zy33 Recovery and Nutritional Evaluation of Proteinaceous Solids Separated from Whey by Coagulation with Chitosan

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1 Reprinted from JOURNAL OF DAIRY SCIENCE, Volume 59, November 1976, pages FKV Recovery and Nutritional Evaluation of Proteinaceous Solids Separated from Whey by Coagulation with Chitosan zy33 =ro W. A. BOUGH and 0. R. LANDES Department of Food Science Universitv of Georsla College of Agricultural Experiment Stations Georgia Station Experiment. GA j ABSTRACT Chitosan, a cationic carbohydrate polymer manufactured from chitin in shrimp and crab wastes, coagulated suspended solids in cheese whey as effectively or more so than ten commercial synthetic polymers. Concentrations of suspended solids after settling were reduced over 90% by coagulation at ph 6.0 with a ratio of chitosan to suspended solids of 2.15%(1:46.5). The yield of coagulated solids was approximately 2,270 mglliter. The proximate composition of the coagulated solids after freeze drying was 73% protein, 6% lactose, 10% ash, and 7% moisture. Without the aid of a coagulating agent, settling for 1 or 3 h reduced suspended solids by only 34% and 49%, respectively. Rat feeding studies showed no significant differences in the protein efficiency ratio of casein, coagulated whey solids containing chitosan, and whey solids containing no polymer. These results provide evidence of utility for coagulated whey solids as a protein supplement. INTRODUCTION Cheese whey has been described as the most concentrated liquid waste that can be found in large quantities (26). The total amount of cheese whey produced annually in the United States is reported to be approximately 13.6 billion kg (28). It contains 30,000 to 50,000 mglliter biochemical oxygen demand (BOD) which accounts for its high polluting potential and cost of waste treatment. Lactose and soluble proteins are the main components Received March Marine lit.sources Extension Center, Box 517, Rrunswick. GA Home Economics Dcpariment. Wylie Ilall. In& ana University, Uloomington of the soluble BOD. Suspended proteinaceous solids also contribute to the BOD unless physically removed. Fermentation of cheese whey with Succhuromyces frugilis results in conversion of lactose into single cell protein. A closed-loop fermentation system with essentially no effluents has been demonstrated on a liter scale (28). Evaporation of the whole fermented mass gave a product containing 35 to 50% crude protein, 12 to 20% ash, 2 to 3% fat, and 3 to 4% moisture. The protein efficiency ratio (PER) of the whole yeast fermentation solubles was 69% of the casein standard. If the yeast was separated from the fermented whey media by centrifugation, in contrast to evaporation of the whole media, the PER of the purified yeast was 91% of the casein standard. While a better quality protein could be obtained by separation of the yeast from the media, evaporation of the whole media containing yeast, whey solids, and minerals was.recommended to minimize waste effluents from the fermentation operation (28). The BOD of whey has been reduced 85% by growth of S. fragilis in a Waldhof aerator to produce single cell protein (26). Electrolysis has been an effective means of reducing the chemical oxygen demand (COD) by over 90% in acid cheese whey and of recovering some of the whey proteins. The COD coum be reduced from approximately 67,000 mg/liter to 5,000 mg/liter by electrolytic oxidation. The carboxylic acids produced by oxidation of carbohydrates could be removed further by charcoal adsorption if desired. During electrolysis, a froth was produced which contained 60% of the original whey protein. The froth residue when dried contained 25% protein and a minor amount of ash (27). The remaining 75% is assumed to be carbohydrate components. Ultrafiltration of cottage cheese whey has been shown to reduce thc BOD by 97% and generate protein and lactose by-products. The

2 COAGULATION OF WHEY PROTEINS WITH CHITOSAN 1875 process was demonstrated in a 4,500-kg-per-day pilot plant (11). The first step of the membrane process generated a protein concentrate which, upon drying, contained 80% protein solids. Deproteinized whey can be concentrated and formed into cattle feed blocks which still contain 25 to 30% moisture. Calves and steers readily consumed this by-product as lick blocks (1 3). The protein concentrates isolated from whey may be used as liquids or dried to give by-products containing from 25% to 80% protein, depending on the method of recovery (27, 11). Whey proteins have been utilized in beverages (16). high protein snacks (17), ground meats (IS), and other food and cosmetic applications (10). Inorganic salts of calcium and iron have been effective coagulants for proteins in cheese whey. dlcium oxide alone or in combination with acetone or methanol precipitated 64 to 91% of the nitrogen-containing compounds from cheese whey (9). The freeze-dried calcium oxide precipitate contained approximately 35% lactose, 5% protein, 2nd 50% calcium oxide. Whey treated with ferripolyphosphate yielded glliter, on a dry weight basis, of a material containing 15 to 50% protein, 8 to 15% iron, 30 to 50% phosphate, and 2 to 5% calcium. The product was reported to be highly nutritional and highly assimilable (20). A subsequent bioassay study of a ferripolyphosphate-protein powder showed it to be 92 to 100% efficient, in comparison to ferrous sulfate, for restoring hemoglobin levels of irondepleted rats and chicks. When used to fortify sterile whole milk, iron from the complex was bioassimilable (21). A ferric-whey protein product containing over 80% protein has been obtained by heating whey treated with ferric chloride at ph 2.5. The protein byproducts contained 81 to 88% protein, 2 to 4% lactose, 4 to 7% ash,.2 to 0.3% iron, and 4% moisture on a dry weight basis. The ferric whey protein products from cottage and cheddar cheese wheys had better balance of essential amino acids than casein, due to considerably higher contents of sulfurcontaining amino acids (1). Treatment of rennet or acid cheese whey with 700 or 1000 mg/liter, respectively, of sodium hexametaphosphate precipitated up to 80% by weight of the total proteins out of these wheys. When cations were removed by elution of the whey through a column of Amerliter IR-120 resin, the precipitation of proteins was increased to over 90%. The dry precipitated solids contained 70 to 85% protein, 10 to 20% sodium hexametaphosphate, and loto 15%lactose (15). A lactalbumin curd product containing 51% protein on a dry weight basis was obtained without any chemical coagulants by heating acidified cheese whey at ph 4.5 (19). Recent studies showed that the coagulated lactalbumin curd could be used satisfactorily as an extender in ground meat balls (18). Chitosan is a polymer manufactured from chitin in shrimp and crab processing wastes. Proteins and minerals are extracted from the crustacean shells, leaving chitin which is hydrolyzed briefly with hot concentrated alkali to remove some or all of the acetyl groups. The product of the deacetylation reaction is chitosan (24, 25). The glucosamine backbone of the polymer imparts to it a polycationic character which places chitosan in a similar class with other polyelectrolytes being used, as flocculating and coagulating agents. Chitosan has been shown to be an effective coagulating agent for suspended solids in processing wastes from vegetable (6), poultry (8), and egg breaking plants (5). It also proved to be an effective aid for coagulation of activated sludge in conjunction with centrifugal dewatering (7). The primary purpose of this study was to investigate the use of chitosan for coagulation and recovery of suspended solids in cheese whey. A recent survey of the dairy product processing industry reported the suspendedsolids load from cottage-natural cheese plants was 1.2 kg per 1,000 kg milk received (29). If 75% of this volume becomes whey, the suspended solids concentration in the raw whey would be approximately 1600 mg/liter. Removal of suspended solids in whey prior to fermentation (26, 28) might be desirable. Removal of these proteinaceous solids would prevent them from being solubilized in the media due to agitation or enzymatic dccomposition. Recovery of suspended solids by coagulation and settling also may be cost effective for reducing surcharges assessed by municipalities for treatment of industrial wastes. Reduced sewage charges and revenues from sale of coagulated cheese whey proteins may induce Journal of Dairy Science Vol. S9, No. 11

3 1876 BOUGH AND LANDES cheese plants to pretreat cheese whey rather than pay waste treatment charges on such concentrated waste. A secondary objective of the study was to compare the nutritional availability of cheese whey solids containg a polymeric coagulating agent (chitosan) with whey solids containing no polymer. The PER of by-products recovered with and without the aid of chitosan are compared to demonstrate the efficacy of these materials as sources of protein in animal feeds. MATERIALS AND METHODS Collection of Whey Samples Cheese whey for laboratory studies was collected and stored at 4 C prior to use within 48 h. The liquid supernant was decanted from solids which formed and settled upon cooling. This decanted whey was warmed to room temperature and used as substrate for evaluation of chitosan and other polymers in laboratory coagulation trials. Samples of hot cheese whey, approximately ph 4.5, for pilot-scale studies were collected in liter barrels lined with double layers of plastic.bags. The whey was allowed to cool at room temperature overnight and coagulation tests were performed on the raw whey during the next day. Laboratory Studies The procedure for laboratory coagulation trials (Jar Tests) to approximate optimum conditions of ph, stirring, and concentrations of chitosan, and other polymers has been described (5). Turbidity (30) was measured as formazin turbidity units (FTU) in the supernatant liquids of treated samples which had been allowed to settle for 1 h. Liquid samples were analyzed for suspended solids and COD (2). Samples of whey solids were freeze-dried prior to chemical analysis. Crude protein, calculated as N x 6.4 (15), was determined by a macro-kjeldahl method (3). Alkali-solubilizable protein was estimated by a biuret method (14). Moisturc, ash, and fat were determined by standard methods (4). Lactose content was measured by the phenol-sulfuric method (12). Pilot-Scale Coagulation and Recovery of Solids Coagulation and settling experiments were in the same liter barrels in which thesamples were collected. A total of 15 barrels, 5 per day, were collected on 3 different days. The volume of wastewater and concentration of suspended solids was determined for each barrel prior to treatment. After cooling overnight and raising the ph to 6.0 with 10 N NaOH, chitosan was added in proportion to suspended solids to give a ratio of chitosan to suspended solids of 2.15%. After mixed for 15 min with a mechanical stirrer, the contents of each barrel were allowed to settle with sampling every 30 min to determine changes in turbidity. Samples were withdrawn via a siphon tube extending 30.5 cm below the surface of the liquid. Suspended solids and COD were determined on samples collected after 1 and 3 h of settling. Solids coagulated with chitosan were collected by centrifugation at 2750 rpm (1300 x g) in a pilot-scale basket centrifuge (Model STM-1000, Western States Machine Co.). Sus-, pended solids in raw cheese whey were collected at 3000 rpm with the basket centrifuge. The solids recovered both with and without chitosan were freeze-dried and stored at -10 C until formulation of the rat diets. The PERs were determined for the cheese whey solids with and without the use of chitosan as a coagulating agent by the A.O.A.C. method (4) with the exception that eight animals wewused per group instead of ten. RESULTS AND DISCUSSION Results in Table 1 on the effect of ph on coagulation and reduction of turbidity in decanted cheese whey show that the lowest turbidity, 190 FTU, was achieved at ph 6.0. Treatment with 30 mg/liter chitosan at ph 4, 4.5, 5, 7, or 8 resulted in turbidity values of 550 FTU or greater. Changes in turbidity in response to different concentrations of chitosan at pll 4.5, the ph of decanted whey, and at ph 6.0, the optimum ph for treatment, are shown in Fig. 1. When coagulated with 30 mg/liter chitosan. the turbidity values in the settled supernatants treated at pll 4.5 and 6.0 were 530 and 150 FlCJ, respectively. The lowest turbidity value (150 1:TU) was by treatment with 30 mg/liter chitasan at ~ At ph 6.0, increasing the chitosan concentration to 60 and 90 mg/liter resulted in restabilization of the coagulated solids and increased turbidity. This Journal of Dairy Science Vol. 59, NO. 1 I

4 ~ ~~ ~~~ ~ COAGULATION OF WHEY PROTEINS WITH CHITOSAN 1877 TABLE 1. Effect of ph on coagulation and reduction of turbidity in cheese whey treated with 30 mglliter chitosan. PH Turbidity of supernatant (FTU) o > 1000 is a common phenomenon, due to overdosing with polymers (23); As the concentration of chitosan was increased further, the turbidity decreased as the floc again stabilized. Chitosan was compared to ten synthetic polyelectrolytes for effectiveness of treatment at ph 6.0. All were applied at 30 mgfliter, which was (Fig. 1) the most effective chitosan concentration for that batch of whey. Coagulation with chitosan resulted in a turbidity value of 150 FTU, the lowest value observed (Table 2). The next lowest values were obtained with Natron 88 and 6082 which gave turbidities of 200 to 220 FTU, respectively. Changing conditions of ph and concentration for the synthetic polymers would be expected to improve their effectiveness, but, under these conditions, chitosan was equivalent or superior in performance to the commercial polymers. For effective reduction of turbidity and suspended solids in raw cheese whey at ph 6.0, the optimum ratio of chitosan to suspended TABLE 2. Comparison of chitosan and various synthetic polyelectrolytes applied at a concentration of 30 mglliter for coagulation of solids and reduction of turbidity in decanted cheese whey at ph 6.0. Coagulating agent None Chitosan (+)a Betz 1160 (+) Betz 1190 (+) Natron 88 (+) Natron 6082 (+) Wt-2640 (+) Wt-2660 (+) Wt-2263 (+) Wt-2870 (+) Atlaxp 1N (N) Atlasep 105c (+) Turbidity a (+) indicates net positive charge on polymer; (N) indicates net neutral charge on polymer. solids was 2.0 to 2.5%. This corresponds to chitosan concentrations of 49 to 62 mgker for cheese whey containing an average concentration of 2470 mglliter suspended solids. A summary of the results on coagiilation of 15 separate 151- to 189-liter samples of cheese whey at ph 6.0 is in Table 3. Chitosan was applied to each barrel of whey at 2.15% of its suspended solids concentration. The average composition of COD and suspended solids in raw whey collected on three occasions is in Table 3, along with the average composition of all 15 barrels, which was 68,500 mg/liter COD and 2,470 mg/liter suspended solids. For this average concentration of suspended solids (2,470 mg/liter), treatment with a ratio of 2.15% chitosan corresponds to 53 mglliter of G c 5 2 ZOO-' \ \ \ ' \ compares closely with values of approximately 67,000 mg/liter COD reponed by Tzeng for raw cheese whey (28). It is also similar to the COD range of 64,188 to 67,213 mg/liter '. reported by Nilson and LaClair (22). However, *... the suspended solids concentration range, to 928 mg/liter, observed by Nilson and LaClair (22) on raw whey was less than the concentrations, 1781 to 2870 mg/liters, encountered in PH ph ,./ "

5 1878 BOUGH AND LANDES TABLE 3. Reduction of COD ad suspended solids in cheese whey coagulated at ph 6.0 with chitosan added in the ratio of 2.15% of suspended solids concentration. ComDosition (m&iter) susp. Reduction (%)a COD solids COD Susp. solids f SD Run raw raw 3h l h 3h 1 75, , Avg.b 68, arepresenu the average of five barrels. 151 to 189 liters each, which were coagulated and sampled. baverage of all 15 barrels which were coagulated and sampled. ' differences are presumed to be due to the processing and draining practices in the plants and the styles of products produced. Coagulation and removal of suspended solids by settling gave little reduction in the COD of the whey. Average COD reductions of 6.7, 3.3, and 2.2% were obtained for Runs 1, 2, and 3, respectively. However, treatment with chitosan and settling of the coagulated solids effectively reduced suspended solids by over 90%. Table 3 shows the average values and standard deviations for runs on three days where five barrels were treated per run. After 1 h of settling, average reductions in suspended solids were 96.6, 90.6, and 81.7 for the three runs. Corresponding reductions after 3 h were 97.1, 92.1, and 86.7% for Runs 1, 2, and 3, respectively. The average reduction and standard deviation in suspended solids concentration at 1 h for all fifteen barrels of whey treated with chitosan was 89.6% f 8%. After 3 h of settling, the overall reduction was 92% f 6%. Thus, approximately 2270 mg/liter could be recovered out of 2470 mg/liter suspended solids in raw cheese whey. These solids could be collected by centrifugation or filtration of the thickened sludge after the clarified supernatant had been decanted. Alternatively, the wet solids could be handled as a solid waste if the plant's only interest was to reduce surcharge payments on suspended solids in the waste discharge. Out of a total volume of 2384 liters of raw whey treated with a ratio of chitosan to suspended solids of 2.15%, the total amount of freeze-dried solids recovered was 3974 g or 1670 mg/liter. This weight corresponds to 68% of the total suspended solids in the whey. The other 32% of the suspended solids were lost in the effluent from the centrifuge and in cleaning out the wet solids from around the side baffles of the centrifuge basket. Scaling up to a commercial centrifuge would no doubt reduce the loss of solids which was inherent in the use of this small basket centrifuge. Without the aid of chitosan as a coagulating agent, the reduction of suspended solids due to settling was ineffective and erratic. For nine barrels, settling for 1 h at ph 4.5 reduced suspended solids by 34%, but the standard deviation was 36%. After 3 h of settling, the suspended solids concentration in raw whey was reduced 49% f 40%. Thus, without the use of chitosan to coagulate suspended solids, settling was ineffective to reduce the suspended solids concentration. The dry coagulated solids contained.15% fat, 9.5% ash, and 6% lactose (Table 4). Crude TABLE 4. Proximate analyses of cheese whey solids coagulated with 2.1 5% chitosan and raw solids containing no polymer. Coag- Component ulated Raw ( %) ( 96) Fat Ash Crude protein Biuret protein Lactose 6.O 6.6 Moisture Journal of Vairy Science Val. 59, No. I I

6 COAGULATION OF WHEY PROTEINS WITH CHITOSAN 1879 TABLE 5. PER of whey solids coagulated with chitosan and of reference samples. Protein content Sample of diet PERF SD (%) Casein Whey solids, no polymer Coagulated whey solids, 2.15% chitosan protein content determined by Kjeldahl analysis was 72.3%; the biuret assay estimated the protein content to be 73.8%. The composition of raw solids collected by centrifugation and without the addition of chitosan approximated that of the solids coagulated with chitosan. The small hifferences in individual components are not considered significant. The protein content of the coagulated solids (Table 4) is comparable to the 80% protein solids obtained by drying an ultrafiltration fraction (11). Similar results also were obtained on a dry ferric whey product reported to contain 81 to 88% protein and 4 to 7% ash (1). Whey proteins precipitated with sodium hexametaphosphate contained 10 to 20% of the phosphate salt and 70 to 85% protein (15). The products evaluated in this study may have wider application as a source of protein than those containing iron and phosphate coagulating agents because their use will not be limited by metal ion content. However, use of polymeric coagulating agents in food or feed applications must have the approval of the Food and Drug Administration in the United States. The results of rat feeding studies (Table 5) showed no significant differences between the PER of coagulated whey solids containing 2.15% chitosan (3.29 *.23) and the values for casein (3.44 f.26) and for whey solids containing no polymer (3.49 *.27). This is the first published demonstration, as best we can determine, that the presence of small amounts of a polymeric coagulating agent had no adversc effects on the nutritional value of proteins recovered from food processing wastes. These results providc evidence of utility for coaplated checse whey solids when contained approximately 73% protein on a dry weight basis. ACKNOWLEDGMENT The technical assistance of Tom Campbell, Chris Kiley, Lanny Stephens, and Stan Donehoo arc gratefully acknowledged. Chitosan, batch 4-74, was obtained from Food, Chemical, and Research Laboratories, Inc.. Seattle, Washington. Synthetic polymers (identified by prefix) were obtained from the following sources: Betz, Betz Laboratories; Natron, National Starch and Chemical Corporation ; W t, Calgo n Corporation; Atlasep, Atlas Chemical Corporation. This work was supported by the University of Georgia College of Agriculture Experiment Stations and by the National Sea Grant Program, U.S. Department of Commerce, Grant No, REFERENCES 1 Amantea, G. F.. C. M. Kason. S. Nakai. D. B. Bragg, and D. B. Emmons Preparation of ferric whey protein by heating. Can. Inst. Food Sci. Technol. J. 7(3): American Public Health Association Standard methods for examination of water and wastewater, 13th ed. Washington, DC. pp. 495, 536, and Association of Official Agricultural Chemists Official methods of analysis, 7th ed. Washington, DC. p Association of Official Analytical Chemists Official methods of analysis, 11th ed. Washington, DC. pp. 123, 300, and Bough, W. A Coagulation with chitosan-an aid to recovery of by-products from egg breaking wastes. Poultry Sci. 54: Bough, W. A Reduction of suspended solids in vegetable canning waste effluenrs by coagulation with chitosan. J. Food Sci. 40: Bough, W. A., D. K. Landes, J. Miller. C. T. Young, and T. R. McWhorter Utilization of chitosan for recovely of coagulated by-products from food processing wastes and treatment systems. Proceedings at Sixth National Symposium on Food Processing Wastes. U.S. Environmental Protection Agency. in press. 8 Bough, W. A., A. L. Shewfelt, and W. L. Saltcr Use of chitosan for the reduction and recovery of solids in poultry processing waste effluents. Poultry Sci. 54: Cerbulis, J Application of Steffen Process and its modifications to recovery of lactose and proteins from whey. J. Agr. Food Chem. 21 (2): Craig, T, W., J. C. Coliney, L. H. b rancis, and N. W. Iferlihy Development and product applications for a high protein concentrate from whey. Pood Prod. Dev. 4(6): Crowley s Milk Company Membrane processing of cottage cheese whey for pollution abate- Journal of Ihtiry Science Vol. 59. Nm 1 I

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