Split Defect and Secondary Fermentation in Swiss-Type Cheeses - a Review David F.M

Split defect and secondary fermentation in Swiss-type cheeses - A review David F.M. Daly, Paul L.H. Mcsweeney, Jeremiah J. Sheehan

To cite this version:

David F.M. Daly, Paul L.H. Mcsweeney, Jeremiah J. Sheehan. Split defect and secondary fermentation in Swiss-type cheeses - A review. Dairy Science & Technology, EDP sciences/Springer, 2010, 90 (1), 10.1051/dst/2009036. hal-00895710

HAL Id: hal-00895710 https://hal.archives-ouvertes.fr/hal-00895710 Submitted on 1 Jan 2010

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Dairy Sci. Technol. 90 (2010) 3–26 Available online at: © INRA, EDP Sciences, 2009 www.dairy-journal.org DOI: 10.1051/dst/2009036 Review

Split defect and secondary fermentation in Swiss-type cheeses – A review

1,2 2 1 David F.M. DALY , Paul L.H. McSWEENEY , Jeremiah J. SHEEHAN *

1 Moorepark Food Research Centre, Teagasc, Fermoy, Co. Cork, Ireland 2 Department of Food and Nutritional Sciences, University College, Cork, Ireland

Received 15 December 2008 – Revised 25 May 2009 – Accepted 4 September 2009 Published online 16 October 2009

Abstract – Split and secondary fermentation defects in Swiss-type cheese varieties are manifested as undesirable slits or cracks that may lead to downgrading of the cheese. Split defect is associated with an excessive production of gas or an unsuitable cheese body that cannot accommodate gas produced, or a combination of both factors. Secondary fermentation is the apparent production of gas after the desired propionic fermentation of the warm room has taken place. No consensus exists as to the definitive causes of the defects, but possible causes are reviewed under factors that are associated with rheological behaviour (including cheese manufacture, acidification, intact protein content and proteolysis, seasonality of milk supply and ripening or storage temperature and duration) or with overproduction of gas (including milk microflora, propionic acid bacteria (PAB) – in particular strains with high aspartase activity and ability to grow at low temperatures, lactic acid bacteria, interactions between starter bacteria, facultatively heterofermentative lactobacilli (FHL) and other sources of gas including butyric acid fermentation). The influence of other parameters such as copper concentration, air incorporation, salt content, rind formation and cheese wrapping materials is also considered. Methods to reduce the prevalence of the split defect and secondary fermentation include addition of water to improve elastic properties by the removal of unfermented carbohydrate and the use of FHL to control PAB activity to prevent the production of excessive gas. Swiss-type cheese / split defect / secondary fermentation / eye formation

摘要 – Swiss 型干酪的开裂和后发酵作用——综述○ 由于后发酵作用引起 Swiss 型干酪出现的开裂和裂纹缺陷使得产品的等级下降○ 开裂和裂纹与过量的气体产生和不适宜的干酪结构形成有关，不适宜的结构无法容纳产生的气体○ 后发酵是指在温室中丙酸发酵后产生的气体○ 至于缺陷产生的原因至今没有一个共识，但可能与干酪流变性 (包括干酪生产、酸化、完整蛋白质含量和水解、泌乳季节，以及干酪成熟或贮存温度和时间) 或与过度产气 (包括牛乳的微生物菌群、丙酸菌特别是一些高天冬氨酸酶活性和低温生长的菌株、乳酸菌、发酵剂菌株之间的相互作用、兼性异型发酵乳酸菌和丁酸发酵产生的气体) 相关○ 其他影响参数，如铜离子浓度、空气进入量、含盐量、外层表皮的形成和干酪包装材料的影响也需要考虑○ 减少裂纹缺陷和抑制后发酵的方法有加水除去未发酵的碳水化合物以改善干酪的弹性，使用兼性异型发酵乳杆菌来控制丙酸菌的活性也可以防止产生过量气体○

Swiss 干酪 / 开裂缺陷 / 后发酵 / 眼状结构形成

*Corresponding author (通讯作者): [email protected]

Article published by EDP Sciences 4 D.F.M. Daly et al.

Résumé – Défaut de lainure et fermentation secondaire des fromages à pâte pressée cuite – une revue. Les défauts de lainure et de fermentation secondaire des variétés de fromage à pâte pressée cuite se manifestent sous forme de fentes ou fissures qui peuvent entraîner le déclassement du fromage. Le défaut de lainure est associé à une production excessive de gaz ou à une pâte fromagère inadaptée pour contenir le gaz produit, ou à une combinaison des deux facteurs. La fermentation secondaire est la production apparente de gaz après que la fermentation propionique désirée dans la cave chaude ait eu lieu. Il n’existe pas de consensus sur les causes définitives de ces défauts, mais des causes possibles sont présentées au regard de facteurs associés au comportement rhéologique (incluant la fabrication fromagère, l’acidification, la teneur en protéines intactes et la protéolyse, la saisonnalité de l’approvisionnement de lait, et la température et la durée de l’affinage et du stockage) ou associés à la surproduction de gaz (incluant la microflore du lait, les bactéries propioniques, en particulier les souches ayant une activité aspartase élevée et l’aptitude à croître à basses températures, les bactéries lactiques, les interactions entre les bactéries du levain, les lactobacilles hétérofermentaires facultatifs et d’autres sources de gaz incluant la fermentation butyrique). L’influence d’autres paramètres tels que la concentration en cuivre, l’incorporation d’air, la teneur en sel, la formation de la croûte et le matériel d’emballage du fromage est aussi examinée. Les méthodes pour réduire la prévalence du défaut de lainure et la fermentation secondaire incluent l’addition d’eau pour améliorer les propriétés élastiques par retrait des glucides non fermentés, et l’utilisation de lactobacilles hétérofermentaires facultatifs pour contrôler l’activité des bactéries propioniques pour prévenir la production excessive de gaz. fromage à pâte pressée cuite / défaut de lainure / fermentation secondaire / ouverture du fromage

1. SWISS-TYPE CHEESE annually in Europe, with France and Germany as the biggest producers and con- Swiss-type cheese is a generic term for sumers of this cheese. In 2004, France pro- hard cheeses that were initially produced duced 252 700 tonnes making it the biggest in the Emmen valley in Switzerland [31]. producer of the Emmental cheese and They are easily identifiable by characteristic accounted for ~ 25% of all French ripened round regular eyes that vary in size from 1 cheese produced [122]. Germany produced to 3 cm in properly produced cheese. The 85 200 tonnes in 2004 [122], while in the body of the cheese is hard to semi-hard. US 142 200 tonnes of Swiss cheese were Examples of Swiss-type cheese include produced in 2007 making it the fifth most Gruyère, Jarlsberg, Comté, Maasdammer, important cheese type in the US accounting Appenzeller, Leerdammer and the most for ~ 3.23% of the total cheese produced commonly produced cheese with large eyes, [112]. Emmental (often referred to simply as The biochemistry and microbiology of “Swiss cheese”)[31, 105], although it Swiss-type cheeses has been reviewed should be noted that French Gruyère con- extensively [30, 76, 79, 105]. In summary, tains a small number of eyes, while Swiss two major desirable fermentations by bacte- Gruyère is blind. There is no internationally ria occur, lactic acid and propionic acid fer- recognised definition of Swiss-type cheeses mentations. Lactic acid fermentation occurs that differentiates them from other varieties; early in manufacture where lactose is con- however, a propionic acid fermentation that verted to lactic acid. Swiss-type cheese star- either occurs spontaneously or through the ter cultures contain thermophilic species application of a culture of selected propionic such as Streptococcus thermophilus, Lacto- acid bacteria (PAB) is characteristic [30]. bacillus helveticus and Lactobacillus del- Swiss-type cheeses are produced widely brueckii subsp. lactis and may also include in Europe and in the US. Approximately mesophilic species such as Lactobacillus 466 000 tonnes of Emmental are produced lactis subsp. lactis and L. lactis subsp. Split defect in Swiss-type cheeses 5 cremoris [1, 30, 105]. Other Swiss-type desired propionic acid fermentation of the cheeses such as Maasdam are made with warm room ripening), causing splitting mesophilic cultures only or with L. del- and variation in eye size of the cheese. brueckii subsp. bulgaricus [48]. Lactose is The result is a cheese, due to structural rea- metabolised by S. thermophilus to galactose sons and/or excess gas production, that can- and L-lactic acid during the first 24 h of rip- not withstand the gas pressure and therefore ening [29, 111]. The lactobacilli ferment the cracks and split form [31, 46]. galactose to L-lactic acid and/or D-lactic There is a certain ambiguity in the pub- acid depending on the species [111]. This lished literature due to the close relationship lactic acid fermentation has a major influ- between secondary fermentation and split ence on cheese quality due to its effects defect. Thus for this review, they are consid- on pH, syneresis, the inhibition of other bac- ered as two separate defects, but with an terial growth, removal of calcium and also inference that secondary fermentation con- in providing the substrate for propionic fer- tributes to causing split defect. mentation [31]. Thus, the selection of cor- There is no consensus on why or how a rect starter cultures for cheese production split defect occurs and how it can be is vital to cheese quality [38]. avoided. Different studies have advanced Propionibacterium freudenreichii is the the issue. Park et al. [82] demonstrated that main species used as ripening culture in gas splitting was dependent on the strain of Swiss-type cheese. The propionic acid fer- PAB used; however, no obvious reason for mentation produces metabolites (propionic this dependency was established. acid and acetic acid) that are essential con- Fröhlich-Wyder et al. [32] investigated tributors to the development of the charac- PAB interactions between starter and non- teristic nutty-sweet flavours of Swiss-type starter lactic acid bacteria (NSLAB) and cheeses. The CO2 produced is responsible found that secondary fermentation is for eye formation [31, 105]. affected by the aspartase activity of PAB, the presence of facultatively heterofermentative lactobacilli (FHL) and the use of 2. SPLIT DEFECT L. helveticus as a component of the starter. AND SECONDARY Jimeno et al. [51] showed that FHL inhib- FERMENTATION: ited the growth of PAB and reduced the AN OVERVIEW prevalence of splits. White et al. [119] reported that the combination of PAB and Split defect manifests as an undesirable Lactobacillus cultures used for the manu- opening and appears as slits and cracks that facture influenced split formation. Studies are visible in the cut cheese loaf and may undertaken to establish a relationship lead to downgrading of the cheese [91]. It between the defect and proteolysis by occurs commonly in Swiss cheese and usu- White et al. [119] were unable to provide ally during the cooling period of the produc- a clear explanation as to the causes and tion process [46, 91]. Generally, split defect control of the defect. is due to increased gas production such as secondary fermentation and/or unsuitable texture within the cheese, which impacts 3. MECHANISMS OF OPENINGS negatively on its openness and elasticity of the cheese [91]. Secondary fermentation is The formation of the characteristic eyes linked with split defect as an apparent of Swiss cheese is coupled with the produc- resumption of gas production and occurs tion of CO2 during the warm room ripening during the cold room ripening (after the stage [79]; the CO2 gas pressure is the 6 D.F.M. Daly et al. mechanical means by which the curd is cheese manufacture, the curds and whey opened up to form eyes [91]. Propionic fer- may be pumped together into a special vat mentation of lactate is the main source of where the curds are allowed to settle and CO2 in Swiss-type cheeses, e.g. in tradi- collect under the whey [79]. The curd is tional Emmental cheese lactate is trans- then pressed and the whey removed. Press- formed into propionate, acetate and CO2. ing should produce a knitted curd that is A number of factors are essential for proper uniform and unbroken with no entrapped eye formation. air within the structure [91].

3.1. The presence of nuclei 3.2. Suitable conditions for the growth of PAB and adequate Nuclei are microscopic bubbles that are gas production trapped in the curd structure and serve as sites into which CO2 dissociates from solu- Itisnecessarytoensurethatconditions tion to accumulate as gas, and thus to (pH, temperature and aw) of the curd are develop into eyes [72]. Originating from sufficient for preferential growth of PAB microscopic bubbles of air, formed from so as to achieve correct and adequate CO2 foam produced by milk handling treat- production [85, 111]. In particular, curd ments or associated with particulate mate- pH at the time of transfer to the warm room rial in the milk, they become attached greatly influences the eye formation in to curd particles and contain nitrogen as Swiss-type cheese [58]. A curd with a low the principal gas component as oxygen pH (< 5.2) at day (d) 1 after manufacture is removed by starter bacteria activity decreases the probability of a correct subse- [72]. The number of eyes developed is quent eye formation due to lack of growth determined by the extent of nucleation of PAB, whereas a high pH (> 5.4) and the shape is determined by the cheese increases the chances of excessive gas pro- consistency with both dependent on gas duction [60]. A pH of 6.5–7 is the optimal production [88]. Physical openness and growth range for PAB [94]. inhomogeneity within the curd lead to a greater number of eyes being formed. 3.3. Gas formation Thenumberofeyescanbeincreasedby and accumulation manufacturing procedures such as thermi- sation and by applying a vacuum to the In traditional Emmental cheese, eye for- cheese during the filling and pressing mation occurs at the nuclei in about 20– processes. Accidental incorporation of 30 days [110]. As concentrations of CO2 H2-producing microorganisms within the increase, the gas will diffuse into any small cheese may also contribute to increase the nuclei present, and as gas production con- number of eyes [105]. tinues an overpressure (1–1.5 bar) will be Similar to the importance of nuclei for established and small eye/holes formed in correct eye formation, it is also necessary the curd [2, 79, 117]. Eye formation is that the dipping (pitching of curds under dependent on the length of time CO2 is whey), draining and pressing processes present, the amount and intensity of CO2 undertaken during cheese manufacture production and the pressure and diffusion should prevent air inclusion in the curd of CO2 within the cheese [105]. After and unnecessary mechanical stress that ~50days,CO2 production has continued could disturb the knitting of the curd should with a subsequent enlargement and increase also be avoided [79, 91]. During Swiss-type in the number of eyes. The appearance Split defect in Swiss-type cheeses 7

of new eyes decreases as the CO2 produc- overpressure in the nuclei is large and the tion declines when cheese is placed in the hole grows relatively fast, after this the hole cold room [105]. The pressure of gas is would ideally expand by flow and form a dependent on diffusion rates from the spherical eye [117]. Split formation in the cheese and on the solubility of gas within cheese mass is likely if the elongational the cheese loaf. The solubility of CO2 is viscosity is high or/and the fracture stress dependent on pH and temperature, with is low (i.e. fracture occurring at low defor- increases in both factors resulting in a mation) [66, 117, 123], particularly if the decreased solubility of CO2 [31]. In an gas production rates are high. 80-kg loaf, ~ 120 L of CO2 gas can be produced before cheese is sufficiently ripened for consumption with ~ 60 L dissolved in 4. FACTORS AFFECTING the cheese paste, ~ 20 L present in the eyes THE FORMATION OF EYES and ~ 40 L diffused from the loaf [31]. The OR SLITS AND CRACKS rind or packaging material functions as a IN SWISS-TYPE CHEESE: physical barrier and, in eye development, RHEOLOGICAL BEHAVIOUR it dictates the rate of CO2 loss from the surface, and hence the loss of the CO2 from the 4.1. Manufacture parameters cheese to the surroundings, thereby influencing the CO2 partial pressure within the Eye formation in Swiss-type cheese cheese [31, 46]. requires appropriate physicochemical and mechanical properties of the protein matrix 3.4. Curd texture and its ability which determine the structural properties to accommodate the gas of cheese thus influencing openness [79]. formed Physicochemical properties are related to cheese composition, while mechanical prop- It is necessary to have a curd that is elas- erties are related to both cheese composition tic and pliable enough to accommodate the and proteolysis [15, 79]. Swiss-type cheeses gas that is formed without any defects have high elastic properties, high deforma- occurring. The relatively low production bility or high cohesion (fracture strain) and of acid before the point of drainage is the high fracture stress expressing high mechan- main reason for the elastic texture that is ical resistance [79]. associated with Swiss-type cheese as a higher pH results in high mineral content 4.1.1. Acidification during cheese of the curd and contributes to elasticity of manufacture the curd [59, 66]. High water and fat content also contribute to the soft elastic structure of Acidification during manufacture of Emmental, which helps in maintaining the Swiss-type cheeses influences the cheese integrity of the curd during eye formation texture [66] due to its effect on bacterial [31]. growth, colloidal calcium phosphate Nuclei will more readily expand into (content and solubility) and its effect on holes if the biaxial elongation rate of the pH and compositional parameters. Lower cheese curd is high, which will occur if pH is associated with a greater probability the biaxial elongational viscosity is low, of slit formation due to the propensity as there is less resistance against the exten- of a cheese to develop slits with short- sion of the hole. The onset of hole forma- ness of the cheese texture [117]. The tion results in an initial fracture, as the model of Horne [47] for casein interactions 8 D.F.M. Daly et al. suggests that the para-casein matrix is sta- with a low pH [30]. This dilution causes a bilised by crosslinking of different casein decreased amount of fermentable lactose molecules and chains by hydrophobic to be present and therefore a decrease in interactions which is mediated by colloidal the overall lactic acid content of the curd. calcium [66]. Emmental contains ~ 1000 mg This results in cheese with a higher moisture calcium/100 g of cheese [105](~50% content and a higher pH that is favourable to more than Cheddar cheese), and these high PAB growth, but also results in a more elas- levels are mainly due to the higher pH at tic texture and the levels of fracture stress separation of curds and whey of Swiss- and strain are increased [50]. Luyten [68] type cheese and confer an elastic texture reported that at a given pH, the rheological to the cheese [66]. Berdagué et al. [6] stiffness of a cheese was decreased by reported a correlation between the occur- increasing the moisture during maturation. rence of split defect in Comté cheeses with Thus, the risk of late fermentation which decreased calcium content at 20 h after is higher in cheeses with inappropriate elas- manufacture. ticity may potentially be reduced by water Low acid development and thus higher addition during manufacture of Emmental cheese pH is associated with high cooking cheese [50]. temperatures that are used during the manufacture of certain Swiss-type cheeses [91]. 4.1.2. Influence of manufacture Syneresis is also influenced by acidification, parameters on protein with decreasing pH increasing the rate of and proteolysis syneresis within the curd [62, 121]. In turn, this determines the moisture content, prote- αs1-Casein is the primary casein olytic activity and other related constituents involved in maintaining the elastic struc- of the cheese, in particular the proportion of ture of Emmental and is vital to maintain- undissolved calcium associated with the ing the integrity of the curd during eye casein matrix [60, 66]. Notz et al. [81] formation as the elastic protein structure reported increased elasticity and decreased will bend and accommodate the accumula- mechanical resistance in Comté-type tion of CO2 [36, 119]. Emmental has a cheeses with increased fat contents due to higher level of native protein (intact αs1 + a higher moisture content resulting from αs2 + β-CN) than other Swiss-type cheese reduced levels of syneresis. Acidification varieties such as Comté and Beaufort and also influences casein hydration and electro- as a result Emmental has higher elastic static and hydrophobic interactions between properties [42]. More native caseins casein molecules [119], and the cohesion in the protein matrix contribute to the and mechanical properties of the cheese cheese having increased firmness and are influenced by interactions between min- deformability, properties that are appropri- erals, water and protein and particularly pH ate to openness [79]. at 24 h which affects the structural state of Research has not shown a strong correla- the protein [79]. The activity of plasmin tion between degree of proteolysis and prev- and chymosin is also dependent on pH (as alence of the split defect [97, 119]. well as on salt to moisture and casein to However, proteolytic action would be a log- moisture ratios) [59]. ical factor that contributes to split defect. Water may be added to milk or the curd/ Fracture strain (longness) decreases as pro- whey mixture (up to 20% of the milk teolysis increases [67] and shortening of weight for Emmental cheese and up to cheese texture due to loss of elasticity by 35% for Masdaam cheese) during cheese proteolysis may contribute to splitting manufacture to avoid producing a cheese occurring due to an inability of the texture Split defect in Swiss-type cheeses 9 to cope with the gas pressure [32]. Berdagué 4.2. Ripening parameters et al. [6] and Grappin et al. [43] related split intensity to higher levels of secondary prote- 4.2.1. Rheological profile during olysis as measured nitrogen levels soluble in ripening phosphotungstic acid. In Swiss-type cheese, plasmin and the Flückiger [27] investigated changes in proteinases and peptidases from starter, rheological properties during ripening of non-starter and secondary starter [34, 35] Emmental cheese using compression (and are the principal proteolytic agents [25, relaxation) tests on cheese samples at ripen- 93]. The activity of proteolytic enzymes in ing and other temperatures. Those authors cheese is influenced by aw, copper content, reported that elasticity decreased during water content, lactic acid concentration, the ripening period independent of temper- pH, storage temperature and time [105]. ature. Measurements of firmness were low- The greater role of plasmin in proteolysis est in cheeses in the warm room, while in Swiss-type cheese is due to the greater those of fracture stress decreased sharply conversion of plasminogen to plasmin, as on entering the warm room. Fracture strain inhibitors of plasmin activators and plasmin increased at the beginning of the warm are inactivated or are lost in the whey by the room and then decreased throughout ripen- use of relatively high cook temperatures, up ing with a rapid change observed on trans- to 55 °C [25]. Chymosin is associated with fer to cold storage. Noël et al. [79] the initial shortening (reduction in yield observed from that study that during the force) of Cheddar cheese texture via hydro- warm room phase of ripening when gas lysis of αs1-casein [15]. However, the coag- production was greatest, the cheeses had ulant is inactivated to a degree dependent on the lowest firmness and a higher elastic the cooking temperatures used during man- modulus supporting a higher deformation ufacture of Swiss-type cheeses. Cooking before breaking and showing a relative temperatures during Emmental manufacture resistance to fracture, and thus attaining exceed 50 °C resulting in little residual appropriate mechanical properties for eye coagulant activity [37, 90], while cooking formation. temperatures of ~ 36–40 °C used in Noël et al. [79] suggested that openness Maasdam/Leerdammer and Jarlsberg manu- is related to local phenomena as CO2 con- facture result in higher residual coagulant tent in Emmental was observed to be activities with greater hydrolysis of αs1- heterogeneous [40]. Akkerman et al. [2] casein or its breakdown products [75]. predicted that a slit would be formed instead There is a greater degradation of αs1-casein of an eye if the local overpressure is higher in cheese manufactured from raw milk than the local fracture stress. Ruegg and which is attributed to the microflora of the Moor [100] studied the size, shape and dis- raw milk and their associated enzymes tribution of curd granules in some hard and [21, 23] and also possibly to the indigenous semi-hard cheeses produced in Switzerland milk enzyme, cathepsin D [41]. Studies and reported that pressing of the cheese have also suggested a putative role for loaves flattened the granules. It was also cathepsin D and for thermophilic lactoba- reported that slit formation occurred in cilli [7, 33] in the hydrolysis of αs1-casein Raclette cheese late in maturation and slits during ripening of Swiss-type cheeses. followed the granule borders, but also cut Low activity of plasmin towards αs1-casein through granules; eye formation occurred compared to β-andαs2-casein also enhances best where the curd granules were appar- the role of αs1-casein in Swiss-type cheese ently completely fused together [100]. structure [5, 114]. Grappin et al. [43] studied splits in Comté 10 D.F.M. Daly et al. cheese and reported that splits are usually Cheese texture may change with the parallel to the surface that is perpendicular physical state of the fats depending on to the press direction. Tests on the samples the storage temperature and time [22]. of cheese perpendicular to the press direction Crystallisation of milk fat influences its showed higher stress and strain values at rheological properties as fat in cheese is fracture compared to the samples tested par- composed of different triglycerides that allel. It was postulated that if there is a large have different melting temperatures span- difference between rheological results from ning from approximately −40 to 40 °C two different directions of a cheese sample, [64]. Lopez et al. [64] monitored the ther- the formation of eyes may result in a slit mal properties and solid fat content in being formed instead of proper eyes [43]. Emmental cheese using differential scanning calorimetry and reported that crystalli- 4.2.2. Temperature and duration sation of fat occurred on cooling to about of ripening and storage 15 °C. Thus, during ripening of Swiss-type cheeses, e.g. Emmental, the ripening tem- In general, the longer the cheese is stored peratures (~ 22–4 °C) greatly influence the greater the prevalence of splits within a the solid fat content, and thus the rheolog- cheese prone to splitting [119]. Once the ical properties of the cheese and its ability cheese has been transferred to the warm to accommodate further gas production. room, few measures can be taken to affect Proteolysis takes place mainly in the the quality of the cheese [91]. The optimum warm room [109, 113]. Thierry et al. growth for PAB is between 25 and 35 °C [109] studied the kinetics of the microbial [94] and temperatures of ~ 20–24 °C are and biochemical changes of the aqueous used for the warm ripening to promote phase of cheese in three Swiss-type cheeses PAB growth and CO2 formation [105]. industrially manufactured and observed that The high temperature used during the warm levels of nitrogen soluble at pH 4.6 and in room phase contributes to maintaining the 12% TCA and levels of free amino acids soft elastic structure of the cheese and (FAA) increased during ripening, at a rate increases the ability of the cheese to support dependent on the ripening room tempera- eye formation [31, 119]. As the temperature ture. An increase in the ripening tempera- increases, the molecules, particles and ture increases proteolytic activity and strands in the cheese possess more thermal promotes greater gas production within the energy and a greater motion causing relaxa- cheese, with even a 1 °C increase in temper- tion of protein interactions and change in ature resulting in a measurable difference the cheese towards a more liquid-like state [43]. Levels of FAA increased, but their rel- [67]. ative proportion remained essentially con- For a cheese to stretch, casein molecules stant, apart from amino acids catabolised must interact with each other and be pliable by bacteria (Arg and Asp, in particular) enough to release stress (i.e. the pressure of [109]. gas is reduced by eye formation), but also After warm room ripening, cheese is maintain sufficient contact between the mol- transferred to maturation rooms at lower ecules to prevent rupture. Time during temperatures or to cold storage (2–13 °C) which the gas is evolved is important, if [91]. Cheese loses some of its elasticity as gas is produced slowly stretch properties at lower temperatures the rheological prop- may be retained, whereas if gas is evolved erties of the cheese change resulting in a quickly the cheese is more likely to crack greater tendency to fracture upon increased or split as the cheese is rapidly stretched CO2 pressure (as fat is in a more solid state) [117, 119]. [43, 46]. At low temperatures, the cheese Split defect in Swiss-type cheeses 11 curd may also become supersaturated with production of cheese from summer milk CO2. An unintended increase in temperature resulted in a higher moisture content com- decreases gas solubility and this increases pared to cheese manufactured in the winter the CO2 pressure possibly resulting in split- season and that the former had a greater ting of the cheese, due to the higher resis- tendency to split compared to the latter. tance to gas compression of the cheese A higher cheese moisture may result in mass. Therefore, it is imperative to ensure higher levels of proteolysis, which could that temperature fluctuations be prevented result in an altered texture and variable rates [46, 86]. of release of small peptides and amino acids Reinbold et al. [92] varied the materials of that could contribute to secondary fermenta- the cheese storage container and temperature tion [30]. Furthermore, cheese with an profiles and showed that rapid cooling pro- increased moisture content due to poor ini- duced uneven temperature in the cheese. This tial acidification may lead to an increased can affect moisture and bacterial activity unfermented carbohydrate content after within the cheese by forming localised areas 24 h in the curd and, by subsequent fermen- of difference. The outer zone of the cheese tation, may result in low curd pH [29] cools down more quickly than the inner zone resulting in undesirable texture and elastic- resulting in more proteolytic microflora in ity of the curd [111]. the outer zones of the cheese, and hence a Rohm et al. [96] observed from large- shorter and firmer body [105]. It is therefore scale experiments that Emmental cheese more desirable to have a uniform cooling and produced during the winter months showed also to arrest propionic fermentation before asignificantly firmer body, accelerated fer- excess gas is produced [92]. mentations of lactate and propionate as well as reduced proteolysis compared to cheese 4.3. Seasonality of milk supply produced in the summer season. Firmness was the parameter that was most heavily Season of manufacture of cheese has influenced by the season of production been linked to variations in milk composi- resulting in differences in fracture proper- tion, cheese moisture, cheese yield and ties. This is consistent with the results quality [12, 60]. Plasmin content of milk obtained by Ginzinger et al. [39]for also varies with stage of lactation [93]. In Austrian Bergkäse and may possibly have countries with seasonal milk supply, cheeses negative consequences in relation to split manufactured from early lactation milk have defect [69]. The change in firmness of the poorer eye development and this may be Emmental with season was attributed to partly attributed to a lower concentration the difference in milk fat composition with of plasmin in the milk [60]. increases in liquid proportions in the fat Fröhlich-Wyder and Bachmann [30] phase of cheese increasing from winter to reported that cheese made from winter milk summer [96]. fromcowsfedonhayandsilagehasa The diet of the cow (e.g. hay and fodder slightly slower rate of acidification and beet) can cause an elevated saturated fatty shows a greater occurrence of secondary acid content in milk used for cheese manu- fermentation compared to grass-fed summer facture and has been shown to be partly milk. Gilles et al. [38] reported that slower responsible for a hardening of the cheese rates of acidification during the manufacture [31]. The diet can be supplemented in order of Swiss-type cheese resulted in a higher to increase the unsaturated fatty acid content moisture content in the final cheese. How- of the milk and produce a softer cheese ever, White et al. [119] reported that the texture [13, 49, 76]. 12 D.F.M. Daly et al.

5. FACTORS AFFECTING associated enzymes [21, 23] and also possi- THE FORMATION OF EYES bly to cathepsin D [41]. OR SLITS AND CRACKS Baer and Ryba [4] showed that the influ- IN SWISS-TYPE CHEESE: ence of raw milk microflora stimulated PAB OVERPRODUCTION OF GAS growth through their proteolytic action, and Beuvier et al. [7] reported that cheeses made 5.1. Milk with raw milk show greater proteolysis and stronger propionic acid fermentation, Milk for the manufacture of Swiss-type although other studies have shown no dif- cheese must be of high quality containing ference in cheese texture between cheeses as few bacteria as possible to ensure made from pasteurised and raw milk optimum starter activity and prevention [9, 39]. Further research is required to deter- of secondary contamination [76, 91]. In mine if greater levels of proteolysis and Switzerland where Swiss-type cheeses are enhanced propionic acid fermentation may made from raw milk, strict measures and promote the occurrence of split defect or laws in relation to the milk quality are secondary fermentation in cheeses made applied [91]. Somatic cell counts (SCCs) from raw milk in comparison to those made are a general indication of the quality of from pasteurised milks. milk and vary depending on the period of lactation. Elevated SCC can correlate with 5.2. PAB a change in milk composition [52]and increased SCC has been associated with Selected PAB of the species P. freud- increased proteolysis during cheese ripen- enreichii are the main and desirable gas ing. Therefore, it is desirable to have a and eye formers in Swiss-type cheese [30]. low SCC to produce quality cheese [14]. However, it has been shown that excessive The heat treatment of the milk before growth of PAB is a major factor that con- cheese manufacture may impact on split tributes to split defect [4]. Low numbers defect. Swiss cheese is prone to split defect of P. freudenreichii may gain access to the whether it is made from pasteurised or raw milk via the environment; however, nowa- milk; however, no detailed work to compare days they are usually added intentionally both treatments in relation to this defect has [30]. Inocula of PAB in cheese milk range − been published. Higher numbers of lactoba- from 103 to 106 cfu·mL 1 [79, 86]. When cilli (FHL), enterococci and coliforms are the desired eye formation has been attained, found in Bergkäse cheese made from raw the PAB have often reached levels of 108– − milk compared with cheeses made from 109 cfu·g 1 in cheese [105].Whiteetal. pasteurised milk [23]. Pasteurisation [119] showed that the selection of the reduces the growth of microorganisms in PAB subspecies and strain is vitally impor- general during ripening of Emmental [7]. tant, as the strain of PAB used for manufac- A greater diversity of microorganisms exist ture had the most obvious effect on split in cheese made from raw milk, but this formation. diversity declines during ripening [41]. The metabolism of PAB involves several Also, raw milk may contain wild PAB and complex pathways and is not fully under- NSLAB strains that may promote secondary stood [84]. PAB can utilise a variety of sub- fermentation [45]. As previously discussed, strates; however, lactate is the main energy there is a greater degradation of αs1-casein source for propionic fermentation. The clas- in cheese manufactured from raw milk sical metabolic pathway of propionic acid attributed to the raw milk microflora, their fermentation in Swiss-type cheese is where Split defect in Swiss-type cheeses 13 lactate is fermented to propionate, acetate, of secondary fermentation [3]. This was CO2 and H2O[30]: attributed to CO2 production by aspartate metabolism and fermentation of lactic acid. 3 lactate ! 2 propionate þ acetate þ CO2 A careful selection of PAB strains is neces- þ H2O þ ATP: sary as high aspartase activity may result in secondary fermentation, but a low aspartase However, the results of these theoretical activity may result in negative effects in equations are rarely found in Swiss-type relation to openness and flavour intensity cheese and the relative concentrations of and prolong ripening times [120]. propionate, acetate and CO2 may be However, it has still not been proven different [84]. conclusively whether aspartase activity is the cause or merely an indicator of secondary fermentation [32]. 5.2.1. PAB with high aspartase activity 5.2.2. Ability of PAB to grow at low The enzyme aspartase, which catalyses temperatures the deamination of aspartate, is found in PAB and aspartase activity varies widely Strains of P. freudenreichii were observed from strain to strain of PAB [31]. Lactate to grow at 3.8 °C in sodium lactate broth is coupled with the fermentation of aspar- [56]. Park et al. [82] demonstrated that some tate, if present, to acetate, CO2,succinate strains of P. freudenreichii grew well at and NH3 with no propionate produced 7.2 °C, a temperature used for the storage [30]. of Swiss-type cheese after warm room ripening. Cheeses made with these strains showed lactate þ 2 aspartate ! acetate þ CO2 a larger eye volume and a greater tendency to þ 2succinate split compared to those made with strains that þ 2NHþ ATP: were inhibited more at low temperature [46]. 3 From experimentation on two PAB cultures, it was shown at 24 °C that there was little dif- This would lead to an increased produc- ference in the growth rate between the two tion of CO2 during lactate utilisation [84]. – L-Lactate is again preferentially used as strains, whereas at 12 14 °C only one strain the aspartate is metabolised [32]. More lac- was capable of growth at the lower temperatures and this was the strain most prone to tate is fermented to acetate and CO2 as opposed to propionate in cheese where causingsecondary fermentation [4].The abil- strains with a high aspartase activity are ity of gas-producing organisms to grow at low temperatures is a crucial parameter in used. Aspartase activity is therefore important as strains with high aspartase activity the cause of secondary fermentation, and this have a greater tendency towards secondary should be taken into consideration when selecting a strain of PAB [4]. fermentation [120]. Piveteau et al. [85] monitored changes in the amino acid composition of whey during PAB fermentation 5.3. Lactic acid bacteria and reported that succinate was only produced when aspartate was present. Compar- Before whey drainage and brining, the ison of 30 Swiss-type cheese samples primary acid-producing starter (S. thermo- revealed that the deamination of aspartate philus) has the greatest influence on pH to succinate by PAB increased the risk [60]. Lactobacillus helveticus theoretically 14 D.F.M. Daly et al. releases a racemic mixture of D- and L-lactic [83]. Piveteau et al. [85, 87] reported that acid in a 1:2 ratio [30]. D- and L-lactate are when the growth rate of P. freudenreichii the substrates for propionic fermentation increased as a result of the stimulation by [94]. PAB utilise L-lactate preferentially, L. helveticus, a greater conversion of lac- and this is explained by the fact that L-lactate tate to propionate and acetate was metabolism results in high intracellular pyru- observed, and thus presumably a larger vate concentration that has a stronger inhibi- CO2 production. Thierry et al. [110] tory effect on D-lactate dehydrogenase reported thermophilic LAB to influence activity than on L-lactate dehydrogenase PAB growth with significant differences activity [84]. Berdagué et al. [6] reported a observed in doubling times and lactate correlation between occurrence of split consumption by the PAB, to a degree defect in Comté cheeses with increased dependent on strain when grown on an D-lactate levels at 20 h after manufacture. Emmental juice-like media [101]. Acidification due to lactic acid bacteria Piveteau et al. [85] studied the interac- (LAB) during cheese manufacture influ- tion between 14 strains of LAB with ences bacterial growth, calcium associated 4 strains of PAB in whey and showed that with the casein matrix, syneresis, pH and none of the LAB strains had an inhibitory composition and enzyme activity. As previ- action on PAB activity. Stimulation of ously described in Section 3.2,correctpHis PAB strains by strains of LAB varied vital to eye formation, while lower pH is widely from strain to strain and strain pair- associated with a greater probability of slit ing used with some showing no apparent formation due to shortness of the cheese interaction. All except one of the LAB texture [117]. strains used stimulated at least one of the four strains of PAB. It was reported from 5.3.1. Interactions between starter that study that L. helveticus was the most LAB and PAB consistent stimulator of PAB as the four strains of PAB were stimulated by the three The interactions and effects of mixed strains of L. helveticus used. However, cultures of some strains of lactobacilli Kerjean et al. [53] reported that the greatest and PAB are well documented [82, 110]. stimulation of five strains of PAB, as mea- White et al. [119] reported that the inci- sured by the production of propionic acid dence of splits can be controlled by a in a whey-model system, was achieved by correct selection of L. helveticus and four of six strains of L. delbrueckii subsp. P. freudenreichii cultures. Different strains lactis tested and that the lowest level of of LAB and PAB can have stimulatory stimulation was achieved by one of five effect on the propionic fermentation [85]. strains of L. helveticus. In general, those Using a whey medium, strains of L. helv- authors reported that stimulation of PAB eticus and S. thermophilus have been was higher with L. delbrueckii subsp. lactis shown to increase the PAB growth with than with L. helveticus, although variations an increase in biomass and fermentation between strains occurred. products; in particular, there was a large increase in propionic acid produced [18]. 5.3.2. Interactions between starters Research on the interactions between LAB and PAB: proteolysis L. helveticus and P. freudenreichii showed that fermentative capability of both micro- LAB are generally weakly proteolytic; organisms was enhanced when grown as however, they do possess an extensive pro- mixed cultures in a complex medium teinase/peptidase system and are capable Split defect in Swiss-type cheeses 15 of proteolysis [103]. Lactobacillus helveticus used by PAB and the greatest stimulator is recognised as possessing efficient prote- of growth [85]. Crow [16] demonstrated ase and peptidase activities with respect to that aspartate was the amino acid that was milk proteins [26, 73]. L. helveticus autolys- most readily metabolised in a Swiss-type es rapidly during ripening and releases cheese environment by PAB. The addition active peptidases in the cheese [35, 113], of aspartate had an apparent stimulatory and increases in the levels of amino acids effect on the growth of P. freudenreichii in andpeptidesinwheyandmostaminoacids control whey and reduced the amount of produced were shown to be utilised by the lactate converted to propionate, but not the PAB [85]. Strains of L. helveticus used for amount of lactate converted to acetate. It cheese manufacture have been shown to is thought that this may have importance have an influence on secondary fermenta- as aspartate may be released due to proteo- tion [30]. White et al. [119] studied split for- lytic activity [85]. mation caused by two different strains of Conversely, Piveteau et al. [87] showed L. helveticus and found that one strain pro- in a simple whey-based model that tetra-, duced more splits. The reason for this was penta- and hexa-peptides produced by pro- not ascertained as the degree of measured teinases associated with the cell wall of proteolysis for both strains was the same, L. helveticus from αs-andβ-casein had a but it was postulated that a difference may greater stimulatory effect on the growth have occurred in small peptides and/or in of P. freudenreichii than amino acids. In the levels of amino acids produced by the this study, no evidence was found that starter cultures. the amino acids produced (proline and PAB possess proteolytic enzymes [57]; alanine were found in the greatest quanti- however, most of them are intracellular ties) had any stimulatory effect on PAB. and are poorly released within the cheese. Attempts to purify the stimulant were Fröhlich-Wyder et al. [32] proposed that unsuccessful. However, nine peptide peaks proteolysis and amino acid catabolism were obtained from HPLC of which six may result in a higher pH, and thus better were stimulatory for P. freudenreichii, growing conditions for the PAB, and hence the stimulation was less than the stimula- a greater gas production. Amino acid catab- tion observed in the unfractioned material, olism may also contribute to further gas which implies that more than one peptide production as decarboxylation produces may be involved. Thierry et al. [110] additional CO2 [105]. reported that propionibacteria were stimu- Research by Baer and Ryba [4] reported lated by high peptide levels and low that under the experimental conditions used levels of FAA as well as low salt levels, for their study, stimulation of PAB by LAB low proportions of L(+)-lactate and other was due to FAA. If FAA are present, they undetermined factors. could result in increased growth rates and as a result more CO2 being produced, which 5.4. FHL is correlated to an increased risk of secondary fermentation. From experimentation on The NSLAB in Swiss-type cheese are 30 Emmental cheeses, it was found that mainly composed of FHL that begin growth at the beginning of ripening [35] and even- there was a correlation between FAA levels − in the cheese and secondary fermentation as tually reach ~ 108 cfu·g 1 [109]. In a study assessed by a professional panel [4]. Aspar- by Demarigny et al. [21], L. paracasei, tate, alanine and serine can be used by PAB L. plantarum, L. rhamnosus and L. brevis [16], but aspartate is the main amino acid were all detected in Swiss-type cheese, 16 D.F.M. Daly et al. but as the cheese ripened L. paracasei by L. rhamnosus with citrate as the sole began to dominate the NSLAB flora. energy source and inhibition was shown at In artisanal cheese manufacture in a value of 0.5 mmol L−1 of milk, which is Switzerland, FHL are often inoculated into a level observed in the cheese during FHL the milk to control PAB activity and reduce growth [51]. secondary fermentation [4]. The suppres- However, higher concentrations of for- sion of the PAB growth by FHL may be mate, diacetyl and acetate compared to explained by the fact that both these micro- those found in Swiss-type cheese are organisms compete for some of the same required in defined growth media for mini- nutrients, which results in less nutrients mal inhibition of PAB [4]. Cheese manufac- available to PAB for growth [118]. How- tured with FHL had less intensive ever, Jimeno et al. [51] attributed the inhibi- proteolysis, as measured by the levels of tion of the PAB by FHL to citrate FAA, compared to cheese manufactured metabolism in Emmental cheese. Martley without these microorganisms, possibly and Crow [72] demonstrated citrate utilisa- due to an inhibitory effect on the proteolytic tion by L. plantarum in Emmental and activity of lactobacilli [4]. Conversely, showed in cheese with citrate-fermenting Jimeno et al. [51] noted that aspartate was organisms that less lactate was fermented present in the cheese manufactured with and less propionic acid was formed com- FHL in much greater quantities (three times pared to cheese manufactured without higher than the cheese manufactured with- citrate-fermenting organisms. Weinreichter out FHL) and that the inhibition effect of et al. [118] reported that propionibacteria FHL on PAB was more pronounced in developed more readily in cheeses made PAB with low aspartase activity. The reason with citrate negative strains of FHL in com- for this observation is unknown [4]. parison to citrate positive strains. Jimeno et al. [51] showed that FHL 5.5. Other sources of gas strains such as L. rhamnosus and L. casei with high peptidolytic activity and fast au- Flückiger [27]reportedthat70–80% of tolysing nature are present in significant the CO2 gas produced in Emmental cheese numbers in Swiss-type cheese, and inhibited is due to the propionic fermentation during the growth of PAB and reduced the preva- warm room ripening. However prior to lence of splits. All citrate initially present warm room ripening, lactic acid fermenta- was consumed during the growth of added tion by native milk and starter microflora L. rhamnosus. Competition for citrate by may contribute 10–15 mL of CO2/100 g itself did not account for the inhibition of of cheese [27]. In other cheese varieties with PAB growth, as PAB do not utilise citrate eyes but without propionic acid fermenta- directly. FHL metabolise citrate to acetate, tion, e.g. Edam, Gouda and Herrgardsöst, formate and CO2, acetate and formate CO2 is produced from citrate by mixed appear to have an inhibitory effect on strain mesophilic starters and Lactococcus PAB growth. Other factors may also con- sp. [91]. CO2 can also be formed from other tribute to this inhibition; a higher copper substrates such as citrate, lactose, urea and level, previously chelated by citrate, was amino acids [72]. Martley and Crow [71] found in juice extracts from cheese manu- demonstrated that citrate metabolism can factured with FHL, and it was observed that occur in Swiss-type cheese resulting in the the ratio of copper to citrate played an complete utilisation of the citrate; it was important role in PAB inhibition. Diacetyl suggested that non-starter Cit+ lactobacilli inhibits the growth of PAB and is produced may be responsible. Certain pathways of Split defect in Swiss-type cheeses 17

amino acid degradation can result in CO2 5.6. Other selected factors related production [57]; decarboxylation of amino to overproduction of gas acids results in the liberation of CO2 and the conversion of amino acids to amines 5.6.1. Copper [17]. For example, decarboxylation of glutamic acid to γ-aminobutyric acid by mixed Manufacture of cheese in copper vats strains of thermophilic cultures is the main results in cheese with a copper concentra- source of CO2 in some Gouda-type cheeses tion of up to 17 ppm, while cheese made [123]. in stainless steel vats contains concentrations of 0.5–1.5 ppm of copper [77]. Cop- 5.5.1. Other sources of gas: butyric per helps to control PAB activity in the acid fermentation manufacture of artisanal Swiss-type cheese [31]. It influences propionic fermentation Butyric acid fermentation in cheese can as it inhibits lactate dehydrogenase of lead to the production of CO2 and H2 gas LAB and PAB, particularly of the latter. [31]. H2 is only sparingly soluble in the A relative increase in D(−)-lactate occurs cheese compared to CO2; therefore, a small as copper inhibits L(+)-lactate dehydroge- production of H2 can lead to large openings nase to a greater extent [54, 55]. An increase and blowing of cheese [72]. Germination of in copper concentration depressed the rate spores from spore-forming anaerobic bacte- of CO2 formation and growth of three ria such as clostridia, which contaminate strains of PAB used in a study by Mueller milk mainly via silage, can cause this defect et al. [77]; however, total CO2 production [19]. The most common species involved after 13 days was approximately the same are C. tyrobutyricum and C. butyricum; for all levels of copper used. Increasing cop- however, research also points to an associa- per concentrations with cupric sulphate tion of C. beijerinckii and C. sporogenes in solution in Swiss-type cheese increased the contributing to and promoting the defect. levels of proteolysis, but decreased gas pro- Recent research by Le Bourhis et al. [61] duction by the PAB, which resulted in a showed the ability of C. beijerinckii to grow longer stay in the warm room [74]. Mueller in cheese and suggested that the species et al. [77] reported that a copper content of could have implications in relation to split 18 ppm in cheese had undesirable effects on defects. cheese quality, particularly in relation to Preventative measures to avoid contami- body, flavour and eye formation. nation with Clostridium spores include pro- hibition of milk from silage fed herds, e.g. 5.6.2. Salt as is practised in Switzerland [105, 115]. Bactofugation can remove 95–99% of Swiss-type cheeses are salted by immer- spores prior to cheese production [116]. sion in brine and brine times are adjusted Addition of nitrate and lysozyme to cheese to achieve desired salt/salt-in-moisture milk prevents the growth of butyric acid (S/M) levels [44]. Salt is added to control bacteria [116]. Germination and growth of microbial growth (including LAB), enzy- the spores can also be inhibited by ensuring matic activity, syneresis, and it also influ- that correct brining is carried out and a low ences cheese texture through physical ripening temperature used [108]. A biologi- changes in cheese proteins [44]. PAB are cal control system may serve as a biological sensitive to salt [30], and inhibition of barrier to control and inhibit the prolifera- P. freudenrichii is expected in Emmental tion of Clostridium spores [11, 80]. cheese even though salt levels are low; 18 D.F.M. Daly et al.

~0.3–0.7% w/w. Growth of PAB at also important as unprotected blocks may pH ~ 5.3 is strongly inhibited above a split and distort the eye shape if handled level of 3% [95]. Increased S/M levels also carelessly [91]. result in reduced production of CO2 as a result of reduced propionic acid fermentation [95]. Similarly, lower levels of acetic 6. TECHNIQUES FOR THE and particularly propionic acid were MEASUREMENT OF CHEESE reported in brine salted cheeses with higher MICROSTRUCTURE, EYE salt concentrations [43]. FORMATION, CO2 CONTENT Richoux et al. [95] suggested that strains AND POTENTIAL INDICATOR of PAB which are more tolerant to salt COMPOUNDS would be more prone to secondary fermentation than strains that are sensitive to salt due to greater activity of the salt-tolerant 6.1. Measurement of the strains at a given salt concentration. These microstructure of cheese authors suggested that the defect may be controlled more effectively by the use of The microstructure of cheese signifi- the PAB with defined salt tolerances. cantly affects its processing characteristics Brining also results in a firm dry rind that and texture properties, and thus may pro- inhibits diffusion of CO2 from the loaf [30] vide understanding and a means of control- and in variations in aw values between dif- ling eye formation and other cheese ferent regions of the cheese (with aw values properties [64]. usually higher near the centre [102]). This Ruegg and Moor [100] examined the can result in zonal variations in cheese com- size distribution, shape and junction pattern position and proteolysis within the cheese, of curd granules in cheeses using light which can result in differences in the tex- microscopy and digital image analysis. tural and functional properties of the cheese The micrographs obtained facilitated visu- [44, 67]. alisation of the fine structure around eyes within the cheese. Confocal laser scanning 5.6.3. Wrapping materials and rind micrography may be used to observe the formation fat and protein microstructure by staining the protein and fat network of thin slices In Swiss-type cheese manufacture where of cheese [20]. Lopez et al. [63, 65]used a permanent rind is produced, the firm dry confocal laser scanning microscopy to rind inhibits the loss of CO2 from the loaf monitor the organisation of fat and disrup- and provides physical protection [30]. In tion of the milk fat globule membrane dur- rindless block Emmental, wrapping material ing the manufacture and ripening of is required. In an experiment to study the Emmental cheese and also to observe the effects of four different wrapping materials, curd junctions formed in Emmental and Hettinga et al. [46] found that impermeable the changes in curd structure during the wrapping (or equivalent rind) would pro- cheese development. Rousseau et al. [99] duce a block Swiss-type cheese that has a studied the structure of Emmental cheese greater disposition to split formation as using scanning electron microscopic tech- CO2 would not be able to diffuse out of niques. Casein and fat globule structure the cheese. Therefore, selection of a wrap- were monitored during manufacture and per with permeability sufficient only to stop curd granule junctions, gas microbubbles mould growth and give proper eye forma- and microcolonies of internal cheese flora tion is required. Handling of the cheese is were also observed. Split defect in Swiss-type cheeses 19

6.2. Measurement and detection of compounds and final products of metabo- eye formation or slits and cracks lism; however, this only gives a very rough estimate of the CO2 levels [45]. Girard and A number of techniques may offer Boyaval [40] measured the CO2 content in a potential for the analysis and detection of Swiss-type cheese by treatment of the sam- both eye formation and defects associated ple by the method outlined by Bosset et al. with eyes such as split defect in cheese. [8] where samples were blended under a Rosenberg et al. [98] investigated the use partial vacuum, sulphuric acid was added of magnetic resonance imaging (MRI) to and the liberated CO2 was analysed by evaluate eye formation in Swiss-type GC. Enzymatic techniques may also be cheese. A sample cheese was subjected to used to determine CO2, but only small MRI prior to evaluating the cheese in a tra- quantities of cheese sample can be used, ditional manner by cutting the cheese and which may not be representative of the photographing the cross-section. Both whole cheese due to variations in CO2 con- techniques were compared, and the MRI centration within the cheese [40]. analysis was shown to be an effective Nelson et al. [78] determined the CO2 non-destructive, high-resolution tool for content of Cheddar cheese by a modifica- the evaluation of structural features of eye tion of the method described by Ma et al. formation. Caccamo et al. [10] photo- [70] using a method of standard addition, graphed the slices of cheese using a digital i.e. determination of the CO2 concentration camera and measured the surface area of of the unknown cheese sample by compari- the cheese slice and the area occupied by son to a set of samples of known concentra- gas holes developed using image analysis tion. Cheese samples were blended and software. Eskelinen et al. [24] proposed that degassed purified water and sulphuric acid ultrasonic methods are capable of monitor- added in an airtight blender. The head space ing gas-solid structure of the cheeses and was sampled by a needle connected to an providing a detection method for eye forma- infrared CO2 analyser. This technique was tion and associated eye defects within the repeated a number of times, using increas- cheese. The advantages of this method are ing increments of sodium bicarbonate that it is non-destructive, relatively inexpen- standard solution that increases the CO2 sive and could aid in quality control. How- content, thus facilitating the formation of a ever, it has limitations for use as there are method of standard addition linear regres- drawbacks in relation to penetration depth sion equation. The CO2 content of individ- and speed in scanning the entire cheese ual samples could be determined by volume. extrapolation of the equation.

6.3. Measurement of cheese CO2 6.4. Potential indicators of split content defect or secondary fermentation An accurate method for the determination of the CO2 content of cheese would No reliable indicators have been deter- facilitate determination of whether correla- mined to predict the occurrence of split tions exist between the CO2 content of defect, although some studies have shown cheese and split defect by comparing cheese correlations between levels of certain bio- with a proper eye formation to those chemical parameters and cheeses with sec- cheeses that have a split defect. Previously ondary fermentation defect. Lower levels CO2 produced by PAB was quantified of D-lactic acid and overall lactic acid were by determination of the intermediate observed by Steffen et al. [104]incheeses 20 D.F.M. Daly et al. with secondary fermentation, and higher on the levels of calcium associated with contents of acetate, succinate, glutamate the casein matrix and on interactions and p-benzoquinone. It was suggested that between minerals, water, protein and curd proteolysis and propionic fermentation were pH at 24 h influence casein hydration, more intensive in cheese with secondary electrostatic and hydrophobic interactions fermentation [106]. Steffen et al. [104] between casein molecules, and thus cohe- reported that a combination of p-benzoqui- sion and mechanical properties of the none and acetate could be used to differen- cheese. Similarly intact casein is important tiate between good quality cheese and those in maintaining the elastic structure of the with secondary fermentation. Steffen et al. cheese and in accommodating eye forma- [107] reported that Emmental cheese sus- tion after accumulation of CO2. Although ceptible to secondary fermentation had high a strong correlation between proteolysis aminopeptidase and low lactate dehydroge- and prevalence of the split defect in nase activities and had elevated acetate, suc- Swiss-type cheeses has not been reported, cinate and glutamate levels. White et al. a shortening of cheese texture and a loss [119] found no direct correlations between of elasticity due to proteolysis may poten- split defect/secondary fermentation and tially contribute to split defects due to an moisture and fat contents, protein, pH or inability of the texture to cope with the D/L lactate ratio. gas pressure, and one report has linked increased slit intensity with increased levels of secondary proteolysis. Season of manu- 7. CONCLUSIONS facture produced a variable response; a firmer body was observed in cheese made Split defects are associated with exces- during the winter months, and secondary sive production of gas or an unsuitable fermentation in cheeses was linked to cheese body that cannot accommodate the slowerratesofacidification in winter milk, gas produced, or a combination of both fac- but split defects were also linked to higher tors, resulting in a cracking or splitting of moisture levels in cheeses made from sum- the cheese. Secondary fermentation is an mer milk. Rheological properties of the apparent resumption of gas production dur- cheese in the warm and cold rooms are also ing cold room storage of the cheese. The of great importance. The high temperatures interrelationship between split defect and used during the warm room phase contrib- secondary fermentation has led to a degree ute to maintaining the soft elastic structure of ambiguity in the published literature con- of the cheese and increase the ability of cerning both defects. The causes of second- the cheese to support eye formation; how- ary fermentation are implicated with the ever if gas is developed at a high rate, the causes of split defect as gas production in cheese may be more prone to split. On the cold room is associated with split defect, transfer to the cold room, cheese loses elas- whereas the causes of split defect not only ticity and may also become supersaturated encompass secondary fermentation, but also with CO2; an unintended increase in tem- inadequacies in the texture and body of the perature at this point decreases gas solubil- cheese. ity, increases the gas pressure and may Rheological and structural properties that result in splitting of the cheese. influence eye or slit and crack formation in Overproduction of gas is the other pri- cheese are dependent on the appropriate mary influence in the formation of eyes or physiochemical and mechanical properties slits and cracks in Swiss-type cheeses. Heat of the cheese protein matrix. Acidification or non-heat treatment of cheesemilk influ- during cheese manufacture and its effect ences milk microflora, their proteolytic Split defect in Swiss-type cheeses 21 activity and their influence on subsequent development of recent and more sophisti- propionic acid fermentation and production cated analytical techniques may offer oppor- of CO2 in cheese. The strains of PAB used tunities to achieve this. are of great importance and in particular their level of aspartase activity and their ability to grow at storage temperatures. Acknowledgements: This review was funded Similarly, stimulation of PAB by some by Tipperary Co-operative Creamery Ltd. LAB may be due to the release of peptides and amino acids resulting from proteolytic activity. This process is very strain specific and the exact mechanisms of this stimula- REFERENCES tion process are unclear. 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