Rennet coagulation properties of heated milk Lucey, J. A., Gorry, C. and Fox, P. F. Lucey, J. A., Gorry, C. & Fox, P. F. 1993. Rennet coagulation properties of heated milk. Agric. Sci. Finl. 2: 361-369. (Teagasc, National Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland and Dept. Food Chem., National Food Biotech- nology Centre, University College, Cork, Ireland.) Heating impaired the rennet coagulationproperties of milk which deteriorated further during storage, i.e. rennet hysteresis occurred. Acidification to pH values < 6.2 or addition of low concentrations of CaCh greatly improved the rennet coagulation properties of heated milk. Acidification of heated milk to pH values < 5.5 followed by neutralization to pH 6.6 to produce reformed micelles, resulted in greatly improved rennet coagulation properties except for severely heated milks (120°C for 10 min) which were not coagulable even after acidification/neutralization. Acidification of heated milk to pH values < 5.5 and storage at the low pH for 24 h before neutralization resulted in a further improvement in the rennet coagulation properties. Dialysis of heated milk that had been acidified and reneutralized against an excess of normal milk resulted in a dramatic deterioration of its rennet coagulability. Reheating milk that had been heated, acidified and reneutralized resulted in little change in RCT or gel firmness. Addition of heated milk to raw milk resulted in an increase in RCT of the latter and a reduction in gel firmness. Key words: rennet coagulation,heated milk, acidification and reneutralization Introduction Severe heating (> 75°C for > 30 sec) of milk, with- out pH adjustment or other treatment impairs its renneting properties and renders it difficult to co- agulate (Morrissey 1969, Van Hooydonk et al. 1987, Singh et al. 1988, Lucey 1992). It has been established that when heated, (3-lactoglobulin ((3- Ig) and K-casein form a complex via sulphydryl - disulphide interchange (Zittle et al. 1962, Saw- yer et al. 1963, Sawyer 1969), although hydro- phobic interactions may play a role in the initial stages of complex formation (Haque and Kin- SELLA 1988). This is the most important factor affecting the rennet coagulability of heated milk (Kannan and Jenness 1956, 1961, Van Hooy- donk et al. 1987, Dalgleish 1990). The rennet coagulation time (RCT) of milk in- creases with the severity of heat treatment (Mor- rissey 1969, Singh et al. 1988, Van Hooydonk et al. 1987, DALGLEISH 1990, LUCEY 1992). How- ever, there are conflicting reports as to whether the increased RCT is due to impairment of the enzy- matic or aggregation reactions, or both. There have been several reports that the total amount of pep- tides released by chymosin is reduced when casein micelles are subjected to extremely high temperat- ures, e.g. 90°C for 1 h, in the presence of (3-lg (Wheelock and Kirk 1974, Shalabi and Whee- lock 1976) or oc-lactalbumin (Shalabi and Whee- 361 Agric. Sei. Fint. 2 (1993) https://www.c-info.fi/en/info/?token=30Dqbh_039J3obDv.FVrVkHUqHHYyKKN64aM9rw.eoz3Oi5zhvbZUssa9TQADbtpkk5N8IpKviWrdjscC4r-wuQtGohhEfXYEVOcL0Ua39E9RpBY5QoZJBxj-F_TzOua5SmZL5HswZdQ7XTfVYU0tkpegoB9suHKmdtarnomKArremlndo3kObxm1H5vScPx1SX7JmyF3Nj9ER7eY84Xx6jLovYg_IGHscS6nz3a82KOWhFWS-Oy4GNCEyaON-DcThy1E050Va48gY_XS1R44yb8zDLMkS4VxE1Q0x50MIHHAHBsUZhiUtPMXBWL2to5uglUMvEXRsvuVtE lock 1976). However, Marshall (1986) reported that the enzymatic stage is hardly affected by heat- ing at 75 or 85°C for 30 min. Van Hooydonk et al. (1987) showed that heating decreased both the ini- tial velocity of K-casein hydrolysis and the amount of hydrolysable K-casein in milk heated up to 120°C for 5 min. Reddy and Kinsella (1990) reported that heating (85°C for 15min) of P-lg with K-casein or with casein micelles inhibitedchymosin hydrolysis, resulting in a reduced initial rate of hydrolysis (37%) and a reduction (26%) in the amount of glycomacropeptide released; heating casein micelles alone did not affect hydrolysis by chymosin. Reddy and Kinsella (1990) suggested that the conformation of the chymosin-susceptible bond of K-casein may be somewhat different and probably less readily accessible to the enzyme after complex formation with p-lg. It is generally agreed (Pyne 1945, Morrissey 1969, Marshall 1986, Van Hooydonk et al. 1987, Singh et al. 1988) that the secondary phase of rennet coagulation is more adversely affected by heating than the enzymatic phase. Denatured whey proteins on the surface ofcasein micelles sterically hinder the aggregation ofrennet-converted micelles resulting in prolonged RCT (Van Hooydonk et al. 1987). Heated milks exhibit a phenomenon known as rennet hysteresis, i.e. the RCT increases when the heated sample is cooled and held following heating (Mattick and Hallett 1929, Powell and Palmer 1935, Pyne 1945, Morrissey 1969, Lucey 1992). Most of the hysteresis effect occurs within 5 h of heating (Mattick and Hallett 1929,Moir 1930). Heating causes the precipitation ofcalcium phosphate with a concomitant reduction in soluble calcium (Mattick and Hallett 1929, Pyne 1945, Hilgeman and Jenness 1951,Tessier and Rose 1958,Demott 1968,GEERTSetal. 1983) which inhibits the aggregation reaction which is sensitive to changes in [Ca2+ ] (Pyne 1945). Some authors (Pyne 1945, Morrissey 1969, Van Hooydonk et al. 1987) have suggested that rennet hysteresis is caused mainly by the slow solubiliza- tionofheat-induced calcium phosphate during stor- age, while others (Kannan and Jenness 1961) consider that the heat-induced interaction between p-lg and K-casein is the principal factor involved. Lucey (1992) showed that heat-precipitated cal- cium phosphates did not solubilize during cold stor- age and was not responsible for rennet hysteresis. It is not clear exactly what causes rennet hysteresis but it is possible that during storage of heated milk, physicochemical changes may occur in the P-lg K-casein complex (Sawyer 1969) which may pro- vide additional steric hindranceand a further reduc- tion in the rate of aggregation. The strength of rennet gels (curd tension) is also adversely affected in heated milk (Dill and Roberts 1959, Mauk and Demott 1959, Keogh 1966, Ashworth and Nebe 1970, Singh et al. 1988, Lucey 1992, McMahon et al. 1993). This reduction in gel strength is presumably caused by the disruption of the continuity of the gel network caused by attachment ofdenatured whey proteins to the casein micelles. The denatured whey proteins may sterically hinder the close approach and con- tact between casein micelles, resulting in a weaker, looser network due to reduced crosslinking (McMahon et al. 1993). The reduced aggregation rate of heated milk means that its gel strength in- creases at a slowerrate than in unheated milk. Thus, the strength of rennet-induced gels from heated milk, determined at a particular time after rennet- ing, will be lower than from unheated milk. The objectives of this study were to investigate the effects of various treatments, i.e. pH adjust- ment, addition of CaCb and acidification/neutrali- zation on the rennet coagulation properties of heated milk. Material and methods Heating Fresh whole milk was heatedat temperatures in the range 70 - 100°C for 10 min in an oil bath, while a direct steam autoclave (Asteli Scientific, Sidcup, Kent), with a thermocouple mounted in the cap of themilk bottle to monitor temperature, was used for heat treatment at I2O°C for 10 min. After heating, milks were cooled rapidly by immersion in ice. 362 Agric. Sd. Fint. 2(1993) Rennet coagulation properties The rennet coagulation properties were determined by the Formagraph, as described previously (Lucey and Fox 1992). Acidification and reneutralization Heated milk was cooledrapidly to 2°C before acidi- fication to pH values in the range 6.5 - 4.6 and reneutralized immediately to pH 6.6 at 2°C or held overnight at 2°C before reneutralization. Milks were warmed to 20°C for 1 h, readjusted to pH 6.6, before renneting at 35°C. pH adjustments were per- formed with an Autotitrator (Mettler DL 21, Greifenese, Switzerland). Effect of reheating acidified/reneutralized heated milk Heated (90°C for 10 min) milk was acidified to pH 5.5 at 2°C and reneutralized to pH 6.6 at 2°C. Milks were warmed to 20°C and readjusted to pH 6.6 and then reheated to 90°C for 10min. The rennet coagu- lation properties were determined at 35°C. Effect of CaCl2 on the renneting properties of heated milk Calcium chloride (1 M) was added to unheated and heated milks at levels of I to 10 mM/L. The pH of the Ca-supplemented milks was adjusted to pH 6.6 and held for I h prior to rennet coagulation. Effect of dialysis on the renneting properties of heated milk Heated milk or heated milk that had been acidified and reneutralized were dialysed against 20 volumes of unheated milk for 2 days at 4°C. Results and discussion The RCT of milk increased (Fig. la) and the firm- ness ofrenneted milk gels decreased(Fig. 1 b) as the severity of heat treatment was increased. Milks heated to very high temperatures (> 100° for 10 min) did not coagulate without pH adjustment. Storage of heated milk caused a further deteriora- tion in the rennet coagulation properties, especially for high heat (> 80°C for 10 min) treatments, i.e. rennet hysteresis occurred (Fig. la). Acidification ofheated (100°C for 10 min) milk resulted in shorter RCTs (Fig. 2a) and increased gel firmness, with a maximum at pH 6.2 (Fig. 2b). Acidification can therefore offset some of the im- paired rennet coagulation properties of heated milks, probably by reducing charge repulsion and increasing (Ca 2+ |. Fig. 1. Rennet coagulation time (RCT) (a) and the firmness (Formagraph reading after 30 min) of the resultant gels (b) of milks immediately after heating (A) and after holding at 4°C for 24 h (A). Control milk (�) had a coagulation time of4.5 min and a gel firmness of 54 mm. 363 Agric. Sei. Fin!. 2 (1993) The addition of low concentrations ofCaCb res- ulted in a reduction in RCT (Fig. 3a) and increased gel firmness (Fig. 3b). Heated milks to which > 2.5 mM CaCb were added did not exhibit rennet hys- teresis or a deterioration in gel firmness during storage. This suggests that the addition of low con- centrations ofCaCh compensates for the additional impairment of the aggregation reaction which ap- parently occurs during storage of heated milk. However, the addition ofCaCh did not completely restore the original gel firmness, suggesting that the incorporation of whey proteins adversely affects the gel network. Van Hooydonk et al. (1987) and Singh et al. (1988) reported that the coagulability of high heat-treated milk could be restored by the addition of low concentrations ofCaCb. Acidification of heated milk (100°C for 10 min) to pH values < 5.5 before immediate neutralization to pH 6.7 resulted in greatly improved RCT (Fig. 4) but gel firmness (Fig. 5) was not completely re- stored to that of raw milk. Acidification and hold- ing at low pH values for 24 h before neutralization further improved the rennet coagulation properties. Acidified and reneutralized milks have a higher [Ca2+ ] than normal milk (Singh et al 1988, Tan and Fox unpublished) which is probably respons- ible for its improved rennet coagulation properties. Dialysis of heated milk that had been acidified and Fig. 2. Effect of pH on (a) rennet coagulation time and (b) the firmness of the resultant gels from unheated milk (□) or from milk heated at 100°C for 10 min (■). Fig. 3. Effect of CaCh on (a) rennet coagulation time at pH 6.6 and (b) the firmness of the resultant gels from unheated milk (□) or milk heated at 100°C for 10 min measured immediately (■) or after 24 h at 4°C (A). 364 Agric. Sd. Finl. 2 (1993) reneutralized against an excess ofraw milk resulted in a dramatic deterioration in its rennet coagulabil- ity (Table 1), probably because dialysis restores the [Ca 2+ ] to the original value of milk (Tan and Fox unpublished). Banks and Muir (1985) reported that the casein in starter made from sterilized milk, which was acidified by lactic acid bacteria and subsequently neutralized on addition to the cheese milk, was incorporated into cheese curd. They suggested that this was due to the disruption of micelles at low pH values, making the buried/masked K-casein avail- able for hydrolysis and capable of participation in gel formation after renneting. However, Van Hooydonk et al. (1987) reported that acidification (to pH values > 5.5) and neutralizationto pH 6.7 did not increase the rate of hydrolysis of K-casein. Reddy and Kinsella (1990) reported that reduc- ing the pH of heatedand cooled mixtures of casein micelles with (3-lg to pH 5.8 and immediately read- justing to pH 6.8 before the addition of chymosin increased the final amount of glycomacropeptide released by about 13%, although there was little change in the rate of hydrolysis. Van Hooydonk et al. (1987) found that acidifi- cation of heated milk to pH values > 5.5, followed immediately by neutralization, had little effect on therenneting properties of heated milks but storage of milk samples for 24 h at pH 5.5 before neutral- ization improved rennet coagulability. VAN HOOY- Fig. 4. Rennet coagulation time of unheated milk acidified to pH values in the range 6.5 to 4.6 and reneutralized immedi- ately to pH 6.6 (A) or heated milk (IOO°C for 10 min) acidified to pH values in the range 6.5 to 4.6 and reneutral- ized immediately to pH 6,6 (■) or heated milk (100°C for 10 min) acidified to pH values in the range 6.5 to 4.6 and maintained at the low pH for 24 h at 4°C before reneutraliza- tion to pH 6.6 (□). Fig. 5. Firmness of renneted milk gels from unheated milk acidified to pH values in the range 6.5 to 4.6 and reneutralized immediately to pH 6.6 (A) or heated milk (100°C for 10min) acidified to pH values in the range 6.5 to 4.6 andreneutralized immediately to pH 6.6 (■) or heated milk (100°C for 10min) acidified to pH values in the range 6.5 to4.6 and maintained at the low pH for 24 h at 4°C before reneutralization to pH 6.6 (□). 365 Agric. Sei. Fint. 2 (1993) Table I. Effect of dialysis on the rennet coagulation proper- ties of acidified and reneutralized milk. Treatment Rennet coagula- Gel firm- tion time ness (RCT)min (aw) mm CONTROL Original milk 4.0 51.0 Acidified to pH 5.0 and read- 1.5 53.0 justed to pH 6.6 Acidified and readjusted milk 15.0 8.0 dialysed against control milk HEATED MILK (100°C for 10min) Heated milk, immediately 11.5 11.0 Heated milk, after storage for 17.5 3.5 2 days at 4°C Heated milk, dialysed against 7.5 22.0 control milk Heated milk, acidified to pH 4.5 28.5 5.0 and readjusted to pH 6.6 Heated milk, acidified to pH N.C. N.C. 5.0 and readjusted to pH 6.6 and dialysed against control milk N.C. No coagulation was observed Table 2. Effect of reheating on the rennet coagulation proper ties of milk that had been previously heated and acidified/re- neutralized. Treatment Rennet coagula- Gel firm- tion time ness (RCT)min (am) mm CONTROL Original milk 6.0 36.5 Acidified to pH 5.5, held 4.5 48.0 for 2 h, readjusted to pH 6.6 HEATED MILK (90°C for 10 min) Original heated milk 11.0 12.5 Heated milk, reheated at 11.0 10.0 90°Cfor 10 min Heated milk, acidified to 5.5 15.0 pH 5.5 held for 2 h and readjusted to pH 6.6 (acidi- fied and readjusted) Heated milk, acidified and re- 6.0 20.0 adjusted before reheating atWCfor 10 min Heated milk, acidified and re- 7.5 19.7 adjusted before reheating at 100°Cfor 10 min donk et al. (1987) suggested that acidification and storage at low pH values was necessary to solubil- ize heat-precipitated calcium phosphate which was reprecipitated on neutralization in a form similar to indigenous CCP which they suggested may be the most important factor responsible for the improved rennetability of heated milks following acidifica- tion and neutralization. They also suggested that heat-precipitated calcium phosphates may solubil- ize more slowly than indigenous CCP on acidifica- tion and that the slow solubilization ofheat-precipi- tated calcium phosphate during storage may con- tribute to rennet hysteresis. However, Van Hooydonk et al. (1987) did not acidify heated milk to pH values < 5.5; the results of the present study demonstrate that acidification to pH values < 5.5 improved therennet coagulation properties of heated milk, even when the samples were reneutralized immediately to pH 6.6. Reheating milk resulted in little change in RCT or gel firmness (Table 2). Acidification and reneut- ralization prior to reheating resulted in an improve- ment in RCT and gel firmness. A similar trend was observed when heated milks were acidified to pH 5.0 and reneutralized prior to reheating (results not shown). After acidification and neutralization, milks should be carefully readjusted to pH 6.6 at 20°C before reheating or precipitation (heat instab- ility) occurred at relatively low temperatures, e.g., 80°C for 10 min. Addition of heated milk (100°C for 10 min) to raw milk resulted in an increase in RCT of the latter and a reduction in gel firmness (Fig. 6). The blend- ing of low levels ofheated milk with unheated milk may provide an acceptable method of utilisizing high heat-treated milk. Severely heated milk (120°C for 10 min) did not coagulate even after acidification to low pH values 366 Agric. Sei. Finl. 2 (1993) and reneutralization to pH 6.6. This suggests that other factors prevent coagulation, e.g., drastic physicochemical changes occur in casein micelles at such high temperatures, including dephospho- rylation of protein residues (Howat and Wright 1934) and conversion of CCP to hydroxyapatite (Visser et al. 1986). Heat-induced changes in ca- sein micelles have been reviewed (Singh 1988). Conclusions The RCT ofmilk increased with increasing severity of heat treatment probably due to inhibition of the aggregation of renneted micelles caused by com- plex formation between (3-lg and K-casein. The in- creased RCT can be offset by pH adjustment or addition of CaCl2, although the original gel firm- ness was not completely restored. Acidification and neutralization resulted in greatly improved rennet coagulation properties probably due to increased [Ca ?+ ], References Ashworth, U. S. & Nebe. J. 1970. Comparison of rennet curd tension with undenatured whey protein as a measure of heat treatment. J. Dairy Sci, 53: 415-419. Banks, J. M. & Muir, D. D. 1985. Incorporation of the protein from starter growth medium in curd during manu- facture of Cheddar cheese, Milchwissenschaft 40: 209- 212. Daloleish, D. G. 1990. The effect of denaturation of (3-lac - toglobulin on renneting - a quantitative study. Milchwis- senschaft 45: 491-494. Demott, B. J. 1968. lonic calcium in milk and whey. J. Dairy Sci. 51: 1008-1012. Dill, C. W. & Roberts, W. M. 1959. Relationships of heat treatment, solids-non-fat and calcium chloride to the curd tension of skim milk. J. Dairy Sci. 42: 1792-1799. Geerts, J. P., Bekhof, J. J. & Scherjon, J. W. 1983. Deter- mination of calcium ion activities in milk with an ion selective electrode. A linear relationship between the logarithim of time and the recovery of the calcium ion activity after heat treatment. Neth. Milk Dairy J. 37: 197-211. Hague, Z. & Kinsel.la, J. E. 1988. Interaction between heated K-casein and (3-lactoglobulin: predominance of hydrophobic interactions in the initial stages of complex formation. J. Dairy Res. 55: 67-80. Hilgeman, M. & Jenness, R. 1951. Observations on the effect of heat treatment upon the dissolved calcium and phosphorus in skimmilk. J. Dairy Sci. 34: 483-484. Howat, G. R. & Wright, N. C. 1934. The heat coagulation of caseinogen. 1. The role of phosphorus cleavage. Bio- chem. J. 28: 1336-1345. Kannan, A. & Jenness, R. 1956. The relation of milk serum proteins to the effects of heat treatment on rennet coagu- lation. J. Dairy Sci. 39: (suppl. 1)911. Fig. 6. Effect ofblending heated and unheated milks on (a) the rennet coagulation time and (b) the firmness of rennet gels (results from two trials). 367 Agric. Sei. Finl. 2 (1993) & Jenness, R. 1961. Relation of milk serum proteins and milk salts to the effects of heat treatment on rennet clot- ting. J. Dairy Sci. 44: 808-822. Keogh, M. K. 1966. Some Factors Affecting Rennet Curd Tension, M.Sc. Thesis, National University of Ireland, Cork. Lucey, J, A. 1992, Acid-base Buffering and Rennet Coagu- lation properties of MilkSystems, Ph.D. Thesis, National University of Ireland, Cork. & Fox, P. F. 1992, Rennet coagulation properties of late-lactation milk: Effect of pH adjustment, addition of CaCh, variation in rennet level and blending with mid- lactation milk. Ir. J. Agric. Food Res. 31; 173-184. Marshall, R. J. 1986. Increasing cheese yields by high heat treatment ofmilk. J. Dairy Res, 53; 313-322. Mattick, E. C. V. & Hallett, H. S. 1929. The effect of heat on milk, (a) On the coagulability by rennet, (b) On the nitrogen, phosphorus and calcium content. J. Agric. Sci. 19:452-462. Mauk, B. R. & Demott, B. J. 1959. Influence of certain salts, whey protein, and heat treatment upon the pH and rennet curd tension ofcasein sols. J. Dairy Sci. 42: 39-48. McMahon, D. J., Yousif, B, H. & Kalab, M. 1993.Effect of whey protein denaturation on structure of casein micelles and their rennetability after ultra-high temperature processing of milk with or without ultrafiltration. Intern. Dairy J. 3: 239-256. Moir, G. M. 1930.The effect of heat upon the rennin coagu- lation. I. J. Dairy Res. 2: 68-75. Morrissey, P. A. 1969. The rennet hysteresis ofheated milk. J. Dairy Res. 36: 333-341. Powell, M. E. & Palmer, L. S. 1935. Behaviour of caseinate sols in a study of a hysteresis-like phenomenon in the rennet coagulation of heated milk. J. Dairy Sci. 18: 401- 414. Pyne, G. T. 1945. Rennet hysteresis and the calcium phos- phate of milk. Biochem. J. 39: 385-390. Reddy, I. M. & Kinsella, J. E. 1990. Interaction ofP-lacto- globulin with K-casein in micelles as assessed by chy- mosin hydrolysis: effect of temperature, heating time, P-lactoglobulin concentration and pH. J. Agric. Food Chem. 38: 50-58. Sawyer, W. H. 1969. Complex between P-lactoglobulin and K-casein. A review. J. Dairy Sci. 52: 1347-1355. —, Coulter, S. T. & Jenness, J. 1963. Role of sulfhydryl groups in the interaction of K-casein and P-lactoglobulin. J. Dairy Sci. 46: 564-565. Shalabi, S. I. & Wheelock, J. V. 1976. The role ofa-lactal- bumin in the primary phase of chymosin action on heated casein micelles. J, Dairy Res. 43; 331-335. Singh, H. 1988. Effects of high temperatures on casein mi- celles. N. Z. J. Dairy Sci. Technol. 23: 257-273. —, Shalabi, S. 1.,Fox, P. F., Flynn, A. & Barry, A. 1988. Rennet coagulation of heated milk: influence of pH ad- justment before or after heating. J. Dairy Res. 55: 205- 215, Tessier, H. & Rose, D. 1958. Calcium ion concentrations in milk. J. Dairy Sci. 41: 351-359. Van Hooydonk, A. C. M., De Koster, P. G. & Boerrioter, I. J. 1987. The renneting properties of heated milk. Neth. Milk Dairy J, 41: 3-18. Visser, J., Minihan, A., Smits, P., Tyan, S. B. & Heertje, I, 1986. Effect of pH and temperature on the milk salt system. Neth. Milk Dairy J. 40: 351-368. Wheelock, J. V. & Kirk, A. 1974. The role of P-lactoglobu- lin in the primary phase of rennin action on heated casein micelles and heated milk. J. Dairy Res. 41: 367-372. Zittle, C. A., Thompson, M. P., Custer, J. H. & Cerbulis, J. 1962. K-casein - P-lactoglobulin interactions in solution when heated. J. Dairy Sci. 45: 807-810. Manuscript received April 1993 J. A. Lucey P. F. Fox Department of Food Chemistry National Food Biotechnology Centre University College Cork, Ireland J. A. Lucey C. Gorry Teagasc National Dairy Products Research Centre Moorepark, Fermoy Co. Cork, Ireland 368 Agric. Sei. Fint. 2(1993) SELOSTUS Kuumennetun maidon Juoksettumisominaisuudet J. A. Lucey, C. Gorry jaP. F. Fox Teagasc, National Dairy Products Research Centre ja University College Cork Kuumennus heikensi maidon juoksetteen saostumisominai- suuksia. Varastointi heikensi edelleen juoksettumista. Hapat- taminen pH 6,2:een tai alle tai CaCb:n lisäys pieninä konsen- traatioina paransivat huomattavasti juoksettumisominaisuuk- sia. Hapattaminen ensin alle pH s,s:een ja sitä seurannut pH:n nosto 6,6:een johti juoksettumismiskyvyn huomattavaan pa- ranemiseen, paitsi voimakkaasti kuumennetuissa maidoissa (120°C 10 min), joissa ei tapahtunut juoksettumista hapatta- misen/neutraloinnin jälkeen. Hapattaminen pH s,s:een tai alle ja varastointi alhaisessa pH:ssa 24 h ennen neutralointia johti juoksetteensaostumis- ominaisuuksien paranemiseen. Kuumennetun maidon (hapa- tettu ja sitten neutraloitu) sekoittaminen normaalin maidon kanssa johti juoksettumiskyvyn huomattavaan heikkenemi- seen. Aikaisemmin kuumennetun, hapatetun ja neutraloidun maidon uudelleen kuumentaminen aiheutti vain vähäisiä muutoksia juoksettumiskykyyn tai saostuman kiinteyteen. Kuummennetun maidon lisääminen raakamaitoon johti jäl- kimmäisen juoksettumiskyvyn paranemiseen mutta samalla saostuman kiinteys heikkeni. 369 Agric. Sei. Fin!. 2 (1993)