Ultrafiltration of Skim Milk at Refrigerated Temperatures

RESEARCH PAPERS UItrafiltration of Skim Milk at Refrigerated Temperatures D. J. KAPSIMALIS and R. R. Z A L L Department of Food Science Cornell University Ithaca, N Y 14853

ABSTRACT

Effects of temperature and membrane pore size on permeation flux rates and microbial quality of retentates and permeates during ultrafiltration of skim milk were investigated. Skim milk was ultrafiltrated at 15 and 45°C with either small pore ( i 0 , 0 0 0 molecular weight cut-off) or large pore (50,000 molecular weight cut-off) membranes. Standard plate counts of the retentates obtained during ultrafiltration at 15°C were lower than standard plate counts at 45°C. Permeation flux rates were almost four times greater at 45 than at 15°C with both small and large pore membranes. INTRODUCTION Ultrafiltration of Skim Milk at Refrigerated Temperatures

Good sanitary practices are mandatory for operating dairy processing equipment to ensure high quality products. Ultrafiltration equipment in the dairy industry is no exception. It is not only necessary to maintain a membrane plant with proper hygiene from a public health point of view but also to remove minute deposits of foreign substances which form upon membrane surfaces and inhibit permeation. Microbiological problems associated with ultrafiltration of skim milk have been documented. Maubois and Mocquot (9) reported that by concentrating skim milk at 50°C bacterial multiplication can be reduced to a factor of less than one, indicating little multiplication. Rash (10) was unable to repeat these results, reporting multiplication factors much greater than one. He found that normal sanitation recommendations from manufacturers of

Received September 19, 1980.

1981 J Dairy Sci 64:1945-1950

cellulose acetate membranes were ineffective in removing bacteria from the membrane, and microbial growth was profuse during the ultrafiltration operation. A more intensive study by Lee (7) into cleaning and sanitizing of membranes showed that while more severe methods of cleaning and sanitizing reduced the potential lifetime of the membranes, they can be cleaned. Kiviniemi and Seuranen (6) found that serious contamination of the ultrafiltration systems was more the rule than the exception. They recommended keeping the temperature of the feed at 48°C or above to prevent microbial growth. However, most cellulose acetate membranes cannot withstand temperatures above 40°C without risk of hydrolysis and compaction. Microbial growth is most abundant from 15 to 40°C. This fact combined with the sensitivity of the cellulose acetate membranes above 40°C suggest that ultrafiltration with these membranes be restricted to temperatures below 15°C. Unfortunately, lower temperatures result in decreased permeation flux rates primarily because of viscosity effects. Because viscosity is inversely proportional to temperature and directly proportional to concentration of the feed, an increase in viscosity of milk increases the thickness o f the laminar layer and, hence, increases deposit formation (11). Permeation rate is decreased by increasing resistance of the boundary (6) and by decreasing the transfer of materials away from the membranes. Decreasing the temperature (and subsequently increasing viscosity) results in a decrease in flux averaging 3% per °C (2). Thus, if the use of lower temperatures is to control microbial growth during ultrafiltration of skim milk, it is important to compensate for loss in flux rates. It was hypothesized that permeation rate could be increased by using large pore cellulose acetate membranes during ultrafiltration of skim milk

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KAPSIMALIS AND ZALL

at refrigerated temperatures. Large pore membranes might compensate for the decreased permeation flux expected by increased boundary resistance and increased viscosity. This study was designed to evaluate effects of temperature and membrane pore size on permeation flux rates and microbial growth in permeates and retentates produced during uhrafiltration of skim milk. MATE R IA LS A N D METHODS Materials

Fresh, pasteurized skim milk in 6-liter amounts were obtained from the Cornell Dairy Plant for all experiments. Apparatus

A bench-top size uhrafiltration system of about 8-liter capacity was designed consisting of one membrane module, a centrifugal pump, inlet and outlet pressure gauges, a reservoir tank, valves, constant temperature circulator, and tygon tubing. Figure 1 shows a schematic drawing of the system. Two cellulose acetate membrane modules (1.1 m 2 membrane area) were obtained from Abcor, Inc. (Cambridge MA): a large pore membrane with a molecular weight cut-off of 50,000 and high permeability and a small pore membrane with a molecular weight cut-off of 10,000 and low permeability. Experiments were at either 15 or 45°C. The refrigerated temperature of 15°C was maintained by placing the entire uhrafiltration system in a walk-in cooler kept at 4°C and by keeping the reservoir tank in an ice bath. It was not possible to run the system at a temper-

Figure 1. Schematic diagram of ultrafihration bench pilot plant model with one membrane module. Journal of Dairy Science Vol. 64, No. 10, 1981

ature below 15°C because of the heat of friction created transferred to the liquid with pumping the milk through the system. A temperature of 45°C was maintained by placing the reservoir tank in a water bath at 45°C, controlled by a constant temperature circulator (Model E l 2 , Haake, Saddlebrook, N J). Analytical Methods

After 15, 30, 60, 120, 180, and 240 min, permeate was collected and the volume measured. Permeation flux rates were recorded as milliliters per minute. Dilutions of retentate and permeate samples were plated on standard plate count agar every hour. Microbiological tests were according to Standard Metbods for the Examination of Dairy Products (1). Total solids were measured by drying permeate and retentate samples in a forced draft air oven at 100°C for 24 h. Chemical oxygen demands (COD) were determined by a rapid COD test (5). Conductivities of permeates were measured at 25°C in an electrolytic conductivity meter (Hach, Ames, IA). The pH was measured by an Accumet pH Meter, Model 120 (Fischer, US).

Experimental Design

A 22 factorial experiment was designed in which we compared treatments that could be formed by combining different factors. The factors were:

Temperature

Membrane pore size (Molecular weight cut-off)

15°C 45°C

50,000 10,000

There were four combinations of treatments in duplicate, each of which was run individually on separate days. The order of runs was random by a program on a Model 9280 Hewlett-Packard Calculator.' Permeate flux rates and microbial counts were recorded for most combinations. Analysis of variance was used to determine which factorial effects were significant according to methods described by Cochran and Cox (4) for 22 factorial experiments. Pressure was at .05 mPa. Permeate was removed and retentate returned to feed tank (batch method).

ULTRAFILTRATION OF SKIM MILK Feed velocity through the system was about 15.1 liters/min. RESULTS Effects of Temperature and Membrane Pore Size on Microbial Growth During Ultrafiltration of Skim Milk

Factors of bacterial multiplication (F) are in Table 1. These F-values are ratios comparing standard plate counts of retentates collected at different time intervals during ultrafiltration to original standard plate counts of skim milk. A concentration factor corrects the F for increasing concentration of retentate over time. These F-values are not to be confused with those in thermal processing calculations. Table 1 shows an increase in microbial counts during ultrafiltration independent of the degree of concentration with time. The Fvalues were greater at 45 than at 15°C. The F-values of retentates during ultrafiltration by the large pore membranes were greater than those for the small pore membranes at 45°C. There was no microbial growth in permeates of small pore membranes and minimal microbial growth in permeates of large pore membranes. Effects of Temperature and Membrane Pore Size Permeation Flux Rates

As in Figure 2, permeation flux rates were almost four times greater at 45°C than at 15°C, an average of 8.10 and 2.24 ml/min. Permeation flux rates decreased with both

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large and small pore membranes at 15°C. Permeates from ultrafiltration runs at 45°C with large pore membranes were cloudy in appearance during the first 2 to 3 h of the run, after which they became clear. Permeates from ultrafiltration runs at 15°C with large pore membranes were virtually clear. Conductivity, chemical oxygen demand (COD), and total solids of the permeates were higher in permeates collected at 45 than at 15°C. The pH were lower at 45 than at 15°C. These data are in Tables 2 and 3. DISCUSSION

Microbial growth in retentates obtained during ultrafihration at 15°C was considerably less than microbial growth in retentates obtained at 45°C. This indicates that refrigerated temperatures (15°C) essentially controlled microbial growth during the 4-h ultrafihration runs. Absence of microbial growth in permeates from small pore membranes was expected since the size of the microorganisms exceeded membrane pore size, preventing them from passing through the membrane. As to the microbial growth in permeates from large pore membrane, the data are unclear because here, too, bacterial size exceeded cut-off size. The counts could have been from inadequate cleaning and sanitation of the membrane backings, illustrating the difficulty in properly cleaning the membranes. Effects of refrigerated temperatures on permeation flux rates were negative. The

TABLE 1. Microbial growth in retentates during uhrafihration runs expressed as a factor o f bacterial multiplication, a Time (min)

Small pore membrane b 15°C 45°C

15°C

45°C

30 60 120 180 240

3.62 3.29 2,74 3.67 2.65

1.72 1.71 2.48 3.28 3.98

2.00 5.60 8.50 14.90 37.10

aFactor of bacterial multiplication

Large pore membrane c

2.00 5.00 2.78 5,96 9.79 Standard plate c o u n t of retentate

Original plate count X concentration

bMolecular weight cut-off = 10,OOO. CMolecular weight cut-off = 50,000. Journal of Dairy Science Vol. 64, No. 10, 1981

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KAPSIMALIS AND ZALL

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Figure 2. Effect of running time on permeation flux rates at 15 and 45°C with large and small pore membranes.

fourfold decrease in permeation flux rates with both small and large pore membranes at 15 compared to 45°C suggests that increasing pore size of membranes to increase flux rates at low temperatures is not a feasible idea. Effects of temperature on viscosity are well-known; decreasing temperature increases viscosity which decreases flow velocity of the feed through the membranes. This decreased flow velocity results in a decreased permeation flux rate. However, the extent of the difference in permeation flux rates and the appearance of the permeates collected during the various runs suggests that other factors were also responsible for the substantial decline in permeation flux rates between runs at 15 and 45°C. Permeates obtained at 45°C were cloudy in appearance compared to the virtually clear

permeates obtained at 15°C from large pore membranes. The cloudy appearance may be from colloidal particles of sufficient particle size to scatter visible light, probably protein molecules or colloidal calcium phosphate. The higher conductivities, COD's, and total solids of the permeates indicate that more solutes and solutes of higher molecular weight passed through the small and large pore membranes during ultrafiltration of skim milk at 45 compared to 15°C. This suggests that temperature of the feed may: 1) alter the membrane's configuration in such a way that the effective molecular size cut-off is changed; 2) alter the grouping of the molecules; or 3) affect material deposit formations on the membrane accumulated during ultrafiltration. Temperature may influence the "tightness" of the membranes. At low temperatures, membranes tend to be tighter, thus prohibiting passage of particles with larger molecular weight. Permeates obtained at 45°C had a higher total solids content than permeates obtained at 15°C. The possibility that the size of molecules may be altered is difficult to discern because of the many unanswered questions involving the structure of the casein micelle. We agreed with the assumptions taken by Webb et al. (12) that the casein micelle is a porous, highly hydrated complex structure with some degree of subunit variations and that the micelle contains colloidal calcium phosphate and is surrounded by a double layer of ions. The outer layer of ions is in equilibrium with those of the solvent. The bulk of inorganic ions is not in equilibrium and is not dispersed readily.

TABLE 2. Select analytical data of permeates obtained from ultrafiltration of skim milk at 15°C.a Process time (rain)

Total solids (%)

Conductiviry (mhos)

COD (mg/Iiter X 103 )

pH

30 60 120 180 240 300 360

1.56 3.33 4.62 5.18 5.41 5.58 5.54

1.90 3.30 4.00 4.40 4.45 4.45 4.50

1.67 ... 4.74 6.03 4.95 5.03 5.12

6.90 6.80 6.75 6.70 6.70 6.70 6.70

aMembrane molecular weight cut-off = 50,000. Journal of Dairy Science Vol. 64, No. 10, 1981

ULTRAF1LTRATION OF SKIM MILK

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TABLE 3. Select analytical data of permeates obtained from ultrafiltration of skim milk at 45°C. a Process time (min)

Total solids (%)

Con ductivity (mhos)

COD (rag/liter X 103 )

pH

15 30 60 120 180 210 265

4-.16 6.11 6.29 6.48 6.13 5.95 5.77

4.20 5.90 6.00 6.10 6.15 5.80 5.95

5.47 7.22 8.38 7.43 7.81 7.43 8.32

6.25 6.25 6.30 6.20 6.15 6.15 6.15

aMembrane molecular weight cut-off = 50,000.

A c c o r d i n g to Carroll et al. (3), at l o w e r t e m p e r a t u r e s s o m e o f t h e beta-casein readily dissociates f r o m t h e casein micelle a n d e n t e r s the serum phase together with kappa-casein and alpha-casein. Lee and M e r s o n (8) s h o w e d t h a t t h e m a j o r d e p o s i t o n m e m b r a n e s in ultrafiltrat i o n o f skim milk was casein micelles l i n k e d b y bridges to f o r m a l a t t i c e ; t h e y r e p o r t e d t h a t c h e m i c a l analysis of t h e s e d e p o s i t s c o n f i r m e d t h a t t h e y c o n t a i n e d a high casein c o n t e n t a n d s h o w e d t h a t clacium p h o s p h a t e was precipit a t e d in t h e d e p o s i t t h r o u g h o u t t h e m e m b r a n e . In t h e s e e x p e r i m e n t s w i t h skim milk, t h e d e p o s i t f o r m a t i o n m a y have b e e n r e s p o n s i b l e for t h e i n a b i l i t y of large pore m e m b r a n e s to c o m p e n s a t e for decreased flux rates at refrige r a t e d t e m p e r a t u r e s . T h e s e data suggest t h a t lower t e m p e r a t u r e s decrease p e r m e a t i o n flux rates d u r i n g u l t r a f i l t r a t i o n of skim milk n o t o n l y b y s u b s t r a t e viscosity b u t also b y t i g h t e n i n g o f m e m b r a n e pores a n d disassociation of betacasein f r o m casein micelles w h i c h increases deposit formation. CONCLUSIONS

T h e decrease in p e r m e a t i o n flux rates overrides t h e i m p r o v e m e n t in m i c r o b i a l q u a l i t y o f r e t e n t a t e s , w h i c h leads to t h e c o n c l u s i o n t h a t using large p o r e m e m b r a n e s d u r i n g ultraf i l t r a t i o n of skim milk does n o t a p p e a r feasible at this time. ACKNOWLEDGMENT

We are i n d e b t e d to A b c o r , Inc. for p r o v i d i n g the experimental apparatus.

REFERENCES

1. American Public Helath Association, 1978. Standard Methods for the examination of daiy products. 14th ed. E. H. Marth, ed. Am. Publ. Health Assoc., Washington, DC. 2. Breslav, B. R. and B. M. Kilcullen. 1977. Hollow fiber ultrafiltration of cottage cheese whey: A performance study. J. Dairy Sci. 60:1379. 3. Carroll, R. J., M. P. Thompson, J. R. Brunner, and C. Kolar. 1967. Aggregation of casein micelles prepared by low temperature centrifugation of skim milk. J. Dairy Sci. 50:941. 4. Cochran, W. G. and G. M. Cox. 1957. Experimental design. 2nd ed. Wiley Intersci., New York, NY. 5. Fenton-May, R. 1., C. G. Hill, C. H. Amundson, M. H. Lopex, and P. D. Auclair. 1972. Concentration and fractionation of skim milk by reverse osmosis and ultrafiltraion. J. Dairy Sci. 55:1561. 6. Jeris, J. 1967. A rapid COD test. Water and wastes engineering. 7. Kiviniemi, L., and A. Seuranen. 1974. Microbial growth during the ultrafiltration of sweet whey and skim milk. Kemia-Kemi 12:791. 8. Lee, A.Y.C. 1975. A study into mehtods of cleaning and sanitizing ultrafiltration membranes. M.S. thesis, Cornell University, Ithaca, NY. 9. Lee, D. N., and R. L. Merson. 1975. Examination of cottage cheese whey by scanning electron microscopy: Relationship to membrane fouling during ultrafiltation. J. Dairy Sci. 58: 1423. 10. Maubois, J. L., and G. Mocquot. 1971. Preparation of cheese from liquid pre-cheese obtained by ultrafiltration of milk. Le Lair 51:495. 11. Rash, K. 1976. Studies on the behavior and prevention of enteropathogenic E. coil and other coliforms in Cammembert cheese made by ultrafiltration processes. Ph.D. thesis, Cornell University, Ithaca, NY. 12. Skudder, P. J., F. A. Gloyer, and M. L. Green. Journal of Dairy Science Vol. 64, No. 10, 1981

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1977. An examination of the factors affecting the reverse osmosis of milk with special reference to deposit formation. J. Dairy Res. 44:293.

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13. Webb, H. B., A. H. Johnson, and J. A. Alford. 1974. Fundamentals of dairy chemistry. 2nd ed. AVI Publ. Co., Inc., Westport, CT.