Rennet Coagulation and Cheesemaking Properties of Thermally

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Rennet coagulation and cheese making properties of thermally processed milk: overview and recent developments Prashanti Kethireddipalli, and Art Hill J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504167v • Publication Date (Web): 21 Jan 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

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Rennet coagulation and cheese making properties of thermally processed milk: overview and

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recent developments

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By

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PRASHANTI KETHIREDDIPALLI AND ARTHUR R. HILL*

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Department of Food Science, University of Guelph, Guelph, ON, Canada, N1G 2W1

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*Corresponding author. Fax: 519 824 6631, e-mail address: [email protected]

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ABSTRACT Thermally induced changes in milk proteins and minerals, particularly

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interactions among caseins and denatured whey proteins influence important properties of

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dairy products in both positive and negative ways. While the extensive protein connectivity

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and increased water holding capacity resulting from such heat-induced protein modification

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account for the much desired firmness of acid gels of yogurt, thermal processing on the other

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hand severely impairs clotting and adversely affects the cheese-making properties of rennet

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coagulated cheeses. In technological terms, the principal ongoing challenge in the cheese

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industry is to take advantage of the water holding capacity of thermally aggregated whey

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proteins without compromising on the rennetability of cheese milk or the textural and

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functional attributes of cheese. Including some recent data from our laboratory, this paper

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will discuss important aspects and current literature on the use of thermally processed milk in

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the production of rennet coagulated cheeses and also some of the potential alternatives

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available for inclusion of whey proteins in cheese, such as the addition of microparticulated

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whey proteins to cheese milk.

15 KEYWORDS: thermally processed milk, cheese, rennet coagulation of milk, casein micelles, 16 whey protein/κ-casein complexes 17 18 19 20 21 22 23 24 25


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1 INTRODUCTION 2

In traditional cheese making milk pre-treatments are commonly limited to

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standardization for consistency of composition and quality and pasteurization for microbial

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safety. Current commercial cheese making is trending towards new and more sophisticated

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strategies, mostly for better ways to control microbes, increase cheese yields, favorably

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manipulate cheese ripening, and optimize cheese texture and functionality.1 Since the

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inception of commercial cheese production, cheese makers have always been interested in

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adopting any such means that can increase the yield of cheese for a given quantity of milk.

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Two such simple treatments that continue to attract considerable attention are (i) heat-

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treatment of cheese milk that increases cheese yield through retention of whey proteins and

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concomitant increases in the moisture holding capacity and ii) addition of denatured whey

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proteins (WPs) to cheese milk. Thermal processing of cheese milk, by far the simplest and

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the least expensive of pre-treatments not only promotes microbial safety, but promises higher

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cheese yields with reduced protein losses in whey mainly due to incorporation of WPs into

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cheese curd. Such heat-treatments however come with serious limitations. The main focus of

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the present article will be to provide an overview of the chemistry, rennet coagulation, and

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cheese making properties of thermally processed milk. The effects of heating on rennet

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coagulation and cheese making properties of milk were reviewed in the past2-4; the present

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discussion will emphasize the significant scientific progress made in this field in recent years.

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Other technological means of enriching cheese with WPs will be discussed briefly.

21 22 OVERVIEW OF CHEESE MAKING PRINCIPLES (RENNET CHEESES) 23

In cheeses that use rennet, the addition of enzyme to pre-warmed milk induces a

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clotting reaction in a matter of minutes. This rapid sol-gel transition is the result of extensive

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destabilization of the colloidal casein-calcium phosphate particles in milk, normally referred 3 ACS Paragon Plus Environment


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to as casein micelles. Several thousand individual casein molecules, αS1-, αS2-, β-, and κ-

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caseins (4:1:3.5:1.5, respectively), held together with calcium phosphate exist as highly

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hydrated aggregates measuring about 150 to 200 nm in average diameter.5 Casein micelles

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remain stable in milk due to steric effects contributed by their surface polyelectrolyte layer

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formed by the C-terminal regions of κ-casein. The proteolytic action of chymosin in rennet

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specifically breaks the peptide bond in κ-casein that removes these C-termini or

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caseinomacropeptide (CMP) hairs. With the elimination of strong inter-micelle repulsive

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forces, i.e., when nearly all of the protein had been hydrolyzed6-8, the para-casein micelles

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come within close proximity of one another and in the presence of ionic calcium (promotes

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calcium bridging), begin to aggregate via hydrophobic interactions to eventually form a

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particulate gel with entrapped serum and fat globules. With increased amounts of ionic

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calcium, aggregation can take place at slightly lower extents of κ-casein hydrolysis than is

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normally required9, but still only when a minimum of about 85‒90% κ-casein has been

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hydrolyzed.10 Increased screening of negative charges by ionic calcium accelerates the close

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approach of the nearly bare micelles and promotes extensive cross-linking via calcium

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bridging.11-13 The end result is the formation of rennet gels with enhanced stiffness which is

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precisely why small amounts of CaCl2 (~ 0.2 gL−1) are routinely added to cheese milk.

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During these early stages of rennet aggregation, the surface attributes of casein micelles and

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concentration of ionic calcium are all that seem to matter, but when micelles begin to fuse

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through interparticle rearrangements, the interior of casein micelles and the presence of

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colloidal calcium become increasingly important.5 Milk that has been depleted in colloidal

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calcium through acidification or addition of EDTA inherently produces weak rennet gels that

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exhibit increased mobility of bonds within the casein network as indicated by their higher loss

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tangent values.14

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Following rennet-induced coagulation in combination with lactic fermentation, the

2 curd is cut, stirred, cooked gently, and/or washed in warm water to expel whey along with the 3 serum proteins and lactose contained within. If the milk was minimally heated and the curd is 4 not washed, the theoretical recovery of whey proteins in cheese is about 9% of cheese 5 moisture. For example, given milk with a casein number of 77% and cheese moisture of 40%, 6 the percentage transfer of whey proteins from milk to cheese is about 3.5%. Following whey 7 removal, the curds are pressed to enable fusion of caseins into a solid viscoelastic mass with 8 entrapped fat globules and stored under conditions that favor important biochemical reactions 9 collectively termed as ‘cheese ripening’. Proceeding concurrently with coagulation and post 10 curd drainage, fermentation of lactose to lactic acid by starter cultures gradually lowers the pH 11 of milk and then curd until a point at which the acid build up along with other factors such as 12 lack of substrate (lactose) and reduced water activity cause cell death and lysis releasing a 13 range of catabolic enzymes that breakdown or otherwise convert milk constituents into a 14 multitude of flavor and aroma compounds. In addition to the starter lactic acid bacteria (LAB) 15 that are added to cheese milk, the non-starter LAB (NSLAB), lipoprotein lipase, and plasmin, 16 all three of which are indigenous to milk together with the residual coagulant (5 – 30% of 17 rennet activity can be retained in cheese), play a significant role in the breakdown of milk fat, 18 lactose, and proteins during cheese ripening. The composition of cheese milk, any pre19 treatments that are applied and the cheese ripening process all influence one another, affect 20 rennet coagulation properties, and strongly determine the overall texture, flavor, and 21 functionality of cheese. 22 23 CHEMISTRY OF MILK HEATED AT TEMPERATURES UP TO 100°C 24

Pasteurization and sub-pasteurization heating may be applied to cheese milk to

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eliminate or reduce pathogenic bacteria that may be present in milk. Even these mild heating



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conditions (72°C/16 s or 63°C/30 min) are sufficient to denature (unfold) a fraction of whey

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proteins, especially the heat-sensitive immunoglobulins, bovine serum albumin, lactoferrin,

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and a small fraction of β-lactoglobulin (β-Lg). The extent of WP denaturation is greatly

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enhanced with increasing temperature and time of heating.15,16 Following denaturation, WPs

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become involved in the classical thiol-disulfide exchange reactions with other WPs, the

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disulfide-bonded polymeric κ-casein residing on casein micelle surfaces, and the κ-casein that

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has diffused into the serum upon heating; hydrophobic interactions are also known to be

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involved. The heat-induced complexes, both micelle-bound and those soluble in the serum

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are typically composed of all three proteins, β-Lg, α-La (α-lactalbumin), and κ-casein and

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range in size from about 30 to 100 nm.17 Presence of small amounts of αS2-casein has also

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been reported.18 The ratio of WPs to κ-casein is reported to be around 2.4 in the serum phase

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and about 1.1 in the colloidal phase of milk heated at 90°C for 20 min; β-Lg/α-La in both

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phases is reported to be about 3.5.19

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The relative distribution of heat-induced complexes of WPs and κ-casein between

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casein micelles and the serum is very sensitive to the pH at which milk was heated. While

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heating milk at its natural pH (~6.7) transfers about a third of the serum WPs to the colloidal

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phase, lowering the pH to about 6.3 increases this proportion to nearly 75% of the total serum

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proteins, and at pH values between 6.8 and 7.1 casein micelles are nearly devoid of WPs,

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nearly all of which remain in the serum phase in complexation with κ-casein.8,19-22 However,

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the total extent of WP denaturation remains constant across pH values and is only affected by

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the temperature and time of heating.23,24 There are conflicting views over the exact sequence

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of reactions leading to the partitioning of heat-denatured WPs between the micelle and serum

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phases, i.e., if micellar dissociation of κ-casein precedes its attachment to denaturing WPs in

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the serum, or if the WPs preferentially bind to κ-casein at the micelle surface followed by

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detachment of WP/ κ-casein complexes into the serum, or in fact if both these reactions 6 ACS Paragon Plus Environment

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coexist. All caseins, αS-, β-, and κ- are known to dissociate from micelles when milk is

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heated and this phenomenon is both temperature and pH dependent; κ-casein is removed to a

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much greater extent than αS- or β-caseins (κ-> αS-> β-casein).20,25 For an extensive review on

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the formation, properties, and technological behavior of heat-induced WP/κ-casein complexes

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in milk, the reader may refer to the work of Donato and Guyomarc’h (2009).26

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Heating influences the mineral balance in milk through a decrease in the

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concentrations of soluble calcium (Casol) and soluble phosphate (Psol) within the first few

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minutes of heating; longer heating times cause little further change. In heated milk, a new

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equilibrium between the soluble and colloidal forms of calcium and phosphate is rapidly

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approached, with the equilibrium shifting towards the latter forms. Casol and Psol are

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transferred to the colloidal phase by precipitation as calcium phosphate: Ca2+ + H2PO4− →

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CaHPO4 + H+, and the released H+ ions contribute to small decreases in the pH of heated

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milk.27,28 Such heat-induced changes in milk’s mineral equilibrium are mostly, but not fully

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reversed upon cooling and the extent of reversal increases with increase in the degree of

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cooling.29 A 10 to 15% loss in diffusible calcium was reported when whey protein free

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(WPF) skim milk was heated at 75-90°C for 10 min and then cooled back to room

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temperature.30 Salt balance is also influenced by the pH at which milk is heated. Increased

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formation of amorphous calcium phosphate was reported when the heating-pH of a synthetic

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milk ultrafiltrate was increased from 6.4 to 7.0.21 The composition of the heat-precipitated

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micellar calcium phosphate is however not affected by the heating conditions and the

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calcium/phosphate ratio is close to unity suggesting that the material is similar to dicalcium

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phosphate, CaHPO4.31-33 Holt34 suggested that native CCP could act as nucleation sites for

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the heat-precipitated calcium phosphate and had based this theory on his observations that

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there were no increases in the amount of casein cross linked by CCP when milk was heated.



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Most likely there are increases in the size of CCP particles caused by the deposition of heat

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precipitated calcium phosphate.35-36 Within the complex array of heat-induced reactions, the casein micelles in milk retain

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their stability and integrity primarily because casein proteins are rheomorphic and therefore

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quite stable to heat. Having said that it becomes questionable if the intricately organized

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interiors and/or the surfaces of these colloidal structures will indeed remain entirely

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unchanged. Evidence for such effects of heat on the internal organization of micelles comes

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from some of our earlier work which will be discussed in the sections that follow.

9 10 IMPAIRED RENNET COAGULATION PROPERTIES OF THERMALLY TREATED 11 MILK 12

It is a common observation that cheese milk that was previously heated to

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temperatures over 75°C takes much longer time to clot and forms curds that are weak and

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difficult to be processed into cheese. The impaired rennet clotting properties of heat-treated

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milk have been routinely attributed to one or more of the following. i) inhibition of primary

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enzymatic phase or the rate at which CMP is released; studies in the past have reported lower

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rates of enzymatic breakdown of κ-casein which was attributed to the attachment of WPs to

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casein micelles causing conformational changes that physically block access of rennet to the

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susceptible bond.37-40 However, with the use of improved analytical techniques later studies

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have demonstrated that no significant differences exist in the breakdown of κ-casein between

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heated and unheated milk.7,22,41 ii) adverse effects of heating on the secondary clotting or

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micelle aggregation phase; micelle-bound WPs are assumed to sterically hinder aggregation

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of casein micelles even after removal of nearly all of the κ-casein, and iii) decreased

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concentration of ionic calcium in heated milk.

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Considering that the amount of ionic calcium is critical to aggregation of para-casein

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micelles, it is logical to assume that coagulation could be impaired due to heat induced

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precipitation of Ca2+ as calcium phosphate. However, there are conflicting reports on the

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effects of heat induced decreases in Ca2+ on enzymatic clotting of thermally processed milk.

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When WPF milk was subjected to ultrahigh temperature heating (100 to 140°C for 30 s; 600 s

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hold time) followed by renneting, the lower gel strength was found to be correlated with

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lower content of soluble calcium and rennet coagulation was prevented when [Ca2+] was

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reduced by about 40%42. On the other hand, when milk was heated between 75 and 90°C,

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precipitation of calcium phosphate was found to have no significant effect on casein micelle

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aggregation41; heat treatment of WPF milk showed no significant effect on the mobility of

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renneted casein micelles as was reflected in the turbidity parameter τ1/2. According to these

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authors, a 10 to 15% loss in diffusible calcium under these heating conditions does not affect

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the onset of micelle aggregation, but may influence gel strength (not examined in this study).

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In any case, the Ca2+ lost upon heating milk could be easily replaced through addition of

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CaCl2 or by more indirect means such as acidification. Of greater concern as it appears is the

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precipitation of calcium phosphate on to CCP which together with casein dissociation from

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the micelles can potentially alter micelle structure in a way that renders the micelle’s interior

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and surface unfavorable to subsequent fusion and rearrangements within the coagulum that is

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formed. These effects are likely to have consequences on the texture and fracture properties

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of cheeses that are produced from such rennet curds.

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The impairment of enzymatic clotting of heated milk can therefore be mostly

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attributed to the adverse effects of heating on the secondary clotting phase, which is neither

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due to reduced activity of rennet or decreased [Ca2+], but appears to be mostly due to coating

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of micelle surfaces by heat-denatured WPs. By heating milk in the presence or absence of

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WPs (WPF milk), Vasbinder et al. (2003)41 have demonstrated that the association of WPs 9 ACS Paragon Plus Environment


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with casein micelles severely inhibits aggregation, but the exact mechanism still remains

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unclear. It is believed that the aggregates of WPs that are attached to micelle surfaces cause

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steric hindrance to micelle aggregation even after the complete removal of CMP.3,30,43,44

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If binding of heat-denatured WPs with casein micelles impairs their aggregation, i.e.

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even after complete breakdown of κ-casein, it is reasonable to assume that by increasing or

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decreasing the amount of WPs associated with micelles (by heating milk at pH values below

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or above its natural pH, respectively) the enzymatic coagulation of heated milk could be

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further impaired or improved with respect to milk heated at its natural pH. However, studies

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based on this assumption have reported no such effects when skim milk was heated at pH

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values lower (6.3-6.5) or higher (6.9-7.1) than milk’s natural pH; in fact all heated milks

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showed extended rennet clotting times and produced very weak gels compared to unheated

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milk.8,22 Anema and coworkers (2011)45 studied the early stages of enzyme-induced micelle

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destabilization by renneting a very dilute suspension of skim milk (20 µL milk was added to

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1 mL of calcium-imidazole buffer) heated at pH values between 6.5 and 7.1. They found that

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compared with native casein micelles from unheated milk, the κ-casein depleted micelles

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(milk heated at pH 7.1) showed rapid destabilization and the WP-coated micelles (milk

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heated at pH 6.5) were the slowest to destabilize. However, upon enzyme treatment of

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undiluted milk, all heat-treated milks exhibited impaired rennet coagulation and formed much

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weaker gels when compared with unheated milk. These results suggest the role of other

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factors, besides the reactivity of casein micelle surfaces, in impaired rennet clotting of heated

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milk. These other factors will be discussed in greater detail in the sections that follow.

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Figure 1 shows the effect of heating at different pH values on the elastic modulus of renneted

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skim milk monitored over time. It was surprising to note that milk heated at pH 7.1 in which

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casein micelles were almost devoid of attached WPs (all of which existed as serum WP/κ-

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casein complexes) either formed very weak gels22 or mostly failed to clot.8 To exploit this 10 ACS Paragon Plus Environment

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interesting protein distribution in milk heated at alkaline pH values in which nearly all of the

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WPs are found in the serum aggregated with κ-casein, Menard and coworkers46 produced

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rennet gels from milk heated at 90°C for 30 s at pH values ranging from 7.1 to 8.3. In spite of

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the negligible amounts of WPs attached to micelle surfaces, they found that milks heated at

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alkaline pH values failed to form a good coagulum. Other studies also reported poor rennet

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clotting properties of milk heated at pH values around 7.5.47 In a 2006 review, Guyomarc’h4

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suggested that alkaline heat-treatment of milk could be a potential means of retaining whey

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proteins in cheese curd. However, it appears from these later studies that severely heated

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milk shows impaired rennet clotting no matter how the denatured WPs are distributed

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between the colloidal and serum phases.8,22

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For a detailed investigation of the individual contribution of micelle and serum

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components of heated milk to its impaired rennet coagulation properties, Kethireddipalli and

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others8 formulated a series of serum exchange experiments (see figures 2 and 3) as follows.

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(i) Casein micelles from milk heated (90°C, 10 min) at one of the three pH values, 6.3, 6.7 or

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7.1 were centrifugally separated and resuspended in the native serum of unheated milk.

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(ii) Native casein micelles from unheated milk were redispersed in each of the sera obtained

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from milks heated at these three pH values; the sera differ in the concentrations of WP/κ-

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casein complexes (pH 6.3 < 6.7 < 7.1) and Ca2+ (pH 6.3 > 6.7 > 7.1). (iii) Native micelles

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from unheated milk were suspended in the ultrafiltrates obtained from each of the above

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heated milks; these ultrafiltrates do not contain serum protein complexes but, being subjected

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to different heating pH values differ in the concentrations of ions, especially Ca2+ (pH 6.3 >

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6.7 > 7.1). Finally, (iv) native micelles from unheated milk were redispersed in each of the

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sera obtained from pH-altered heated milks after dialyzing these sera against unheated milk,

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mainly to restore the ionic equilibrium that was altered by heating; these sera are similar in

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[Ca2+] and differ only in the concentration of WP/κ-casein complexes (pH 6.3 < 6.7 < 7.1). 11 ACS Paragon Plus Environment


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From the results of this study it was concluded that impaired rennet clotting properties of

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heated milk can be attributed to complex interactive effects among the following three

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factors: (i) casein micelles with surfaces (and possibly interior) modified by heat due to

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attachment of WPs (at pH 6.3 and 6.7), due to heat-induced dissociation of caseins, or due to

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precipitation of calcium phosphate onto micelles; the latter two effects are predominant in

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milk heated at pH 7.1; (ii) serum WP/κ-casein complexes; and (iii) other dialyzable serum

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components. A very interesting finding of this study was that complete removal of serum

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protein complexes (by ultrafiltration) restored clotting of native casein micelles to a large

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extent, but surprisingly clotting was also restored when serum was dialyzed against unheated

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milk even if these complexes were still present. The reason(s) however are poorly

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understood.

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The Guelph study for the first time established the direct role of serum protein

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complexes on impaired rennet clotting of heated milk.8 To examine the exact mechanism

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underlying this phenomenon, native casein micelles were separated from unheated milk and

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resuspended in the serum of heated milk (contains WP/κ-casein complexes), treated with

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rennet, and at different extents of CMP removal the concentration WP/κ-casein complexes in

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the serum was determined using size-exclusion chromatography.48 As the enzyme reaction

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progressed, the soluble complexes of WPs and κ-casein were found to increasingly bind to

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native micelles so that just before the onset of clotting nearly 50% of the complexes were

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micelle-bound (see figure 4). It did not seem to matter if the serum was previously dialyzed

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(versus unheated milk) or not; both these micelle/ heated serum mixtures exhibited this

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phenomenon. Paradoxically, the rennet clotting times and elastic moduli of the gels were

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almost completely restored (to values close to that of unheated milk) when the serum was

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simply dialyzed against unheated milk.8 This raises the question if steric hindrance from the

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bound WPs in fact causes clotting impairment of these micelles or if there are distinct ways in 12 ACS Paragon Plus Environment

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which heat-induced and rennet-induced binding of WPs influence aggregation of casein

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micelles. Not only native casein micelles from unheated milk, but also micelles from milks

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heated at any of the three pH values were found to be capable of binding more serum protein

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complexes during the course of renneting. Further studies will be needed to determine the

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importance of these interactions between casein micelles and WP/κ-casein complexes on the

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overall renneting behavior of heated milk systems.

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It now seems reasonable to assume that casein micelles that were subjected to higher

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temperatures, say ≥ 75°C, are not the same structural entities that they were in the original

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fresh unheated milk even if they were heated in the absence of WPs. Precipitation of calcium

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phosphate possibly onto CCP nanoclusters, dissociation of caseins (or their deposition onto

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micelles as is known to happen when heated milk is dried into powder), and other possibly

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unknown reactions could all significantly change the micelle from its native conformation so

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that its aggregation behavior in the presence of rennet is also altered. Milk that was

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reconstituted from low heat skim milk powder (SMP) was found to produce weaker rennet

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gels compared with those of raw milk of similar overall composition48,49 and similar whey

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protein nitrogen index values.50 Using diffusing wave spectroscopy, Kethireddipalli146 found

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that casein micelles in reconstituted SMP did not fully recover their native conformation (as

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in raw milk) even after prolonged rehydration times. The light scattering properties and the

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specific manner of aggregation of the reconstituted micelles were significantly different from

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those of native micelles in fresh cow’s milk. The scientific knowledge gained so far on

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enzymatic coagulation of thermally processed milk should form the basis for developing

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better strategies and technological means for production of good quality and novel cheese

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types from such milk.

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1 MEANS TO RESTORE RENNET COAGULATION PROPERTIES OF THERMALLY 2 PROCESSED MILK 3

Various attempts to restore rennet clotting properties of heated milk date back to the

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1980s and 1990s. A considerable gap therefore exists between these past efforts and current

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knowledge on the renneting chemistry of heated milk. Earlier attempts were mostly

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concerned with manipulation of the mineral balance in milk with particular emphasis on

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increasing the concentration of ionic calcium. A dynamic equilibrium exists between the

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minerals and caseins associated with the colloidal and serum phases in milk and this

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equilibrium is quite critical to cheese making and affects nearly all aspects of cheese

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production. We have already discussed how both Ca2+ and CCP influence rennet coagulation

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of milk. Preacidification of cheese milk by lactic cultures, the pH of whey at drainage, and

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other curd handling procedures (such as cooking and washing) together determine the mineral

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content of cheese and therefore its texture and functionality. A considerable proportion of

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calcium and phosphate in cheese are present in insoluble/colloidal forms and determine the

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buffering properties of cheese.51

16 17 Calcium chloride supplementation to cheese milk 18

Addition of CaCl2 to cheese milk is a routine practice and the simplest of

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technological means available to speed up rennet clotting and increase curd firmness.

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Calcium ions favor aggregation by screening the negative charge on casein micelles and

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increase gel firmness by cross linking the phosphocasein molecules. Up to 0.1-0.2 g L−1 of

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CaCl2 is sufficient to obtain desired clotting parameters in pasteurized milk, but it is common

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to add up to 0.3-0.6 g L−1 to milk that was subjected to more intense heat treatments.52,53

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Maintaining adequate concentrations of ionic calcium is especially important in milk heated

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at alkaline pH in which there is considerable precipitation as calcium phosphate.4 Addition of 14 ACS Paragon Plus Environment

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up to 17.5 mmol kg‒1 of Ca2+ (~ 0.2 g L−1 CaCl2) causes an increase in buffering capacity

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between pH 4.5 and 5.5.54 The added Ca2+ reacts with the serum phase phosphate and citrate

3

and the ions are transferred to the micelle phase as salts of calcium phosphate and calcium

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citrate.13,55

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Manipulating the pH of cheese milk Changes in milk pH both unheated and heated affect its mineral balance. In general,

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acidification of milk by bacterial activity, addition of acid, or by treatment with CO2

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progressively demineralizes casein micelles due to protonation of acid groups and eventually

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releases individual casein molecules (at temperatures < 20°C) that are naturally bound to

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colloidal calcium and magnesium through phosphoserine and carboxyl groups.56-58

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Depending on the extent of acidification there are decreases in solubility, hydration, and zeta

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potential of caseins which eventually lead to their precipitation if acidification is rapid or

14

gelation if it proceeds more gradually without agitation as in yogurt production.

15

Several studies based on the manipulation of pH of thermally processed cheese milk

16

have reported at least a partial restoration of its rennet coagulation properties. The various

17

means that were adopted include: (i) acidification to pH values below 6.2, (ii) pH cycling, and

18

(iii) a combination of heating milk at alkaline pH, pH cycling, and CaCl2 addition.3 When

19

heated milk is acidified to pH below 6.2, clotting is faster and curds are firmer.53,59 This

20

effect is attributed to increased ionic calcium concentration from partial solubilisation of

21

CCP, reduced electrostatic repulsion between casein micelles due to their decreased net

22

negative charge, and increased activity of chymosin at lower pH values (being highest at pH

23

5.5). One of the drawbacks of this method is the retention of higher amounts of rennet in

24

cheese curd due to increased association of the enzyme with para-casein micelles60 and this 15 ACS Paragon Plus Environment


1

unfavorably enhances proteolysis during cheese ripening causing problems of flavor and

2

texture development.61-63 Retention of rennet is especially significant in conventional

3

Cheddar procedures where acidification by cultures is very slow and begins post renneting

4

unlike in soft cheeses where acidification begins prior to renneting and mostly continues

5

throughout63. Also, when Cheddar cheese was produced from milk heated at 90°C for 0.5 ‒ 1

6

min and renneted at pH 5.8 or 6.2 clotting improved and cheese yields were higher, but the

7

suppressed growth of lactic acid bacteria lead to insufficient flavor development.64,65 Any

8

effects of acidification on casein micelle structure may also have consequences on rennet

9

clotting and cheese making properties of the acidified heated milk.

10

pH cycling was proposed as an efficient alternative to increasing ionic calcium levels

11

in heated milk without causing enzyme retention and proteolysis in cheese curd.53,66 Milk is

12

acidified to pH values between 5.1 and 6.3, stored between 5 and 20°C for a time period

13

ranging from 2 to 24 h, and then reneutralized to pH 6.5 ‒ 6.6 prior to renneting.3 During the

14

acidification step of the pH cycle considerable amounts of both the original colloidal and the

15

heat-induced forms of calcium phosphate are solubilized and are only partly reformed upon

16

subsequent neutralization, probably with composition and properties similar to the original

17

CCP.57 Increased concentration of Ca2+ reported in the reformed milk suggests that not all

18

Ca2+ is returned to the colloidal phase upon reneutralization.53 The higher [Ca2+] and

19

reformation of CCP are indicated as factors contributing to the partial restoration of rennet

20

coagulation in heated milk that was pH cycled; clotting time was found to be more responsive

21

to the cyclic pH treatment than curd firmness. By heating milk at alkaline pH ~7.3 combined

22

with pH cycling, faster curd firming rates and slightly firmer gels could be obtained, but the

23

clotting properties of the treated milks were far from those of unheated milk.67

24

Lucey and coworkers68 studied the acid-base buffering and rennet coagulation

25

properties of unheated milk that has been cold acidified with 0.5 M HCl to pH values 16 ACS Paragon Plus Environment

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between 6.5 and 4.6 and reneutralized with 0.5 M NaOH to a final pH of 6.6. The findings of

2

this study may be applicable to heated milk as well. The reformed milks, those that were

3

acidified to pH values < 5.5, showed a reduction in buffering peak at pH ~ 5.1 and an

4

elevated Ca2+ activity, both of which suggest reduced content of CCP and/or formation of

5

calcium phosphate with a different chemical composition. The pH induced structural changes

6

in the casein micelle were therefore not totally reversible. The shorter clotting times and

7

higher gel firmness of the reformed milk upon renneting were mainly attributed to the

8

elevated Ca2+ activity.

9

Interestingly, in contrast to pH cycling by acidification, a pH reversible CO2 treatment

10

was found to completely restore the mineral and protein balance in reformed unheated milk

11

without causing any changes in the size or zeta potential of casein micelles and without

12

forming any insoluble salt complexes.69-71 There were however irreversible changes in the

13

buffering properties (the maximum buffering value dB/dpH was depressed and the peak was

14

slightly wider) and micelle water hydration, and a significant improvement in rennet

15

coagulation when acidification of milk with CO2 was carried to pH < 5.8. Unlike in the case

16

of milk cold acidified to pH 6.6 with 0.5 M HCl in which the altered buffering properties are

17

mainly due to formation of calcium phosphate precipitates, 68 no insoluble salt complexes

18

were formed during carbonation. It was therefore suggested that the original CCP was

19

changed to other salt forms possibly leading to a re-organization of the casein micelle surface

20

and thereby affecting their colloidal stability.72 To date CO2 reversible acidification has not

21

been applied to heated milk but appears to have the potential to improve rennet-clotting

22

properties of the heat-altered micelles either alone or in combination with other technological

23

adaptations.

24 25



1 Concentration by ultrafiltration (UF) 2

Membrane processing methods are widely applied to milk for a number of purposes

3

such as concentration, protein separation and standardization, demineralization, and removal

4

of bacteria. Of these, ultrafiltration (UF) and to a lesser extent microfiltration (MF) may be

5

used as a pre-treatment for cheese milk.1 UF of cheese milk concentrates the fat globules,

6

caseins, colloidal minerals, and whey proteins as the aqueous phase containing ions and

7

lactose (referred as permeate) is pushed through the membrane. Factors such as pH and

8

temperature of filtration influence the extent of changes in CCP, aqueous phase composition,

9

and therefore micelle structure and function. These effects of UF are well documented.73-75

10

UF technology is successfully used in the commercial production of Feta, fresh acid cheeses

11

such as Quark, and soft ripened varieties like Camembert and Blue. In the production of so

12

called cast Feta, milk is concentrated 4.0 – 5.5 fold to a composition similar to the final

13

cheese to be made (pre-cheese), acidified with starter culture, and set with rennet with no

14

subsequent cutting and drainage. This process creates a closed cheese with no mechanical

15

openings but otherwise produces a good quality product with the advantage of greatly

16

increased yield due to retention of whey proteins. Lower UF concentrations combined with

17

heating are used to produce a more typical open structured Feta with yield advantages due to

18

retention of denatured whey proteins. Brie and Camembert in large scale operations are often

19

made from milk concentrated 1.5 to 2.5 times. The general principles of UF concentration

20

and its effects on milk’s physico-chemical and rennet coagulation properties can be applied to

21

both unheated and heated milk. This section will therefore address the fundamental aspects

22

of enzymatic coagulation of UF concentrated milk with specific reference to thermally

23

processed milk.

24

Rennet gels produced from UF concentrated milk are considerably stronger and

25

exhibit faster gel firming rates. Although reports on the effects of UF on rennet clotting times 18 ACS Paragon Plus Environment

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are not consistent, it is generally agreed that flocculation in UF retentates when compared

2

with skim milk is initiated at relatively smaller extents of κ-casein hydrolysis.76-78 However,

3

based on recent observations from our lab (unpublished data), no significant differences were

4

found between UF concentrated milk and the original skim milk (at natural pH) either in

5

clotting times or in the extent of CMP removed at the onset of flocculation. These findings

6

emphasize that the faster rate of gel formation and the increased firmness of gels from UF

7

milk are mainly due to increased micelle concentration and the altered mineral balance and

8

buffering capacity. The buffering capacity of retentates increases proportionally with protein

9

concentration and micellar calcium phosphate content79 and is shifted towards acidic pH

10

values at concentration factors >5.80,81 This is why longer fermentation times are required to

11

reduce pH in UF retentates, but slower fermentation of cheese milk often increases residual

12

lactose which is undesirable as it is later fermented during early cheese ripening thus

13

producing acid flavored cheese.82,83 Following extensive microsyneresis, UF rennet gels

14

were found to be less coarse/thick and made of fine stranded protein network and this

15

microstructure was preserved during a 60-day storage at 13°C.76 The rapid strengthening of

16

bonds between casein strands was thought to prevent further rearrangements due to the high

17

volume fraction of casein micelles, and the large surface to volume ratio was implicated in

18

the higher water retention of UF cheeses. Besides concentration-induced effects, the casein

19

micelles can be irreversibly altered during the ultrafiltration process. Using diffusing wave

20

spectroscopy, Ferrer and coworkers84 have studied the effects of ultrafiltration on the

21

physico-chemical properties of casein micelles. UF concentration, about 5-fold, induced

22

losses in insoluble calcium phosphate (up to 5 mM Ca2+ lost from CCP)84,85 with no increases

23

in the amount of soluble caseins.80,84 Changes in refractive index of casein micelles from the

24

concentrated milk were attributed to a localized spatial redistribution of mass within the

25

micelle in response to a decreased level of CCP. Such protein re-arrangements within the



1

casein micelle were implicated in their slightly altered surface reactivity as reflected by

2

changes in renneting functionality.87 Upon reconstitution of the UF-altered micelles (5X

3

concentration) to similar volume fraction and ionic environment as in original skim milk, the

4

aggregation rate of renneted micelles was enhanced but the firmness of the gel was reduced.84

5

The authors mainly attributed this effect to loss of calcium from the micelle and subsequent

6

changes to its internal structure as evidenced by light scattering studies. At higher

7

concentrations when the UF retentate forms a soft solid, the micellar structure can be

8

distorted and irreversibly damaged.86-88 UF cheeses are also reported to have insufficient

9

flavor development due to reduced rates of casein proteolysis possibly resulting from the

10

11

retention of inhibitors of chymosin and plasmin in the UF concentrates.89,90 The improved rennet coagulation and cheese making properties of UF concentrated

12

milk are mostly offset by heating effects and vice versa (i.e, the impaired enzymatic clotting of

13

heated milk is improved by UF concentration). Prior heat-treatment of milk has the additional

14

benefit of inclusion of WPs in the UF cheese due to concentration of both micelle-bound and

15

serum phase denatured WPs. In hard and semi-hard cheese varieties produced from UF

16

concentrated heated milk, there are problems associated with higher moisture retention, poor

17

ripening, and related flavor and texture defects; these effects are well documented.44,77,78,91-99

18

Therefore, even from heated milk the successful development of UF cheeses has been limited

19

to high moisture, low-pH, and unripened cheeses such as Quarg and Cast Feta. Feta made

20

this way produces a smoother curd and its processing directly into retail packages prevents

21

losses of fines.100 In the production of Thermoquarg, heat treatments up to 90°C that can

22

integrate up to 70% of WPs into cheese are known to produce superior quality product in

23

terms of texture, flavor, and mouthfeel. The concentration of heat-denatured WPs is also

24

positively correlated with the increased viscosity of fresh cheeses.101-103 The effects of

25

combined UF and heat treatment on rennet coagulation and cheese making properties of milk 20 ACS Paragon Plus Environment

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differ depending on whether milk is heated before or after UF. Heat treatments prior to UF

2

were found to produce weaker rennet gels with slower curd firming rates when compared

3

with heating the UF concentrate.78 This suggests important differences in protein and mineral

4

interactions between heated milk and heated UF retentate. Contextually, heat treatment of

5

milk (above 75°C) that was recombined from milk protein concentrate and containing 20%

6

total solids was also found to lower the elastic moduli and fracture stress of the corresponding

7

rennet gels.104 pH cycling of heated milk either before or after UF was reported to further

8

impair its rennet coagulation properties. Complex mechanisms were implicated, probably

9

involving equilibrium of milk salts, state of colloidal calcium phosphate, and nature of

10

interactions between whey proteins and κ-casein.78 Optimization of heat treatment, the UF

11

concentration factor, and other processing treatments could be used with some benefit in the

12

production of WP enriched cheese with desired eating quality and functionality. Using a

13

combination of ultrahigh temperature (UHT) treatment and UF/MF concentration, Bulca and

14

coworkers105 have demonstrated that it is possible to optimize protein composition of UHT

15

milk to obtain rennet coagulation times and gel firmness similar to those of pasteurized milk.

16

They have found two discrete combinations of casein and heat-denatured WP concentrations

17

that fit these requirements: 3.4% casein/0.01% WP and 6.4% casein/0.65% WP. It was

18

suggested that a broader range of protein combinations may be feasible if gentler heating

19

conditions are used.

20 21 CHEESE PRODUCTION FROM THERMALLY PROCESSED MILK 22

As discussed in the previous sections, the prolonged rennet clotting times and

23 formation of weak curds from thermally processed milk could be alleviated to a great extent by 24 adopting one or more of the technological means, albeit with the means themselves posing 25 severe limitations during the subsequent cheese making steps. Of greater concern are the



1 problems associated with the processing ability of curds, curd structure, and cheese ripening all 2 of which unfavorably influence cheese composition, functionality, and organoleptic qualities. 3 Table 1 shows the characteristics of various cheeses produced from heat-treated milk that has 4 been subjected to one or more of the additional treatments to restore rennet clotting. It can be 5 seen that by using milder heating conditions together with careful selection of restorative 6 treatments and suitable process adaptations, it is possible to produce cheeses with desirable 7 organoleptic qualities while taking advantage of the higher protein and yield recoveries. 8 Greater success in this respect can be achieved with respect to fresh acid and softer cheese 9 varieties than the traditional firmer types. Below, we will briefly discuss some of the common 10 product defects and problems that come up during the production of cheese from thermally 11 processed milk. 12 13 Longer set-to-cut times 14

The delayed curd cutting times encountered during cheese production from heated

15

milk are due to prolonged rennet clotting times and lower curd firming rates which can be

16

corrected to a large extent through manipulation of renneting pH and/or Ca2+ supplementation

17

to cheese milk. But this is only a part of the solution. Curd firmness/structure at the point of

18

cutting is known to affect cheese composition, (e.g., moisture and fat contents), cheese

19

quality and ripening.106 In spite of restoring gel cutting times and firmness, the

20

microstructure of heated milk curds could differ significantly from that of unheated milk

21

which could further result in less than desirable cheese composition, ripening characteristics,

22

and eating quality.

23

24


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1 Increased curd moisture retention 2

Poor syneresis or the reduced ability of the cut rennet curds to expel serum causes

3

increased retention of moisture in heated milk cheeses.107-110 The heat-denatured whey

4

proteins not only have higher water binding capacities but their attachment to casein micelles

5

further impedes micelle fusion and shrinkage of the para-casein matrix.111,112 Increased curd

6

water retention results in reduced firmness and fracture stress.94,107,108,110 This is especially

7

undesirable for the texture of hard and semi-hard cheeses such as Cheddar, Provolone, Gouda

8

or Havarti113, but has been used with some advantage in reduced-fat Cheddar in moderating

9

its otherwise undesirably firm texture.114 On the same lines, softer cheese varieties also

10

benefit from the higher moisture contents. For example, in fresh acid cheeses like Quarg or

11

Fromage Frais, the increased water-binding capacity, the aggregation of WPs which introduce

12

strong covalent bonds, and a low proteolytic activity were found to be especially beneficial to

13

cheese yield, texture, and taste.103,115,116 Retention of higher amounts of moisture in cheese

14

has further consequences on the development of cheese pH and ripening.

15 16 Decrease in cheese pH and buffering capacity 17

Due to impaired syneresis, higher amounts of lactose are retained in the curd of heated

18

milk and its conversion to lactic acid by starter cultures makes these curds more acidic.

19

Cheddar made from milk pasteurized at higher temperatures (82 to 87°C, 26 s) showed

20

significant reduction in pH and retained higher amounts of total lactate and D (‒) lactate114,

21

the latter form being the precursor of calcium lactate crystals in aged cheese. The appearance

22

of a white calcium lactate crust on the surface of cheese is sometimes considered a visual

23

defect, but has no health or flavor implications. Cheese pH can be controlled to some extent

24

by intensifying cooking and/or curd washing procedures to remove lactose. Higher content of

25

lactic acid together with reduced concentrations of protein and calcium (due to higher 23 ACS Paragon Plus Environment


1

moisture content) also decrease the buffering capacity of cheese.117 These effects on cheese

2

pH and buffering capacity have further negative consequences on proteolysis during the

3

ripening process.

4 5 Effects on cheese texture and functionality 6

The binding of heat-denatured WPs with caseins and associated increases in curd

7

moisture retention result in curds that are soggy and ragged in appearance with poor matting

8

ability.3 Due to impaired fusion of curds, the resulting cheese tends to have an increasingly

9

porous matrix. Cheddar curds produced from heated milk are generally known to be crumbly

10

and hard to process mechanically.52,65,108 Poor melting and stretching properties and reduced

11

oiling off in cheeses like Cheddar and Mozzarella made from heated milk are attributed to

12

increased interaction between fat and protein52, altered protein matrix of the curd, and binding

13

of calcium and water by the denatured WPs.118 The coarse and mealy texture of semi-hard

14

cheeses is attributed not only to the interaction of denatured WPs with casein micelles but

15

also to the decreased cheese pH when these cheeses are produced from high heat-treated

16

milk.113

17 18 Heat-induced changes in cheese ripening 19

Any effects of heating or other technological treatments on the microbiological and

20

biochemical characteristics of milk/cheese are reflected in the protein degradation processes

21

and flavor formation during cheese ripening. Lipases and proteases from the indigenous milk

22

microflora, NSLAB, and the lysed starter cells contribute significantly to the cheese ripening

23

process. Heat-treatment of cheese milk decreases the diversity and numbers of raw milk

24

microflora and NSLAB both of which are extensively involved in the breakdown of caseins.

25

Additionally, the lipoprotein lipase present naturally in raw milk is partially or completely 24 ACS Paragon Plus Environment

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inactivated by thermization and pasteurization, respectively, and the esterases from starter

2

bacteria are the major lipolytic agents in cheeses produced from such milk.119 The

3

acceleration or inhibition of proteolysis in heated milk cheeses affects attributes such as curd

4

fusion, melting properties, bitterness, and flavor. The thermophysical properties of cheese

5

(meltability and flowability) are dependent not only on its fat content120 and melting point,121

6

but are also strongly influenced by the breakdown of para-casein matrix during cheese

7

ripening.122

8 9

Heat-induced biochemical changes in cheese such as increased moisture content, reduced pH, and altered mineral balance and buffering capacity all influence the ripening

10

process in complex ways. Any additional pre-treatments to cheese milk such as UF or pH-

11

cycling further increase the complexity of these effects on texture and flavor during ripening.

12

For example, the high moisture content of heated milk cheeses is suggested to enhance the

13

activity of plasmin and residual chymosin in the curd.123 The residual chymosin retained in

14

the cheese curd contributes to the slow breakdown of αS1-casein which has several chymosin

15

susceptible bonds and to some extent αS2- and β-caseins. Chymosin mainly contributes to

16

primary proteolysis that generates large and intermediate sized peptides which are

17

subsequently hydrolyzed by proteases from starter and non-starter LAB.124,125 Heat-treatment

18

per se does not influence chymosin activity, because the enzyme is added to cheese milk after

19

heating. However, the presence of both native and denatured WPs in the curd can cause

20

decreased levels of hydrolysis of αS1-casein126,127 resulting in atypical maturation in some

21

cheese types.128 Plasmin, which readily hydrolyzes β-casein, is part of a complex enzyme

22

system in milk which is comprised of the precursor called plasminogen (PG), plasmin, and

23

plasminogen activator (PA) all associated with casein micelles and incorporated into the

24

rennet-coagulated casein curd. Being relatively heat-stable, plasmin survives pasteurization

25

temperatures and in fact is further activated due to inactivation of an inhibitor of PA, but 25 ACS Paragon Plus Environment


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1

heating milk at 80°C for 10 min mostly inactivates plasmin due to interaction of the

2

components of plasmin system with β-Lg129. The proteolytic action of plasmin on β-casein

3

contributes to improved cheese flavor and overall quality130 and its heat-induced inactivation

4

affects breakdown of β- and to some extent αS2-casein.131 In cheeses produced from heated and UF concentrated milk, the whey proteins that are

5 6

integrated into cheese matrix in denatured and native forms, respectively, are most likely not

7

susceptible to proteolysis during cheese ripening.65,118,132,133 Heat-treatment of milk is also

8

known to induce desirable changes in the flavor profile of cheeses.113,134-136 It may be useful

9

for the reader to refer to the general biochemical aspects of cheese ripening which have been

10

extensively reviewed.137-143

11 12 ALTERNATIVE MEANS OF INCORPORATING WHEY PROTEINS INTO CHEESE 13 14 Addition of whey protein particles

Whey protein products are increasingly recognized as valuable nutritional and

15

16 functional ingredients with their steadily increasing prices now on par with casein-based 17 ingredients. In addition to thermal processing and membrane concentration of cheese milk, 18 used alone or in combination, cheese whey could be recycled back into cheese milk or curd 19 matrix following thermal modification and mechanical shearing to generate microparticulated ®

20 whey. Commercial WP particulates (WPP) such as Simplesse and Dairy-Lo

TM

are finding

21 increasing applications especially in the production of soft cheeses and processed cheese 22 products.

Most importantly, microparticulated whey proteins mimic the structural and

23

24 functional properties of fat in cheese by acting as non-interacting fillers within the cheese 144

25 matrix.

WPP ranging in size range between 1 and 10 µm could be readily integrated into 26 ACS Paragon Plus Environment

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115

1 cheese matrix with a typical network pore size of about 10 µm.

Addition of 1% Simplesse

2 was found to significantly improve the texture and thermophysical properties (meltability and 3 flowability) of fat-reduced semihard Gouda cheese.145 The same authors have suggested that 4 WPP (here, 0.01 to 3 µm in size) in cheese mainly functioned by structurally weakening the 5 casein matrix by acting as ball-bearing points between protein strands thereby minimizing 6 protein-protein interactions. This is unlike milk fat which acts both as structure breaker and as 7 lubricant (once liquefied) between the attached casein layers. In model processed cheese 8 spreads, the addition of predenatured and microparticulated WPs produced significantly softer 9 cheese with increased meltability in contrast to native whey proteins which contributed to 10 increased cheese firmness and reduced meltability;146 the latter become heat-denatured during 11 processing and become actively integrated with the casein matrix. When adding WPP 12 especially to firm cheese types, certain adjustments in cheese processing should be made to 13 mitigate increases in moisture and also to remove other milk constituents such as lactose that 14 get recycled with WPP. Examples of such process modifications would be to increase 15 fermentation/renneting temperature, decrease curd grain size, intensify curd washing and 16 cooking conditions, or choose shorter brining times.

115

Depending on the dry matter content of

17 cheese, a maximum of 0.7, 0.5, and 0.3 g of WPP per 100 g of milk has been suggested for 18 soft, semi-hard, and hard cheese types, respectively.115 Reports on the effects of WPP on 19 rennet coagulation properties of milk are contradictory,106,147,148 but any negative effects could 20 be mitigated by adopting one or more of the restorative processes discussed earlier. 149

21 Particulated whey proteins were found to be stable during the ripening of cheese. 22 23 High pressure processing of cheese milk 24 25

Commercial applications of high pressure processing (HPP) emerged as possible means of food preservation in the 1990s in Japan. HPP exerts antimicrobial effects without



1

impairing nutritional quality. In fact, in a variety of products including jams, jellies, juices,

2

sauces, guacamole, and cooked ham, HPP has become the only appealing processing

3

alternative to heat treatment.150 But, there are not as yet commercially available HPP dairy

4

products mainly due to the high capital investment required to set up HPP equipment.151

5

Considerable progress was made in recent years on the effects of HPP on milk

6

proteins and enzymes and its rennet and acid coagulation properties. Subjecting milk to static

7

pressures up to 250 MPa increases the size of casein micelles by about 30% mainly due to

8

aggregation of casein micelles; above this limit micelles disintegrate with up to 50%

9

reduction in size depending on the magnitude of applied pressure.152-154 Pressure-induced

10

fragmentation of casein micelles has been attributed to (i) solubilisation of colloidal calcium

11

phosphate (including heat-precipitated calcium phosphate in heated milk), and (ii) hindrance

12

to hydrophobic, hydrogen and/or van der Waals interactions.155 Whey proteins when treated

13

with sufficiently high pressure become denatured and aggregate with casein micelles. β-Lg is

14

the most pressure-sensitive whey protein denaturing at pressures under 200 MPa.152,156-158

15

Renneting properties of pressure-treated milk have been studied by a number of

16

authors. Subjecting milk to lower pressures from about 100 to 250 MPa gradually decreases

17

rennet coagulation and set-to-cut times. But, when treatments exceed 250 MPa the RCT and

18

cutting times begin to increase gradually so that at about 400 MPa, the RCT and cutting times

19

are similar to those of untreated milk, and at even higher pressures, coagulation and cutting

20

times are delayed beyond those of control milk.159 Two opposing mechanisms were

21

suggested: disruption of casein micelles at treatments up to 250 MPa decreases coagulation

22

and cutting times whereas above this limit these times are prolonged due to pressure-induced

23

denaturation of WPs and their association with casein micelles. When pressure treatments are

24

high and sufficiently long, increased yields of cheese curd can be obtained through

25

incorporation of WPs and concomitant increases in moisture content.160,161 Huppertz and 28 ACS Paragon Plus Environment

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1

coworkers161 also showed that impaired rennet coagulation properties of heat-treated milk

2

(90°C, 10 min) can be restored and up to 15% increases in cheese curd yield can be obtained

3

by HP treatment of heated milk at economically feasible treatment times.

4

A few cheese varieties have been produced from HP-treated milk: Cheddar, 400 and

5

600 MPa162,163, Cheddar, 345−676 MPa164, goat’s milk cheese, 500 MPa165,166, reduced-fat

6

cheese, 400 MPa167, Camembert, 500 MPa168 and Queso Fresco, 400 MPa.169 In general, HP-

7

treatment of milk which is in a way similar to heating, produced cheeses with increased yields

8

and WP content, higher moisture contents, decreased numbers of raw milk microflora,

9

reduced activity of indigenous milk enzymes, and increased rates of proteolysis. In a recent

10

Cheddar ripening study with HP-treated milk, Voigt and coworkers163 have reported initial

11

decreases in NSLAB counts with complete recovery and growth within 60 days of ripening,

12

decreased plasmin activity, increased activity of lipoprotein lipase unlike in heated milk, and

13

an overall enhanced rate of primary proteolysis with no significant changes in the later stages

14

of ripening. The key question still remains, that is with its cost and scale limitations, if HP

15

treatment of cheese milk could be made commercially viable. An assessment of cheese

16

flavor and consumer acceptability is also warranted.

17 18 Other means of enriching cheese with whey proteins 19

One way of adding WPs to cheese would be to reconstitute creams (or create fat

20 globules) from milk fat or any other vegetable oil for that matter by using whey proteins as 21 emulsifiers and add these creams back into cheese milk. This provides a unique opportunity to 22 incorporate whey proteins into cheese milk with none of the adverse effects on its rennet 23 coagulation properties that are commonly encountered with the use of thermally processed 24 milk. However, for the production of good quality cheese (comparable to traditional whole 25 milk cheese) from the recombined milk, it is of utmost importance that the created WP-



1 stabilized fat globules have size distributions nearly identical to the native fat globules in cow’s 2 milk, i.e., roughly ranging from 1 to 10 µm with a 4 µm average diameter. In our Guelph 3 laboratory, we have successfully formulated such creams containing inert fat globules using 4 butter/butteroil and commercially available whey protein concentrates; studies are ongoing on 5 the characterization of these creams and production of Cheddar cheese from milk that was 6 recombined from skim milk and the prepared creams. These creams are unlike those that are 7 conventionally produced by homogenizing blends of skim milk powder or milk protein 8 concentrates with fat which have poor cheese making properties because the globules are much 9 smaller than native fat particles and also because they are stabilized with caseins that actively 10 participate in cheese gel formation. We are also currently experimenting to determine the 11 maximum concentration of whey proteins which can be loaded onto the fat interface without 12 affecting droplet sizes or their functionality in cheese. 13 14

To conclude, production of cheese from thermally processed milk is not

15 straightforward due to the complexity of physicochemical events that occur during heating and 16 which influence rennet coagulation and cheese making properties of the heated milk in further 17 complicated ways. Casein micelles, the principal clotting material in cheese milk and the main 18 structural and functional components in cheese are significantly altered when milk is heated or 19 subjected to any of the corrective measures thereof. Research in this area has progressed and 20 much knowledge is now at hand about the fundamental aspects of enzymatic coagulation of 21 heat-treated milk, but there are considerable gaps in applying this knowledge to understand the 22 effects on cheese texture, functionality and ripening. The current knowledge base should be 23 exploited for creation of cheeses with specific attributes and functional applications.

24


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167. Molina, E.; Alvarez, M.D.; Ramos, M.; Olano, A.; Lopez-Fandino, R. Use of high

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168. Linton, M.; Mackle, A.B.; Upadhyay, V.K.; Kelly, A.L.; Patterson, M.F. The fate of

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169. Sandra, S.; Stanford, M.A.; Meunier Goddik, L. The use of high-pressure processing in

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the production of Queso Fresco cheese. J. Food Sci. 2004, 69, 153-158.

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170. Kethireddipalli, P. The physico-chemical aspects of the impaired rennet coagulation

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properties of heat-treated milk. Dissertation, University of Guelph. 2011, 53-54.

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171. Banks, J.M. Elimination of the development of bitter flavor in Cheddar cheese made

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from milk containing heat-denatured whey protein. J. Soc. Dairy Technol. 1988, 41, 37-41.

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172. Banks, J.M.; Law, A.J.R.; Leaver, J.; Horne, D.S. The inclusion of whey proteins in

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cheese−an overview. In Cheese Yield and Factors Affecting its Control, Special issue,

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International Dairy Federation: Brussels, 1993; pp. 387-401.

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173. Guinee, T.P.; Pudja, P.D.; Mulholland, E.O. Effect of milk protein standardization, by

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ultrafiltration, on the manufacture, composition and maturation of Cheddar cheese. J. Dairy

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Res. 1994, 61, 117-131. 51 ACS Paragon Plus Environment


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174. Rynne, N.M.; Beresford, T.P.; Kelly, A.L.; Tunick, M.H.; Malin, E.L.; Guinee, T.P.

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Effect of exopolysaccharide-producing adjunct starter cultures on the manufacture,

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175. Marshall, R.J. Increasing cheese yields by high heat treatment of milk. J. Dairy Res.

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1986, 53, 313-322.

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curd for manufacture of reduced fat, Havarti-type cheese. J. Dairy Sci. 1998, 81, 16-24.

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177. Hougaard, A.B.; Ardo, Y.; Ipsen, R.H. Cheese made from instant infusion pasteurized

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milk: rennet coagulation, cheese composition, texture and ripening. Int. Dairy J. 2010, 20,

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449-458.

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178. Chromik, C.; Partschefeld, C.; Jaros, D.; Henle, T.; Rohm, H. Adjustment of vat milk

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treatment to optimize whey protein transfer into semi-hard cheese: A case study. J. Food Eng.

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2010, 100, 496-503.

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179. Benfeldt, C.; Sorensen, J. Heat treatment of cheese milk: effect on proteolysis during

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cheese ripening. Int. Dairy J. 2001, 11, 567-574.

16

180. Schafer, H.W.; Olson, N.F. Characteristics of Mozzarella cheese made by direct

17

acidification from ultra-high-temperature processed milk. J. Dairy Sci. 1975, 58, 494-501.

18

181. Kumar, S.; Kanawijia, S.K.; Kumar, S. Effect of different degree of heat treatments on

19

sensory and biochemical characteristics of buffalo Feta type cheese during ripening.

20



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182. Ghosh, B.C.; Steffl, A.; Hinrichs, J.; Kessler, H.G. Effect of heat treatment and

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homogenization of milk on Camembert-type cheese. Egyptian J. Dairy Sci. 1999, 27, 331-

3

343.

4

183. Shammet, K.M.; Mcmahon, D.J.; Ernstrom, C.A. Effect of acidification and heat-

5

treatment on the quality of white soft cheese from ultrafiltered whole milk retentate.

6


7

8

9

10

11

12

13

14

15

16

17

18

19

20



1

FIGURE CAPTIONS

2

3

Figure 1. Elastic moduli (Gʹ) of heated skim milk samples monitored for 3.5 h after rennet

4

addition: unheated milk (○), milk heated at pH 6.7 (□), 7.1 (∇), and 6.3 (◊). Insert shows all

5

heat-treated milk samples, but not unheated control. All samples were adjusted back to pH

6

6.7 before renneting. All treatments were replicated thrice, but results of a single sample are

7

shown.8

8

9

Figure 2. A schematic diagram showing the preparation of the micelle/serum re-dispersed

10

systems through the exchange of the serum fractions between unheated and heated skim

11

milk.170

12

13

Figure 3. A schematic diagram showing the preparation of milk mixtures containing native

14

casein micelles from unheated milk (UP) suspended in ultrafiltrate of heated milk (HUF) and

15

the undialyzed (HS) or dialyzed serum (HS) of heated milk.170

16

17

Figure 4. The WP/κ-casein complex peak in the elution profile obtained by SEC. (A) the

18

renneted serum of heated milk (HS) and (B) the centrifugal supernatant obtained from a

19

renneted suspension of native casein micelles from unheated RSMP in HS (UP/HS).

20

Renneting times were, 0 min (○), 30 min (●), 60 min (□) and 75 min (■). (C) The relative

21

area of the WP/κ-casein complex peak plotted as a function of percent CMP released during

22

renneting, for UPHS (●) and HS (■).48


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1


Table 1. Characteristics of cheeses produced from thermally processed milk Cheese type Cheddar64

Heating conditions 110°C, 60 s

Restorative treatments pH 5.8 prior to renneting

Composition and Yield +4.7% yield (dry basis)

Texture/ Flavor

Cheddar171

110°C, 60 s

pH 5.8; reduced amount of rennet

+7.5% yield

firmness comparable to pasteurized milk Cheddar; bitterness eliminated, but impaired Cheddar flavor

Cheddar52,172

85 or 90°C, 1 min

Cheddar66

91°C, 16 s

+3.61% yield at 85°C; +3.93% yield at 90°C +2.5% moisture +3.0% protein

poor melting and stretching; reduced oiling off; some flavor defects no information

Cheddar128

91°C, 16 s

pH 6.2; rennet reduced to 90% pH 5.5, stored overnight at 4°C, back to pH 6.2 Heating pH 7.5

Cheddar65

90°C, 30 s

Cheddar173

90°C, 30 s

reduced fat Cheddar107

72 to 88°C, 15 s

half fat Cheddar110

72‒87°C, 26 s

minor defects in moisture and texture; bitterness

+ protein yield no information despite higher losses of fines heating pH higher protein no information from 6.5 to recovery and 8.7 slight increase in moisture heating pH less crumbly than from from 7.1 to heated milk at normal 7.5 pH; typical Cheddar flavor with peptide profiles nearly similar to control Cheddar reduced firmness, but increased pH at little influence on milling 5.75 moisture and reduced calcium proteolysis or cheese or 5.35 and protein with grading scores heat and higher milling pH increased lower fracture stress, moisture and strain, and firmness; lower levels of reduced flowability and protein, fat and stretchability; altered degradation profiles free oil with heat treatments 55

ACS Paragon Plus Environment


72‒87°C, 26 s

increased moisture (~ 45% at 72°C to ~50% at 87°C), total lactate, and D(‒) lactate; reduced cheese pH

Cheddar119

raw, thermized and pasteurized cheese milk

Cheshire175

97°C, 15 s

decrease in diversity and numbers of indigenous microflora and free amino acids during ripening; lipoprotein lipase partly or wholly inactivated in thermized and pasteurized milk, respectively. no increase in texture and flavor moisture; +6.7% comparable to control protein, +0.7% fat, +4.5% yield

semi-hard cheeses113

72‒100°C, 15 or 120 s

reduced fat Havati176

85°C, 17 s

Havarti-type177

instant infusion at 72‒120°C for 0.2 s

4.5 to 9 mM increased Ca2+ added moisture; reduced pH

Gouda178

up to 85°C, 60 s

Danbo179

72 to 90°C, 15 to 60 s

up to 2X MF; Ca2+ added; coagulant adjusted CaCl2 added

half fat Cheddar174

pH 6.4; +Ca2+; intense cooking; Cheddaring UF after heating (18.5% protein and 14% fat); renneting and processing conditions adjusted

increased moisture, salt in moisture, and whey protein in cheese; lower ex-brine pH values; reduced rates of pH increase during ripening +2.5% protein +3.0% moisture

+6% protein in curd; 15‒30% decrease in permeate/whey protein loss


no significant effect on starter LAB or NSLAB; suited to higher moisture, short ripened, mild flavored Cheddar or Cheddar-like cheeses

poor curd fusion and lower yield (fracture); higher degree of primary proteolysis, but lower levels of small peptides, amino acids, and free fatty acids (due to lack of endogenous microflora) No information shorter texture compared to HTST; altered patterns of casein breakdown and peptide formation delayed proteolysis due to heat and MF; sensory quality and slicing behavior rated satisfactory lower levels of plasmin peptides with heat treatment

Page 56 of 62

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direct acidified Mozzarella180

80 to 130°C, 2 s

buffalo Feta type181

63‒70°C, 30 min

Camembert182

80°C, 3 min

Fresh acid cheese103

72‒92°C, 15 to 60 s

white soft cheese183 (precheese, 38% solids)

decreased moisture; up to +3.4% yield; no effect on fat recovery good quality Feta from 65°C for 30 min

pH 5.6

homogenize or not prior to heating

added WP and UF prior to heating 71.7°C, 16 s preacidified 76.7°C, 16 s at 2°C to 71.7°C, pH 6.0 prior 15 min to UF

higher moisture and whey protein content higher yield

76.7°C/ 16 s gave most acceptable cheese with higher calcium content

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16


enhanced firmness; no adverse effects on proteolysis and rheological aspects; mild flavored weak/pasty body, slightly acidic/bitter, and higher lipolysis rate with heat higher soluble protein during ripening; homogenization reduced sensory scores favorable consistency and texture from denatured WPs extended heating caused increased mealiness


1

FIGURES

2

Figure 1.

100

2.0 1.5 1.0

80

G' (Pa)

0.5 0.0

60

0

50

100

150

200

40

20

0 0

3

50

100

150

200

Rennet reaction time (min)

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19


Page 58 of 62

Page 59 of 62

1


Figure 2.

2

3 4 5 6 7 8 9 10 11 12 13 14 15



1

Figure 3.

2

3 4 5 6 7 8 9 10 11 12 13


Page 60 of 62

Page 61 of 62

1


Figure 4.

A.

0.12

0.09 100

% original peak area

Absorbance at 280 nm

0.06

0.03

0.00

B.

0.12

80

60

C.

0.09 40 0

0.06

20

40

60

% CMP released 0.03

0.00 40

2 3

60

80

100

120

Elution time (min)

4 5 6 7

8 9 10 11 12 13 14 15 16 17


80

100


1 2 3 4 5

The relative area of whey protein/κ-casein complex peak (in the elution profile obtained by sizeexclusion chromatography) plotted as a function of percent caseinomacropeptide released during renneting of the serum of heated milk, alone or with native casein micelles (from unheated milk) suspended in this serum.

6

135x90mm (300 x 300 DPI)

7


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Rennet Coagulation and Cheesemaking Properties of Thermally

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