketed, stainless steel vats with automated variable speed cutting and stirring equipments

equipment (APV Schweiz AG, Worb, Switzerland). Starter blend was added to the cheese milk.

Cheese milk (454 kg per vat) was inoculated with 0.003% (w/w) St. thermophilus, 0.0061%

(w/w) Lb. helveticus, 0.0061% (w/w) Propionic acid bacteria and 0.0061% of Yeast

kluyveromyces lactis (Table 3.1). After a 60 min ripening period, chymosin (Chymax plus),

diluted in 1:6 with de-ionised water, was added at a level of 18 mg/kg. A coagulation period of

50 min was allowed, prior a cut program of 5 min duration which produced curd particles of

approximately 5 mm3. After a 10 min healing period, the curd/whey mixture was stirred and

cooked by steam injection into the jacked of the vat. Curds were cooked at a rate of 1°C per

1.5 min until reach 53°C of maximum scald for treatment S3 and at a rate of 1°C per 1.5 min

until reach 48°C of maximum scald for treatment S4. The process variable applied in each trial

was that one vat was cooked to a maximum scald of 53°C (S3) and the second vat to 48 °C

(S4).

At pH 6.15 the whey was drained and the curds were cheddared, milled at pH 5.4, salted at a

level of 1.5% (w/w), mellowed for 15 min and moulded into 24 kg moulds. The moulds were

pressed in a vertical press at 3 kPa for 30 min and pressed overnight on a horizontal press at

265 kPa. Cheeses were vacuum packed next day and stored at 15°C for 35 days and at 8°C

thereafter.

Table 3.1 Details and differences between make procedures of Swiss T1 vs. Swiss T2.

Treatment Cheese code

S3 S4

Milk volume 454 kg 454 kg

Pasteurization 72°C * 15 seg 72°C * 15 seg

Standardization 0.80:1 0.80:1

Starter cultures (w/w) 0.003% St. thermophilus,

0.0061% Lb. helveticus

0.0061% Propionic acid bacteria

0.0061% Yeast kluyveromyces

lactis

0.003% St. thermophilus,

0.0061% Lb. helveticus

0.0061% Propionic Acid

Bacteria

0.0061% Yeast Kluyveromyces

lactis

Rennet Standard Standard

Curd Formation Firm Firm

Cook 1 °C per 1.5 min 1 °C per 1.5 min

Max scald 53 °C 48 ° C

Drain pH 6.15 6.15

Curd handling Cheddaring Cheddaring

Milling pH 5.4 pH 5.4

Salting method Dry salting 1.5% Dry salting 1.5%

Mellow time 15 min 15 min

Cheese size 2 blocks of 24 kg 2 blocks of 24 kg

Ripening regime 15 °C 35 d

8 °C up to 6 months.

15 °C 35 d

8 °C up to 6 months.

3.3.3. Enumeration of starter bacteria, non starter bacteria, propionic acid bacteria and

yeast

Cheese samples were aseptically removed at 30 d, 60 d, 90 d, and 120 d of ripening. The

cheese samples were placed in a stomacher bag diluted 1:10 with sterile trisodium citrate (2%

w/v) and homogenised in a stomacher (Stomacher, Lab-Blender 400, Seward, Thetford,

Norfolk, UK) for 5 min at room temperature. A serial dilution of the resultant slurry was

performed in 9 ml sterile maximum recovery diluent as required. Independent duplicate

samples were taken at each sampling point and the bacterial groups were enumerated on the

following agars: Streptococcus thermophilus on LM17 agar (Becton Dickson and Company,

Cockeysville, New Jersey, USA), incubated at 45°C for 3 d (Terzaghi and Sandine, 1975);

starter, Lactobacillus helveticus cells on MRS 5.4 agar after anaerobic incubation for 3 d at

42°C (IDF, 1998B). Non-starter lactic acid bacteria (NSLAB) on LBS agar (Becton Dickson and

Company, Cockeysville, New Jersey, USA) incubated aerobically with an overlay for 5 d at 30

°C (Rogosa, Mitchell and Wiseman, 1951); Propionic acid bacteria on sodium lactate agar after

incubation at 30°C for 7 d (Drinan and Cogan, 1992); Yeast kluyveromyces lactis enumerated

on YGC-agar incubated at 21°C for 7 d.

3.3.4. Cheese Analysis

3.3.4.1. Cheese sampling

Cheeses were sampled at various times throughout ripening at 1 d for gross compositional

analysis; at 30 d, 60 d, 90 d and 120 d for pH4.6 SN, individual free amino acids, and short

chain fatty acids; at 30 d, 90 d and 120 d for texture and at 120 d for volatiles compounds. At

each sampling time, a 7 to 6 cm slab of cheese was cut from the exterior face of the block; the

outer layer (1-2 cm) of the slab was discarded and the remainder was used for analysis.

3.3.4.2. Gross composition

Cheese samples were grated to yield particles of <1mm, using a food processor. Samples

were analyzed at one d ripening in triplicates for pH (British Standards Institution, 1975),

moisture (International Dairy Federation, 1982), fat (International Dairy Federation, 1996),

protein (International Dairy Federation, 1993), ash (International Dairy Federation, 1964),

calcium (International Dairy Federation, 1992) salt (International Dairy Federation, 1988),

lactates were measured using the Megazyme kit (D-/L-Lactic Acid Kit, Megazyme International

Ireland Ltd., Bray, Ireland) and sugars (lactose and galactose) were analysed by HPLC as

described by the method of Zeppa et al. (2001).

3.3.5. Assessment of Proteolysis in Cheese

3.3.5.1. Primary proteolysis

pH 4.6 soluble nitrogen (SN) was determined by the macro-Kjedahl method (International Dairy

Federation, 1993). The levels of pH 4.6 SN were determined in triplicate using the method of

Kuchroo and Fox (1982) and expressed as a percentage of total nitrogen (TN).

3.3.5.2. Secondary proteolysis

Individual free amino acids (FAA) were determined in duplicate on the pH 4.6 SN extracts of

cheeses after 30 d, 60 d, 90 d and 120 d of ripening prepared by a modification of the method

of Kuchroo and Fox (1982) as described by Fenelon, O’Connor and Fox (2000). Samples were

deproteinised by mixing equal volumes of 24% (w/v) trichloroacetic acid (TCA) and samples

were allowed to stand for 10 minutes before centrifuging at 14400 x g (Microcentaur, MSE, UK)

for 10 min. Supernatants were removed and diluted with 0.2 M sodium citrate buffer, pH 2.2 to

give approximately 250 nmol of each amino acid residue. Samples were then diluted 1 in 2 with

the internal standard, norleucine, to give a final concentration of 125 nm/ml. Amino acids were

quantified using a Jeol JLC-500/V amino acid analyser (Jeol (UK) Ltd., Garden city, Herts, UK)

fitted with a Jeol Na+ high performance cation exchange column.

3.3.6. Functionality

3.3.6.1. Texture

Cheese samples (25 mm3 cubes) were cut from the slab of cheese (Cheese Blocker; Boos

Kaasgreedschap, Bodengraven, Netherlands) and stored at 4°C overnight before analysis. Six

cheeses cubes were analyzed by compression on a TA-HDi Texture Profile analyzer (model

TA-HDI, Stable Micro Systems, Godalming, UK) with a 5mm compression plate and a 100 kg

load cell at room temperature. Each sample was subjected to 2 consecutive compressions at a

speed of 1 mm/s, each to 30% of original sample height, as described in Rynne et al., (2004).

Texture profile analysis parameters were calculated. Hardness (N) was measured as the force

at maximum compression on the first bite. Fracture stress (kPa) was measured as the force per

unit area at the point of fracture on the first bite. Cohesiveness (dimensionless) was calculated

as the ratio of the area of the second bite to that of the first bite. Springiness (dimensionless)

was calculated as the ratio of the area of the second bite to that of the first bite. Chewiness (N)

was calculated as the product of harness x cohesiveness x springiness. Adhesiveness (N * S)

was calculated as the negative area after the first bite in the texture profile curve as described

in Van Vliet (1991), Bourne (1978) and expressed in absolute values.

3.3.7. Flavour

3.3.7.1. Assessment of short chain volatile fatty acids

Acetate, Propionate and n-butyrate (C2:0, C3:0, C4:0) contents were determined in cheeses by

steam distillation and quantified by ligan-exchange, ion-exclusion HPLC as described by

Kilcawley et al. (2001).

3.3.7.2. Volatiles profile

The volatile profiles of the headspace of each sample was analysed by solid phase micro-

extraction (SPME) gas chromatography mass spectrometry (GCMS).

For volatile analysis, 5 g of sample was added to a 20 ml amber screw capped SPME vial and

equilibrated at 40°C for 5 min with pulsed agitation of 4 s at 400 rpm. Sample introduction was

accomplished using a CTC Analytics CombiPal Autosampler. A single DVD/Carboxen/PDMS 1

cm fiber was used for all analysis. The SPME fiber was exposed to the headspace above the

samples for 25 min at depth of 1 cm with pulsed agitation of 4 s at 350 rpm. The fiber was

retracted and injected into the GC inlet at 250°C and desorbed for 2 min. Injections were made

on a Varian 450 GC with a Perkin Elmer Elite DMS (60 m x 0.25 mm ID x 0.25 DF µm) column.

The detector used was a Varian 320 triple quad mass spectrometer. Individual compounds

were identified using mass spectral comparisons to the NIST 2005 mass spectral library.

Individual compounds were assigned quantification and qualifier ions to ensure that only the

individual compounds were identified and quantified, especially in the case of co-eluting or

semi-co-eluting samples. Compounds were quantified by calculating the area under the peak of

each compound and are expressed in arbitrary units. An autotune of the GCMS was carried

out immediately prior to analysis to confirm that the GCMS was operating under optimal

conditions. Each sample was analysed in duplicate.

3.3.8. Statistical analysis

All statistical analyses were carried out using SAS (version 9.1.3, SAS Institute, Cary, NC).

Analysis of variance was carried out on data using the general linear model procedure of SAS

(SAS Institute). The Tukey honestly significant difference test was used to determine the

significance of difference between the means. The level of significance was determined at P <

0.05.

For variables analysed at several times during ripening, analysis of variance for the split-plot

design was carried out on data using the mixed procedure of SAS (SAS Institute).

Statistically significant differences (P < 0.05) between different treatment levels were

determined by using Tukey honestly significant difference.

Principal component analysis (PCA) of the individual amino acids, short chain fatty acids,

texture parameters, and volatiles compounds were performed by using the statistical software

The Unscrambler (v 9.7, CAMO, Norway).

3.4. Results

3.4.1. Gross Composition

The mean composition of S3 and S4 cheeses are given in Table 3.2.

Table 3.2. Cheese composition of novel Swiss type cheeses made with yeast adjunct differing

in their cooking temperature.

Composition Treatment* S3 SD** S4 SD** Moisture% (w/w) 36.70a ± 0.08 40.25b ± 0.12

Fat % (w/w) 32.53a ± 0.27 30.52b ± 0.33 Protein %

(w/w) 25.98a ± 0.2 23.84b ± 0.31 Salt % 1.18a ± 0.07 1.17a ± 0.12 Ash% 3.53a ± 0.02 3.61a ± 0.02 Ca mg/100g 848a ± 15 843a ± 17 Ca : Protein

mg/g 32.45a ± 0.01 35.56b ± 0.25 P/F 0.80a ± 0.01 0.78a ± 0.04 pH 5.34a ± 0.01 5.24b ± 0.03 ¹MNFS% 54.39a ± 0.25 57.93b ± 0.38 ²FDM% 51.39a ± 0.37 51.08a ± 0.29 ³S/M% 3.22a ± 0.02 2.91a ± 0.02

a, b Values within a row not sharing a common superscript, differ significantly (P < 0.05) *S3= cheese cooked at 53ºC; S4=cheese cooked at 48ºC * Values presented are the means of 2 replicates ** Standard deviation of 2 replicates ¹MNFS= Moisture in the non-fat substance ² FDM= Fat in dry matter ³ S/M= Salt in moisture

3.4.2. Lactates and sugars

Mean levels of lactates and sugars are shown in Table 3.3. The mean levels of lactic acid

expressed as total lactates (D-lactate and L-lactate) and protein:lactate, were significantly lower

(P < 0.05) in cheese cooked at 53°C (S3), while the levels of residual lactose and galactose

were significantly (P < 0.05) higher.

Table 3.3 Lactates and sugars contents of novel Swiss type cheeses made with yeast adjunct

differing in their cooking temperature.

Composition Treatment*

S3 S4

d-lactate g/100g

0.17a

0.15b

l-lactate g/100g

0.65a

0.80b

Total lactate g/100g

0.83a

0.95b

Protein: lactate

31.45a 25.02b

Lactose g/100g

0.44a 0.35b

Galactose g/100

0.25a 0.07b

a, b Values within a row not sharing a common superscript, differ significantly ( P < 0.05) *S3= cheese cooked at 53ºC; S4=cheese cooked at 48ºC. ¹MNFS= Moisture in the non-fat substance ² FDM= Fat in dry matter ³ S/M= Salt in moisture

3.4.3. Viability of starter bacteria: Streptococcus thermophilus, Lactococcus lactis and

Lactobacillus helveticus during cheese ripening.

Viability of starter bacteria is shown in Figure 3.1. Cook temperature had significant effect on

mean viable cell number of St. thermophilus, and Lb. Helveticus. Cheeses cooked at 48°C had

significantly higher counts (P <0.001) of viable St. thermophilus than cheeses cooked at 53°C,

and they decreased significantly (P < 0.001) in both cheeses over ripening (Fig. 3.1).

Cheeses cooked at 53°C had significant (P < 0.001) higher counts of Lb. helveticus (from the

period 60d and thereafter), than those cooked at 48°C. Mean viable cell numbers of Lb.

helveticus decreased significantly (P < 0.001) during ripening for both cheeses (Fig. 3.1).

0.0

2.0

4.0

6.0

8.0

10.0

30 60 90 120

Lo

g1

0 c

fu/g

ch

ee

se

Time (days)

St. thermophilus

S3

S4

0.0

2.0

4.0

6.0

8.0

10.0

30 60 90 120

Lo

g1

0 c

fu/g

ch

ees

e

Time (days)

Lb. helveticus

S3

S4

Fig. 3.1 Effect of varying cook temperature during cheese manufacture on novel Swiss type cheeses: S3= cheese cooked at 53ºC; S4=cheese cooked at 48ºC, on viable cell count of Streptococcus thermophilus enumerated on LM17 agar @42ºC and viable cell counts of Lactobacillus helveticus enumerated on MRS 5.4 agar during cheese ripening.

3.4.4. Viability of Lactobacillus during cheese ripening

Mean counts of undefined lactobacilli (Lb. helveticus and NSLAB) are shown in Figure 3.2.

Viable lactobacilli numbers increased significantly (P <0.05), throughout the ripening period.

(Fig 3.2). No significant differences of viable counts of lactobacilli were found between

cheeses cooked at 53°C and those cooked at 48°C, throughout the ripening period in study.

0.01.02.03.04.05.06.07.08.09.0

30 60 90 120

Lo

g1

0 c

fu/g

ch

ees

e

Time (days)

Lactobacillus

S3

S4

Fig 3.2 Effect of varying cook temperature during cheese manufacture in novel Swiss type cheeses: S3= cheese cooked at 53ºC; S4=cheese cooked at 48ºC, on viable cell counts of non starter lactic acid bacteria enumerated on LBS agar during cheese ripening.

3.4.5. Viability of adjunct culture: yeast and propionic bacteria

Viability of yeast and propionic bacteria is shown in Figure 3.3. Cook temperatures significantly

(P < 0.05) affected viable cell counts of yeast Kluveromyces lactis and propionic acid bacteria.

Counts of viable yeast was significantly (P < 0.05) higher in cheeses cooked at 48°C than

those cooked at 53°C (Fig. 3.3).

Counts of viable propionic acid bacteria was significantly higher (P < 0.05) in cheeses cooked

at 48°C than those cooked at 53°C.

Viable counts of yeast K. Lactis decreased significantly (P < 0.01) over ripening in S1 and S2

cheeses, while viable PAB counts increased significantly (P < 0.05) over ripening in S1 and S2

cheeses (Fig. 3.3).

.

0.0

1.0

2.0

3.0

4.0

30 60 90 120

Lo

g10 c

fu/g

ch

eese

Time (days)

Yeast

S3

S4

0.0

2.0

4.0

6.0

8.0

10.0

30 60 90 120Lo

g1

0 c

fu/g

ch

ee

se

Time (days)

Propionic acid bacteria

S3

S4

Fig 3.3 Effect of varying cook temperature during cheese manufacture on novel Swiss type cheeses: S3= cheese cooked at 53ºC; S4=cheese cooked at 48ºC, on viable cell counts of Kluveromyces lactis, enumerated on yeast extract agar and propionic acid bacteria (PAB) enumerated on sodium lactate agar (SLA).

3.4.6. Proteolysis

Means levels of primary proteolysis expressed as pH 4.6 SN are shown in Figure 3.4. The

mean levels of pH 4.6 SN increased significantly (P < 0.05) during ripening in both cheeses..

Cook temperatures significantly (P < 0.001) affected primary proteolysis (Fig. 3.4, Table 3.4).

The cheese cooked at 48ºC had significant higher (P < 0.001) levels of pH 4.6 SN than the

cheese cooked at 53 ºC.

Different cook temperatures had no significant effect on secondary proteolysis (Fig 3.5, Table

3.4). However, levels of total free amino acids increased significantly (P < 0.001) in all cheeses

during ripening (Fig 3.5, table 3.4). Individual free amino acids are shown in Figure 3.6.

Table 3.4. Levels of pH 4.6 soluble nitrogen and total free amino acids during ripening of novel

Swiss-type cheeses.

Ripening time (d)

Treatment

S3 S4

pH 4.6 SN (% of total N)

30 8.00a,A 12.02b,A

60 11.93a,B 16.13b,B

90 15.04a,C 17.38b,C

120 16.24a,D 20.05b,D

Total free AA (mg/kg of cheese)

30 8059 a, A 9606 a, A

60 12771 a, BC 12906 a, B

90 14755 a, CD 15489 a, C

120 16082 a, D 16470 a, C

a, b Values within a row not sharing a common superscript, differ significantly (P < 0.05) A,B Values within a column not sharing a common superscript, differ significantly (P < 0.05). S3= cheese cooked at 53ºC; S4=cheese cooked at 48ºC.

5

10

15

20

25

30 50 70 90 110

pH

4.6

-SN

/ %

TN

Time (days)

Primary proteolysis (as determined by pH4.6-SN levels) over time

S3

S4

Fig 3.4. The effect of different cooking temperatures on primary proteolysis in novel Swiss type cheeses: S3= cheese cooked at 53ºC; S4=cheese cooked at 48ºC, on levels of pH 4.6 soluble nitrogen, expressed as % of total nitrogen, during ripening. Values presented are the means from two replicates.

0

5000

10000

15000

20000

30 60 90 120

To

tal fr

ee a

min

o a

cid

s m

g/k

g

of

ch

ee

se

Time (days)

Secondary ProteolysisTotal free amino acids

S3

S4

Fig 3.5 The effect of different cooking temperatures on secondary proteolysis in novel Swiss type cheeses: S3= cheese cooked at 53ºC; S4=cheese cooked at 48ºC, on levels of total free amino acids in pH 4.6 soluble nitrogen extract during ripening. Values presented are the means from two replicates

Secondary Proteolysis determined by Individual free amino acids

0

200

400

600

800

1000

1200

1400

1600

1800

Asp

Thre Se

r

Glu

Gly

Ala

Cys

Val

Met Ile Leu

Tyr

Phe

His

Lys

Arg

Pro

Fre

e am

ino

aci

ds

mg

/kg

ch

eese

30 d of ripening

S3

S4

0

400

800

1200

1600

2000

2400

2800

Asp

Thre Se

r

Glu Gly Ala

Cys

Val

Met Ile Leu

Tyr

Phe

His

Lys

Arg

Pro

Fre

e a

min

o a

cid

s m

g/k

g

chee

se

60 d of ripening

S3

S4

0

500

1000

1500

2000

2500

3000

Asp

Thre Se

r

Glu Gly

Ala

Cys

Val

Met Ile Leu

Tyr

Phe

His

Lys

Arg

Pro

Fre

e a

min

o a

cid

s m

g/k

g

ch

ee

se

90 d of ripening

S3

S4

0

500

1000

1500

2000

2500

3000

3500

Asp

Thre Se

r

Glu Gly

Ala

Cys

Val

Met Ile Leu

Tyr

Phe

His

Lys

Arg

Pro

Fre

e am

ino

aci

ds

mg

/kg

ch

eese

120d of ripening

S3

S4

Fig 3.6. The effect of different cooking temperatures on secondary proteolysis in novel Swiss type cheeses: S3= cheese cooked at 53ºC; S4=cheese cooked at 48ºC on individual free amino acids in pH 4.6 soluble nitrogen extract at 30 d, 60 d, 90 d and 120 d of ripening. Values presented are the means from two replicates.

3.4.7. Functionality

3.4.7.1. Texture (TPA)

Figure 3.7 shows the texture profiles of S3 and S4 cheeses over ripening. Texture parameters

of hardness and gumminess were significantly (P < 0.001) higher in cheeses cooked at 53°C

while the texture parameter of adhesiveness was significantly (P < 0.001) higher in cheese

cooked at 48°C. No significant differences were found in the parameters of springiness,

cohesiveness and chewiness.

Texture Profiles

0

100

200

300

400

500

30 90 120

N

Time (days)

Hardness

S3

S4

0.00

1.00

2.00

3.00

30 90 120

-

Time (days)

Springiness

S3

S4

0

20

40

60

80

30 90 120

N

Time (days)

Gumminess

S3

S4

0

50

100

150

200

30 90 120

N

Time (days)

Chewiness

S3

S4

0.0

2.0

4.0

6.0

8.0

10.0

30 90 120

N m

m

Time (days)

Adhesiveness

S3

S4

0.000.050.100.150.200.250.30

30 90 120

-

Time (days)

Cohesiveness

S3

S4

Fig 3.7. Evolution of texture parameters over ripening of novel Swiss-type cheese: S3= cheese cook at 53ºC; S4=cheese cook at 48ºC

3.4.8. Flavour

3.4.8.1. Short chain fatty acids (acetate, propionate and butyrate)

Levels of short chain fatty acids (acetic, propionic and butyric acids) are shown Fig. 2.6. Cook

temperature significantly (P < 0.05) affected the levels of acetate, propionate and butyrate, and

they increased significantly (P < 0.001) during ripening in S1 and S2 cheeses (Fig. 3.8).

Short chain fatty acids

0

1000

2000

3000

4000

5000

30 60 120 150

mg

/Kg

o

f c

he

ese

Time (days)

Acetic acid

S2-T1

S2-T2

0

1000

2000

3000

4000

5000

30 60 90 120

mg

/kg

of

ch

eese

Time (days)

Propionic acid

S2-T1

S2-T2

0

500

1000

1500

30 60 90 120

mg

/kg

of

ch

ee

se

Time (days)

Butyric acid

S2-T1

S2-T2

Fig 3.8. The effect of different cooking temperatures on short chain volatile fatty acids in novel Swiss type cheeses: S3= cheese cooked at 53°C, and S4= cheese cooked at 48°C. Values presented are the means from two replicates.

3.4.9. Volatiles compounds.

Relevant chromatograms (Fig 3.9) show the volatile compounds found in S3 and S4 cheeses.

The PCA scores clustered the S3 and S4 cheeses into two different positions. S3 cheese

located on the positive dimension of PC1, and S4 cheese located on the negative dimension of

PC1, which means that volatiles compounds in S3 cheeses are quite different to those of S4

cheeses (Fig 3.10)

A second PCA was performed on a dataset comprised all treatments S1, S2, S3 and S4

cheeses in order to identify any correlation in volatiles compounds. PCA (Fig 3.11) grouped S1

and S3 cheeses together on the positive dimension of PC1. S4 cheeses were grouped on the

negative dimension of PC2 and S2 cheeses on the negative dimension of PC2. Volatiles

compounds in S1 and S3 are similar to each other but differ significantly with those of S2 and

S4 cheeses.

S3 and S4 cheeses differed significantly to each other. PCA explained 64% of total variance.

Fig 3.9. GC-MS chromatograms of the headspace volatiles for S3 and S4 cheeses at 120d of ripening. S3= cheese cook at 53ºC during manufacture; S4=cheese cook at 48ºC during manufactur

S3

S4

Fig. 3.10 Result of principal component analysis of volatile compounds of S4 and S3 cheeses at 120 d ripening. S3= cheese cook at 53ºC during manufacture; S4=cheese cook at 48ºC during manufacture

Fig. 3.11 Result of principal component analysis of volatile compounds of S1, S2, S3, and cheeses at 120 d ripening. S1= cheese produced with recombinant rennet, with curd washing step and std. milk 0.99:1 of protein: fat ratio; S2 = cheese produced with microbial rennet, without curd washing step and std. milk 0.90:1 of protein: fat ratio; S3= cheese cook at 53ºC during manufacture; S4=cheese cook at 48ºC during manufacture.

3.5. Discussion

In agreement with the results of Sheehan et al. (2007), cheeses produced from curds cooked to

48°C (S4) had significantly higher levels of moisture, moisture in nonfat substance (MNFS),

and ca: protein ratio, but significant lower levels of protein, fat, fat in dry matter and pH than

cheeses cooked to 53 °C (S3). The reduction in cheese moisture caused by the increased

cook temperature is most likely due to the increased syneresis (Turner et al., 1983). Gel

syneresis controls cheese moisture and hence regulates the growth of bacteria (Fox et al.,

2004). The high cook temperature applied to S3 cheeses reduced the viability of the starter

cultures and also its acidification rate (as consequence of thermal stress and moisture

reduction). Hence, the pH at d1 of S3 cheese was significantly (P < 0.05) higher, despite the

fact that the curds for both cheeses were drained and milled at similar pH values. Increases in

the concentration of moisture are parallel to the reduction in fat content and fat in dry matter.

The mean levels of lactic acid expressed as total lactate, were significantly lower (P < 0.05) in

cheese cooked at 53°C; the direct effect of the elevated cook temperature and reduced growth

rate of St. thermophilus, slowing down the consumption of lactose and hence the production

of lactic acid. Values of residual lactose were significantly higher in the cheese cooked at

53ºC; due to less lactose being converted into lactic acid. Galactose from lactose breakdown is

not utilised by the St. themophilus, but is metabolised by the Lb. helveticus (Fox et al., 2004)

and according to Martley (1983), the temperature at which most rapid acid production for 38

strains of lactobacilli occurred in the range of 41.8ºC-46.6°C with a mean temperature for

maximum acid production of 44°C (Giraffa et al., 1993). Residual galactose content was

significantly (P < 0.05) lower in cheese cook at 48°C, presumably Lb. Helvetius adapted better

to that temperature and thus consumed more galactose at 1 d after cheese manufacture.

Cheeses cooked at 48°C had significantly ( P < 0.001) higher counts of viable St. thermophilus

than cheeses cooked at 53°C, the results are similar to those obtained by Turner et al. (1983)

who observed that increasing cook temperature from 48 to 54ºC in Swiss type cheese

manufacture reduced St. thermophilus counts. Similar observations have been obtained in

Swiss cheeses (Thierry et al., 1998; Valence et al., 2000 and Sheehan et al., 2007). Mean

viable cell number of St. thermophilus decreased significantly (P < 0.001), from 108 cfu/g at 30d

of ripening to 105.5 cfu/g at 120d of ripening.

Contrary to the results obtained by Sheehan et al. (2007), cheeses cooked at 53°C (S3) had

significant (P < 0.001) higher counts of Lb. helveticus (from 60d of ripening and thereafter),

than those cooked at 48°C (S4). Mean viable cell numbers of Lb. helveticus decreased

significantly (P < 0.001) during ripening in S3 and S4 cheeses. This trend is in agreement with

the studies of Thierry et al. (1998), Valence et al. (2000). There was significant interaction

between the effect of treatment and ripening time (P < 0.001).

Viable lactobacilli numbers as enumerated in LBS agar (Lb. helveticus and NSLAB) increased

significantly (P < 0.05), from 107.6 cfu/g at 30d to 108.3 cfu/g at 120d of ripening, while the

numbers of Lb. helveticus on their own decreased. The increase in lactobacilli is an indication

of the increasing levels of NSLAB. Split plot analysis showed that the greatest increase in the

counts of lactobacilli in S3 and S4 cheeses occurred early in the ripening period of 30 d. Viable

lactobacilli numbers were slightly higher to that observed by Thierry et al. (1998); Sheehan et

al. (2007) is Swiss-type cheese and similar to those reported by Demarigny et al. (1996) and

Beuvier et al. (1997) in a Swiss-type cheese manufactured from raw milk. NSLAB may

originate from many sources including cheese milk, manufacturing equipment and the cheese

making environment as reviewed by Beresford and Williams (2004). Although lactobacilli

counts increased at varying rate, there were no significant differences of viable counts of

lactobacilli between cheeses cooked at 53°C and those cooked at 48°C, throughout the

ripening period in study.

Cook temperature had a significant effect on counts of the yeast Kluveromyces lactis. Cheese

cooked at 48ºC (S4) had significant (P < 0.05) higher counts of viable yeast than cheese

cooked at 53°C (S3) at 30d and 60d of ripening; thereafter the difference was no longer

significant. The differences may be attributed to sensitivity of yeast to high cook temperatures.

Viable yeast count decreased significantly (P < 0.01) from 103.5 cfu/g (S4) and 102.9

cfu/g (S3)

at 30d of ripening to 102.6 cfu/g (S4) and 102.5 cfu/g (S3), at 120d of ripening. These results are

similar to those obtained by Ferreira and Viljoen (2003), where the yeast number of D. hanseii

and Y. lipolitica decreased gradually alter 6 months of maturation to a minimun value of 102.3

cfu/g. In the study D. hanseii and Y. lipolitica were added as adjunct cultures in the

manufacture of mature Cheddar. According to Fleet (1990) and Welthagen & Viljoen (1998),

survival of yeasts in the cheese might be attributed to the utilisation of organic acids produced

by the lactic acid bacteria, and the proteolytic and lipolytic abilities of yeast. Furthermore,

yeasts grow particularly well during the initial period of ripening, due to their tolerance to low pH

values and high NaCl concentrations (Eliskases-Lechner and Ginzinger, 1995) and according

to Bartchi et al. (1994), a decrease in yeast counts towards the end of the ripening period is

observed in cheeses.

Cheese cooked at 48°C (S4) had significant (P < 0.05) higher counts of viable PAB compared

to cheese cooked at 53ºC (S3) during the period: 30d, 60d and 90d. At 120d both treatments

had similar values on viable PAB. Counts of viable PAB were 106.46 cfu/g (S4) and 105.44

cfu/g

(S3) at 30d of ripening and increased significantly (P < 0.05) until 108.1 cfu/g (S4) and 107.8 cfu/g

(S3) at 120d of ripening. The greatest increment occurred during the hot room period (35 d). A

significant interaction occurred between treatment and ripening time (P < 0.001) on viable PAB

counts. Thierry et al. (1998), Gilles et al. (1983) and Sheehan et al. (2007) reported similar

trends in numbers of PAB during the ripening of Swiss-type cheeses.

Cook temperature had a significant effect on primary proteolysis expressed as mean levels of

pH 4.6-SN. Fox et al. (2000) stated that primary proteolysis in low-cooked cheese, in which the

chymosin is not inactivated during cooking, is due mainly to chymosin.

Cheese cooked at 48ºC had significant higher (P < 0.001) levels of pH 4.6 SN and it might be

attributed to a greater activity of residual chymosin (Delacroix-Buchet & Fournier, 1992), which

presumably was less inactivated by the cook temperature applied to S3 cheeses (48°C) along

with its higher moisture content. Recombinant chymosin was used in S3 and S4 treatments,

and as discussed in Chapter 2, it is heat liable, probably it was partially inactivated by the high

cook temperature (53°C) applied during the manufacture of S3 cheeses.

Secondary protelysis, assessed as levels of individual and total free amino acids was not

affected by different cook temperatures. However, total free aminoacids increased significantly

from 8000-9000 mg/kg to 16000-16500 mg/kg during ripening. These levels were much greater

than those reported, at comparable ages, in full fat Cheddar (Guinee et al., 2000) and Gouda

(Fox and Wallace, 1997), but similar to those observed by Lawlor et al. (2000) and Sheehan et

al. (2007) in Swiss-type cheeses.

An increase in levels of total free amino acids during ripening is associated with the release of

intracellular peptidases, particularly from starter lactic acid bacteria (LAB) as a result of cell

lysis as reviewed by Khalid and Marth (1990). When the population of starter LAB declines, the

adventitious non-starter lactic acid bacteria (NSLAB) ultimately becomes the dominant bacterial

population in the maturing cheese (Peterson and Marshall, 1990; Martley and Crow, 1993; Fox

et al., 1998). The proteolytic activity of NSLAB could be supplementing that of the starter,

producing peptides with generally similar molecular weights, and free amino amino acids (Lane

and Fox, 1996; Lynch et al., 1997). As discussed previously, cook temperature had no

significant effect on mean viable lactobacilli (including NSLAB) counts and thus levels of total

amino acids were not markedly different between S3 and S4 cheeses. Secondary proteolysis

might be attributed as well to the peptidolytic activity of adjunct cultures. Propionibacterium spp.

are weakly proteolytic but strongly peptidolytic and they are particularly active on proline-

containing peptides during the ripening of Swiss-type cheeses, which may contribute to the

characteristic flavour of these cheeses (El-Soda et al,. 1991; Ezzat et al, 1994; Tobiassen et

al., 1996; Stepaniak et al., 1998b). On the other hand, Kluveromyces lactis is known for its

enzymatic activities mainly amino peptidase activity (Kagkli et al., 2006). Although no

significant differences occurred, S4 cheeses had numerically higher levels of amino acids.

Texture parameters of hardness and gumminess were significantly (P < 0.001) higher in

cheeses cooked at 53°C (S3). Adhesiveness was significantly (P < 0.001) higher in the cheese

cooked at 48°C (S4) and no significantly differences were found in the parameters springiness,

cohesiveness and chewiness. Differences in hardness might be attributed to the partial

inactivation of chymosin by high cook temperatures (Fox et al.,2000), reducing the proteolytic

activity of residual chymosin (Sheehan et al., 2007; Hayes et al., 2002). Residual chymosin

acts on intact casein, thus its partial inactivation reduce the hydrolysis of intact casein, resulting

in a harder texture, such as in S3 cheeses. Moisture levels also may affect hardness, the

decrease in moisture is considered to increase hardness (Costa et al., 2010).

Gumminess correlated well with hardness, similarly to previously reported results (Fox et al.,

2000). Hardness and gumminess decreased significantly (P < 0.05) from the period 90 d to 120

d, due to softening of the cheese structure with maturity (Awad et al., 2005). Adhesiveness,

defined as the force required for removing cheese that adheres to the mouth (generally the

palate) during the normal eating process, was significantly higher in cheeses cooked at 48°C,

this might be attributed to its higher moisture content and increased primary proteolysis.

Levels of short chain fatty acids (acetic, propionic and butyric acids) were affected by cook

temperatures and increased significantly (P < 0.001) during ripening in S3 and S4 cheeses.

Cheeses cooked at 48°C (S4) had significant higher levels of acetate (P < 0.01) than cheeses

cooked at 53°C during all the ripening period, owing to higher count of viable PAB in S4

cheeses. Levels of acetic acid in S3 and S4 cheeses, at 120d of ripening were 2800mg/kg and

4100mg/kg, respectively. Acetic acid is produced in cheeses that undergo a propionic acid

fermentation where lactate is transformed by PAB into propionate, acetate, carbon dioxide and

H2O (FrÖhlich-Wyder and Bachman (2004). Acetate may also be produced by NSLAB from

citrate or lactate (Thomas, 1987), from amino acids (Nakae and Elliot, 1965), or from lactose

(Thomas et al., 1979; Bouzas et al., 1993).

Mean levels of propionate in cheeses cooked at 48°C were significantly higher (P < 0.01) than

cheeses cooked at 53°C, correlating with the higher numbers of propionic acid bacteria and in

agreement with others(Sheehan, 2007). Propionate levels in the S4 ( 5000 mg/kg) were similar

to those reported by Sheehan et al. (2008) in a Swiss-type cheese and to those reported for

Emmental cheeses (5000mg/kg) by FrÖhlich-Wyder and Bachman (2004). Propionate levels in

S3 (3300 mg/kg) were greater than those reported for Cheddar by St Gelais et al. (1991) and

McGregor and White (1990) (120-750 mg/kg and 1000mg/kg) and similar to those reported by

Marsili (1985) (3200mg/kg).

Cook temperature had a significant effect on the levels of butyrate, with cheeses cooked to

53°C having significantly higher levels than cheeses cooked to 48°C. The difference started to

be significant from 60d of ripening. Butyrate is released by lipases present in cheese and/or is

synthesised by cheese microflora (Bills & D, 1964; McSweeney and Sousa, 2000). Esterolytic

and lipolytic enzymes have been identified in a diverse range of lactic acid bacteria including

Lb. helveticus (El-soda El Wahab, Ezzat, Desmazeud et al., 1986; Khalid & Marth, 1990) and

St. thermophilus (Liu, Holland & Crow, 2001). Mean levels of butyrate increased significantly (P

< 0.01) during ripening in S3 and S4 cheeses.

Principal component analysis and chromatograms showed that cheese samples S3 and S4

significantly differed to each other in terms of volatile profiles. S3 cheese samples were highly

correlated with ethyl propionate (fruity), 3-methyl butanoic acid (swiss cheese, waxy, sweaty,

old socks), 2 methyl-1-butanol (fruity) and ethyl hexanoate (fruity), while S4 cheeses were

strongly associated with 1-Propanol (pungent), benzaldhyde, (almond like), ethanol, diacetyl

(buttery), ethyl butyrate (fruity), 2-phenylethanol (fruity), 2,3-Butanediol (fruity), dymethil

trisulphite and methanethiol (cabbage like).

The strong association between ethanol and S4 cheese might be attributed to the significant

higher viable counts of Kluveromicyes lactis in cheeses cooked at 48°C (S4). K. lactis

produces ethanol directly from lactose (Rogosa et al., 1947; Mawson, 1994), and it is able to

produce volatile compounds (Arfi et al., 2004), that are associated with the formation of fruity

flavours such as alcohols (e.g. isoamyl alcohol, isobutyl alcohol, 2- phenyethanol), aldehydes

(e.g. 2-phenyacetaldehyde) and esters (e.g. ethyl acetate, 2- phenyacetate) (Law, 2001).

Diacetyl a by-product of yeast fermentation was strongly associated as well to S4 cheeses.

All of the compounds identified are typically present in dairy products.

3.6. Conclusion

Increasing the maximum cooking temperature from 48 °C to 53°C during the manufacture of

the semi-hard novel Swiss cheeses significantly reduced viable cell counts of Streptococcus

thermophilus and K. lactis, while it increased viable cell counts of propionic acid bacteria and

Lb. helveticus. Lactobacilli numbers including NSLAB were not significantly affected by the

increased temperature. Increased cook temperature significantly reduced levels of moisture,

MNFS, and ca: protein ratio, and also resulted in significant differences in mean pH, acetate,

propionate, butyrate, primary proteolysis, texture and volatiles compounds. Overall, the study

showed how the cook temperature process variable may be used to create new cheeses with

varied composition, functionality and flavour profile on existing manufacturing plants.

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CHAPTER 4

General discussion

4. General Discussion

The overall objective of this thesis was to undertake research to provide technical information

to facilitate diversification of the Irish cheeses industry. The Irish cheese industry is making

efforts to diversify its product portfolio from its traditional commodity product, Cheddar. Three

approaches have been used: (1) utilisation of existing facilities with the addition of down-stream

processing plant, (2) development of new plants or modification of existing plant dedicated to

continental-type cheese production such as Emmental and Grana-type and, (3) development of

novel hybrid cheeses incorporating the characteristics of diverse cheese types such as Swiss

and Cheddar but which are capable of manufacture, wholly or in part, on existing commercial

Cheddar plant.

The objectives of this thesis were defined as follows:

(1) to evaluate the combined effect of altered rennet-type, milk composition and curd wash

level on the composition, proteolysis, functionality and flavour characteristics of a novel Swiss-

type cheeses and

(2) to investigate the effects of increased cook temperature during manufacture, on cheese

composition, growth of primary and secondary micro flora and in turn the impact on proteolysis,

texture and flavour of a novel Swiss type cheese.

As reported in Chapter 2 the combined effect of rennet-type, milk composition and curd

washing affected primary and secondary secondary proteolysis, functionality (texture,

flowability) and flavour (short chain fatty acid and volatiles compounds), but otherwise had little

impact on cheese composition. Curd washing reduced lactose content in the resultant cheese

and thus increased the pH due to less lactose being converted into lactic acid. Increases in

cheese pH were correlated with increased count of PAB and consequently higher levels of

propionate and acetate were produced. The texture was significantly affected by the treatment,

with the cheeses containing microbial rennet, undergoing curd wash and more fat content

being softer, less gummy and more adhesive. Consequently, the latter cheeses had also

significantly higher flowability.

While all cheeses developed in Chapter 2 shared volatile compounds typical of a Swiss type

cheeses, there were significant differences in the amounts between the two treatments.

As reported in Chapter 3, increasing cook temperature from 48 °C to 53°C during the

manufacture of the semi-hard cheeses significantly reduced viable cell counts of St.

thermophilus, and K. lactis, while it increased viable cell counts of propionic acid bacteria and

Lb. helveticus. Lactobacilli numbers including NSLAB were not significantly affected by the

increased temperature. Increased cook temperature significantly reduced levels of moisture,

MNFS, and ca: protein ratio, and also resulted in significant differences in mean pH, acetate,

propionate, butyrate, primary proteolysis, and texture. Volatile compounds were affected by the

different cook temperatures. However, both cheeses (either subjected to 48°C and 53°C

cooking) presented aromatic compound with fruity notes typical of yeast, suggesting that yeast

was able to withstand high cook temperatures.

Overall, this thesis provides a knowledge base related to the application of process variables

as rennet type, milk standardization, curd wash and high cooking temperatures during the

manufacture of novel Swiss-type cheeses with thermophilic cultures.

It has shown that the use of different rennet types, wash level and milk standardisation level

can be a useful tool to diversify the flavour and functionality of cheeses, especially for those

cheeses cooked at high temperatures, where chymosin is heat labile.

It demonstrated that propionic acid bacteria and yeast Kluveromyces lactis may be used in

modified Cheddar manufacture conditions to produce novel Swiss-type. Yeast may be used to

enhance aroma especially fruity notes in cheeses subjected to high cook temperatures.

The manipulation of processing variables may be used to create novel cheeses with varied

composition, functionality and flavour profile on existing manufacturing plants.

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