A. Coloring Agents
The color of cheese is determined by the carotene color of the milkfat, which may vary with the seasons and with type of milking animals. Jersey milkfat, for example, is usually high in carotene whereas sheep milkfat is low. Where coloring is permitted, the color of cheese may be adjusted by means of annatto extract or carotene dye.
All cheeses are salted, either by addition of salt to milled curd after acidification and before molding, or by absorption of salt by diffusion after the cheese has been formed. A special case is Domiati cheese, where 8-15% salt is added to the milk, yielding coagulum and resulting cheese that is very soft.
For cheese of the Gouda/Danbo type, there is a risk of Clostridium tyrobutyricum spore growth during ripening, resulting in excessive gas production and the development of an unpleasant smell. The spores can be inhibited by means of nitrate added to the milk (0.10.2 g KNO3/L); the nitrate is reduced to nitrite (NO2_), which inhibits the spores. This reduction is catalyzed by the milk enzyme xanthin oxidase, which is active in milk heated to about 72 °C for 15 sec but inactive at a slightly higher heat treatment. Other possible additives for inhibition of spores are the enzyme lysozyme (from hen's egg white) and nisin (bacteriocin). The use of spore-inhibiting additives is legally restricted in cheese-producing countries.
For most cheese varieties, starter cultures of lactic acid bacteria are added to the cheese milk. Where the curd is either not heated or heated to about 40 °C, starters with mesophilic Lactococcus sp. and Leuconostoc sp. are used. If formation of carbon dioxide is undesirable (e.g., for Cheddar cheese, and for Feta cheese destined to be put in tins), mesophilic starters without citrate-fermenting Leuconostoc sp. and Lactococcus lactis subsp. lactis biovar. diacetylactis ( = Lactococcus diacetylactis) are utilized. In cheeses scalded at higher temperatures (e.g., Grana, Emmental), lactic acid fermentation is performed by thermophilic cultures of Streptococcus thermophilus and Lactobacillus helveticus.
Starter cultures can be local natural starters, or laboratory cultures from culture manufacturers either for propagation in the dairy or, as frozen or lyophilized starter concentrates, for direct inoculation in the cheese milk. The amount of starter culture added to the milk is typically about 1% for starter propagated in milk and about 0.01-0.05% for starter concentrates.
Slow acidification due to antibiotics in the milk or the development of bacteriophages can cause serious problems. Bacteriophages will not be destroyed by heat treatment of the milk at about 72 °C for 15 sec, so there will always exist a risk of development of bac-teriophages, along with the starter bacteria, in a cheese factory. Counteractive measures include careful cleaning and disinfection with chlorine or disinfectants with peracetic acid (hydrogen peroxide has no effect on bacteriophages), preventing any contamination of milk and curd with whey, propagation of starter culture in highly heated milk in a separate room, the use of starter cultures with good bacteriophage resistance, and if necessary, change of starter culture. Fast development of lactic acid bacteria is of utmost importance for cheese quality. For each type of cheese there is an optimal range for minimum pH; Table 5 gives some examples.
The effects of lactic acid bacteria are:
1. Coagulation and syneresis. The lactic acid promotes the action of rennet enzymes and the acidification of the curd increases syneresis. In the production of sour milk cheese (quarg, etc.) the milk is coagulated only by the acid produced by the starter bacteria.
2. Inhibition of detrimental bacteria. Lactic acid bacterial growth inhibits harmful bacteria by fermentation of all the lactose and by the formation of lactic acid. Some of the lactic acid bacteria also produce bacteriocins that may inhibit other bacteria.
Table 5 Optimal pH Minima for Various Cheeses
Cheese pH minimum
Gouda/Danbo group 5.20-5.25
Feta, Danablu 4.65-4.7
3. Control of consistency. The acidification controls the consistency and texture of the cheese. If the pH becomes too low, much of the calcium is dissolved from the casein network and the cheese will be brittle, less coherent. If the acidification is weak, little calcium is dissolved and the cheese mass will be rubbery. For semihard, sliceable cheese, the best consistency is obtained with a fresh cheese pH at about pH 5.2.
4. Control of enzyme activity. pH determines the activity of the various enzymes of the cheese during ripening.
5. Taste. Lactic acid is important for the fresh acid taste of young cheese.
6. Low redox potential. The lactic acid bacteria lower the redox potential to about —150 mV, yielding cheese that can be kept for a long time without the development of off-flavors due to oxidation of milkfat.
7. Formation of proteolytic enzymes. The lactic acid bacteria have proteolytic enzymes important for the breakdown of proteins during ripening. Intracellular peptidases, releaed after autolysis of the cells, are responsible for the formation of free amino acids; starter bacteria may also contribute to conversion of amino acids into various flavor compounds.
8. Formation of CO2 and diacetyl: If the starter contains citric acid-fermenting bacteria such as Leuconostoc sp. and Lactococcus diacetylactis, the citric acid will be converted to acetic acid, diacetyl/acetoin, and CO2.
As described above, acidification is of fundamental importance for the quality of cheese. The velocity of the acidification is also important. A too-rapid drop in pH may cause the cheese to become too sour and too hard (increased syneresis); on the other hand, slow acidification gives the opposite effects and there will be a risk of growth of detrimental bacteria. Fig. 2, left, illustratesthe rate of acid development during the first 24 hours for a typical semihard cheese such as Gouda/Danbo, for a blue-veined cheese (Danablu), and for milk, all starting with inoculation with 1% mesophilic starter culture at 30 °C. Fig. 2, right, shows the rise in pH for both types of cheese during ripening due to release of ammonia by the breakdown of proteins and by degradation of lactic acid. The faster increase in pH during ripening for Danablu, compared to Gouda/Danbo, is a result of very active proteolytic enzymes from the mold and aerobic degradation of lactic acid. In milk, acidification stops at about pH 4.4 because the bacteria are completely inhibited by lactic acid at low pH althoug there will still be surplus of nonfermented lactose. In cheese, the inverse situation prevails: the growth of the bacteria and the decrease in pH ends when all the lactose is used up, not because pH had become too low.
For the Gouda/Danbo cheese, the pH levels off at about pH 5.2; for the blue-veined Danablu cheese, pH continues to decrease until a minimum pH at 4.7. This difference is due to a difference in the lactose/buffering capacity ratio. The amount of lactose determines the amount of lactic acid formed, and the amount of buffers (i.e., protein and inorganic phosphate) determines the change in pH for a given amount of acid. In the young blue-veined cheese, compared to the Gouda/Danbo cheese, there is a higher moisture content, hence a higher content of lactose. At the same time, the blue-veined cheese has a lower content of buffers (protein and inorganic phosphate) than the Gouda/Danbo cheese.
A further important factor that contributes to higher pH minimum in Gouda/Danbo cheese compared to the blue-veined cheese is that for Gouda/Danbo, water is added to the
Figure 2 The course of acidification for a Gouda/Danbo cheese, for a blue-veined cheese (Da-nablu), and for milk, respectively, during the first 24 hr after inoculation with 1% mesophilic starter culture, all starting at 30 °C (left). The graph to the right show pH changes during storage and ripening. The fall in pH during the first 24 hr is caused by the fermentation of lactose into lactic acid by the lactic acid bacteria. The rise during storage is caused by release of amino acids and ammonia by breakdown of proteins and by degradation of lactic acid. The bottom curve applies to milk inoculated with lactococci and which is protected from infection by yeasts and molds during storage. (From Ref. 1.)
mixture of whey and cheese grain in order to dilute the whey, thereby reducing the lactose concentration.
However, the pH minimum is not solely a function of the ratio of lactose to buffers. As long as the curd grains are dispersed in the whey, lactose can diffuse from the whey into the grains in replacement of that which has been fermented into lactic acid during stirring. (About 90% of the lactic bacteria are being concentrated in the curd grains, hence the lactose fermentation mainly takes place here.) Consequently, the total decrease in pH has to be looked upon as two phases: phase one, the decrease of pH in the curd grains until their final separation from the whey, and phase two, the decrease in pH in the cheese curd after it has been finally separated from the whey; in this phase, the decrease in pH is determined solely by the ratio of lactose to buffers.
An approximate calculation of pH minimum:
pH minimum = pH1 - 0.8298 x (L/B) x (pH1)2 + 24.89 x pH1 x (L/B)2
where pH is pH at molding/start of pressing (when cheese grains are finally separated from the whey); L is % lactose hydrate of the pressed cheese, and B is buffering capacity = % protein of the pressed cheese +19 x % inorganic phosphate (5). For Cheddar cheese, the addition of salt to the milled curd, depending on the concentration of salt, may delay or stop the acidification before all lactose has been completely fermented (5). In the model it is assumed that all lactose is converted to lactic acid. Lactose is calculated here as hydrate, therefore one weight unit of lactose corresponds to one weight unit of lactic acid.
Lm(100-A(100 - Tw- h * Pw)/(100 - Tm - h* Pm)) * ((100 - Tc - h* Pc)/100)
" (100 -Tm - h* Pm) - A(100 - Tw - h* Pw)/100 + W '
where Lm is % lactose hydrate in milk, Tm and Pm are % total solids and % protein, respectively, in milk, Tw and Pw are % total solids and % protein in whey, and Tc and Pc are % total solids and % protein in cheese (24 hr). A is the amount of whey (% of milk by volume) removed during stirring, W is water (as % of milk by volume) added to the mixture of whey and curd grains during stirring. h [exclusion factor (2)] is amount of water bound to protein in such a way that it cannot function as solute for lactose. h can be set to 0.3 g H2O per g protein (6).
In some countries, it is allowed for certain types of cheeses (e.g., quarg, cottage cheese, and mozzarella) to acidify the curd by phosphoric-, acetic-, or citric acid, or by glucono-delta-lactone, which slowly is converted to gluconic acid.
1. Propionibacteria. For Emmental cheese and other types with large eyes, cultures of Propionibacterium sp. are added in order to initiate fermentation of lactic acid to propionic acid, acetic acid, and CO2; the latter contributes to the formation of the large holes.
2. Molds. For blue-veined mold cheeses, spores of Penicillium roqueforti are added to the milk. For white mold cheeses, spores of Penicillium camemberti and other Penicillium species are added to the milk or onto the surface of the cheese.
3. Secondary flora of lactic acid bacteria. In semihard cheeses, the number of starter bacteria culminates at about 109 cells per gram after 1-2 days. Thereafter these bacteria gradually die out and autolyse. During the first month of ripening, 99% of the lactococci may die out, and simultaneously a secondary flora of lactobacilli develops and may grow to a number of 107-108 cells/g (7). These lactobacilli, mainly belonging to the group of facultatively heterofermentative lactobacilli (e.g., Lactobacillus casei/paracasei), develop spontaneously in the cheese and are found only in low numbers (e.g., 1-10 per milliliter, or less, in pasteurized cheese milk); they may originate from the flora in the cheese factory or they may be lactobacilli surviving pasteurization. Pediococci have also been found in the secondary flora. Although the presence of a secondary flora in cheese has been known for more than one hundred years, knowledge of their growth and their effects on the cheese is still relatively scarce. There are indications that the secondary flora may have positive effects on the quality of cheese: for example, by inhibition of detrimental bacteria such as heterofermentative lactobacilli (8) and Clostridium tyrobutyricum (9) and by the consumption of oxygen diffusing into the cheese (10).
The primary effect of rennet is coagulation of the milk. Later, rennet enzymes play an important role in the hydrolysis of proteins during ripening. The coagulating activity of calf rennet, or standard rennet, is due to the enzyme chymosin. Traditionally, rennet is made from the fourth stomach of calves, the abomasum, which is sliced and extracted in a weak acid salt solution. The extract is filtered and the pH adjusted to 5.5. Finally, 15-20% salt and benzoic acid is added. Milk-coagulating enzymes can also be extracted from stomachs from oxen or from other animals, by the fermentation of certain molds, or by microorganisms into which the gene for chymosin has been cloned.
Coagulation takes place in two phases. First, a negatively charged part of the k-casein (one-third of the K-casein molecule) is split off by hydrolysis (catalyzed by the rennet enzyme) of one peptide bond (no. 105, phenylalanin/no. 106, methionin) in K-casein. The casein micelles thereby lose a part of that negative charge that otherwise prevents the micelles from coagulating. The casein (paracasein) is now insoluble in the presence of Ca2+. In the second phase of renneting, the paracasein micelles aggregate by hydrophobic attraction between hydrophobic amino acid residues in the caseins. With 30 mL standard rennet per 100 L and at 30C, the first phase takes about 10 min and the second phase about 1 min. It then takes about 20 min before the coagulum is sufficiently firm for cutting.
J. Addition of Calcium Chloride
Addition of CaCl2, 5-20 g per 100 L, can increase the rate of renneting partly because the addition of CaCl2 gives a slight reduction in pH.
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