Microbial Production of Lysine and Threonine from Whey Permeate

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1983, p Vol. 45, No /83/ $02.00/0 Copyright 1983, American Society for Microbiology Microbial Production of Lysine and Threonine from Whey Permeate YOUNG T. KO1 AND JOHN R. CHIPLEY2* Ohio State University, Department of Food Science and Nutrition, Columbus, Ohio ; and United States Tobacco Company, Research and Development Division, Nashville, Tennessee Received 19 March 1982/Accepted 31 October 1982 Extracellular accumulation of lysine and threonine was investigated in modified whey permeate by using Brevibacterium lactofermentum ATCC and Escherichia coli ATCC Whey permeate was prepared from whey by membrane ultrafiltration, and lactose was hydrolyzed by treating permeate with HCI or 3-galactosidase. The highest amount of lysine (3.3 g/liter) was produced from a mixture of acid-hydrolyzed whey permeate and yeast extract (0.2%). The highest amount of threonine (3.6 g/liter) was produced from a mixture of whey permeate, (NH4)2SO4 (1.4%), yeast extract (0.1%), and Na2CO3 (0.3%). Annual whey production in the United States was estimated to be 30 x 109 pounds and 34 x 109 pounds in 1975 and 1976, respectively (5). In 1975, about 60% of the whey solids was utilized in human food and animal feeds, and the rest was wasted (4). Current trends indicate a steady increase in the availability of whey in the future, and new technologies need to be developed to enhance whey solids utilization. By the more recent processing method of ultrafiltration, whey can be fractionated into a protein concentrate and a deproteinized whey permeate fraction. Although utilization of protein concentrate appears to be commercially feasible, the disposition of large volumes of whey permeate still presents a serious problem. The permeate fraction represents about 90% of the original whey volume and contains from 80 to 85% of the original whey solids and most of the lactose in whey. The major component of whey permeate is lactose. Thus, the biological oxidation demand of the permeate is only slightly lower than that of the original whey (9). Studies related to the utilization of whey permeate, such as alcohol production (7, 15), conversion into animal feedstuffs (8, 12), production of oil and single-cell protein (14), production of xanthan gum (3), and utilization in canned beans (17) have been published. However, no studies have been reported on the use of whey permeate as a substrate for microbial production of amino acids. The objective of the present study was to establish a method for microbial production of two essential amino acids (lysine and threonine) from modified whey permeate. The effects of different methods of lactose hydrolysis and fortification of permeates with selected nutrients and buffering agents were also investigated. MATERIALS AND METHODS Preparation of whey permeate. Sweet whey powder (Teklac) was purchased from Foremost Foods Co., San Francisco, Calif. Twenty-eight grams of whey powder was reconstituted with 100 ml of demineralized double-distilled water (DDD water). Whey permeate was prepared by ultrafiltration through an Amicon UM-10 membrane (molecular weight cutoff, 10,000; diameter, 7.6 cm) at room temperature. The flow rate of the permeate was about 12 ml/h under a pressure of 40 lb/in2. Higher concentrations of whey powder reduced the flow rate significantly. Ultrafiltration was continued until the volume of concentrate reached 20% of the original volume. Preparation of AHWP. Hydrolysis of lactose in whey permeate was accomplished by HCI treatment. Whey permeate was mixed with concentrated HCI (final concentration, 1.3 N) and placed in a water bath at 60 C (6). After 24 h, the solution was cooled rapidly, and excess HCI was neutralized with concentrated NH40H to form NH4Cl (ph 7) and decolorized with carbon (2 g/100 ml). The final product, acid-hydrolyzed whey permeate (AHWP), was a light yellow liquid. Preparation of EHWP. To prepare enzyme-hydrolyzed whey permeate (EHWP), the lactose in whey permeate was hydrolyzed by p-galactosidase. The conditions of lactose hydrolysis were as follows (19). The incubation temperature and ph were 45 C and 6.9, respectively. The activity of,-galactosidase (from Escherichia coli) was 120 U/mg. The ratio of enzyme to lactose was 1:200. After 5 h of incubation, the mixture of enzyme and whey permeate was heated in a boiling water bath for 2 min to inactivate the enzyme and then filtered through Whatman no. 1 filter paper. The extent of hydrolysis was measured by determining the concentration of glucose. 610

2 VOL. 45, 1983 AMINO ACID PRODUCTION FROM WHEY PERMEATE 611 TABLE 1. Lysine production from whey permeate, AHWP, and EHWP by B. lactofermentum ATCC no. Mediuma Klett units Final ph glucoses (g/liter) Lysine (glliter) 1 Whey permeate (5.4% lactose),c 118d e % NH4Cl 2 AHWP (5.4% glucose)c (50 ml) AHWP (50 ml), 0.05% yeast extract AHWP (50 ml), 0.1% yeast extract AHWP (50 ml), 0.2% yeast extract AHWP (50 ml), 0.5% yeast extract AHWP (50 ml), 0.5% peptone AHWP (50 ml), 0.5% tryptone AHWP (50 ml), 0.1% yeast extract, NDf o CaCO3 10 AHWP (50 ml), 0.5% soytone EHWP (2.3% glucose),c 1% (NH4)2SO4, 0.2% yeast extract 12 EHWP, 1% (NH4)2SO4, 0.3% yeast extract 13 EHWP, 1% (NH4)2SO4, 0.5% yeast extract 14 EHWP, 1% (NH4)2SO4, 0.5% peptone a Media of treatments 2 through 10 contained 50 ml of demineralized, double-distilled water. b For this and all subsequent tables, residual glucose is the amount of glucose remaining in the medium at the end of the fermentation. c Initial concentration of lactose or glucose. d For this and all subsequent tables, 17 Klett units = 1 mg (dry weight) of cells. Unhydrolyzed lactose. f ND, Not determined. Microorganisms. For lysine production, Brevibacterium lactofermentum ATCC 21086, an auxotrophic mutant requiring threonine, isoleucine, and valine, was used throughout this work. The addition of methionine to the culture medium was also necessary for increased accumulation of lysine (Kubota et al., U.S. patent 3,527,672, 1970). For threonine production, E. coli ATCC 21151, an auxotrophic mutant requiring diaminopimelic acid and isoleucine, was used throughout this work (Nakayama et al., U.S. patent 3,684,654, 1972). Stock cultures of both organisms were maintained on nutrient agar slants. Culture medium. Whey permeate, AHWP, or EHWP was supplemented with one or more selected nutrients and buffering agents. The prepared substrate was sterilized by membrane filtration (Gelman Metricel membrane filter; 0.45-,.m pore size, 25-mm diameter) and placed in presterilized 500-ml flasks in 60-ml quantities. Cultivation method. The sterilized substrate (60 ml) was inoculated with 1 ml of B. lactofermentum ATCC or E. coli ATCC which had been previously cultured in nutrient broth for 48 h at 33 C. The fermentation was conducted in a controlled environmental shaker incubator at 33 C with agitation at 180 rpm. Depending upon the components of the medium, the maximum levels of lysine were produced within 5 to 7 days (18), whereas the maximum levels of threonine were produced within 4 to 6 days (11). All experiments were conducted in triplicate, and the results are expressed as averages. Analytical methods. Cell mass was estimated by measuring optical density of the broth at 660 nm and verified by determination of dry cell weight (13). An average value of 17 Klett units was found to be equal to 1 mg (dry weight) of cells. Glucose was determined by a glucose oxidase-peroxidase method with a glucose determination kit (Worthington Diagnostics, Freehold, N.J.). Lactose was determined by the anthrone method (1). The amount of lysine (or threonine) accumulated in the culture broth was determined by thin-layer chromatography (2, 16) as follows. Thinlayer chromatography plates were prepared with silica gel 60 G (E. Merck AG, Darmstadt, Germany). A 25-,ul sample (filtrate of the culture broth that was prepared by removal of microbial cells with membrane filtration) was applied to a thin-layer chromatography plate. The thin-layer chromatography plate was developed twice in a solvent system of n-propanol-concentrated NH40H-water (70:27:3), dried, and sprayed with ninhydrin reagent (0.5% ethanol solution). Lysine (or threonine) was identified by standards cochromatographed with each run. The band corresponding to lysine (or threonine) was removed and placed in a test tube containing 4 ml of 70%o ethanol. After centrifugation, the optical density of the supernates was read at 570 nm. The amount of lysine (or threonine) was estimated by comparison with standard curves. Chemicals. All amino acids were purchased from Sigma Chemical Co., St. Louis, Mo. Nutrient agar, nutrient broth, phenol red broth, yeast extract, peptone, tryptone, and soytone were purchased from Difco Laboratories, Detroit, Mich. Glucose, ninhydrin, and (NH4)2SO4 were purchased from Matheson, Coleman & Bell, Norwood, Ohio. Lactose, CaCO3, and Na2CO3 were purchased from Mallinkrodt, Inc.,

3 612 KO AND CHIPLEY TABLE 2. Effect of amino acids on the production of lysine, cell mass, final ph, and residual glucose KlettFinalResidual Lsn Supplementationa Suppemetaton' Kl.et t Fina lucs Lysine units ph glucose (g/liter) (g/liter) a Based upon previously reported values (11) for threonine (50 mg/100 ml), isoleucine (10 mg/100 ml), valine (30 mg/100 ml), and methionine (300 mg/100 ml) supplementation. The data for the control are taken from Table 1. Paris, Ky. Decolorizing carbon was purchased from Fisher Scientific Co., Fair Lawn, N.J. 1-Galactosidase was purchased from Worthington Diagnostics. Anthrone was purchased from Nutritional Biochemical Co., Cleveland, Ohio. Silica gel 60 G was purchased from E. Merck. NH4Cl was purchased from. Allied Chemical Co., Morristown, N.J. RESULTS Lysine production from whey permeate and AHWP by B. lactofermentum ATCC (i) Lysine production. Table 1 (treatment 1) shows lysine production in unhydrolyzed whey permeate. NH4CI (3.2 g/100 ml) was added as a nitrogen source since it occurred as a byproduct in AHWP. Unhydrolyzed whey permeate was not a good substrate since growth of the organism and production of lysine were minimal. The ph did not change significantly from an initial value of 6.7. Unhydrolyzed whey permeate was not used in subsequent experiments because it was apparent that lactose was not used by this organism (verified by inoculation of phenol red broth base plus lactose; data not shown) and that the nutrients in unhydrolyzed whey permeate were not sufficient for this organism to produce high levels of lysine. This organism cannot metabolize galactose (verified by inoculation of phenol red broth base plus galactose; data not shown). Lysine and cell mass production were very low from AHWP but were higher than in unhydrolyzed whey permeate (Table 1, treatment 2). AHWP was used in subsequent experiments because it appeared that lysine production could be stimulated by fortification with selected nutrients. (ii) Effect of selected nutrients and buffering agents. To determine whether AHWP could be improved as a substrate for lysine production, selected nutrients and CaCO3 were added to AHWP. CaCO3 was added as a buffering agent because the optimum ph during fermentation has been reported to be 5.0 to 9.0 (Kubota et al., U.S. patent 3,527,672, 1970). APPL. ENVIRON. MICROBIOL. The addition of yeast extract, peptone, tryptone, soytone, and CaCO3 improved AHWP as a substrate in all cases (Table 1, treatments 3 through 10). The addition of yeast extract (0.2%) resulted in the highest amount of lysine production (3.3 g/liter). This was not due to the presence of lysine in the nutrients since contribution of lysine by nutrients could account for less than 1% of the observed production. The addition of CaCO3 with yeast extract resulted in a relatively constant ph and increased depletion of glucose, but reduced lysine production. In the presence of CaCO3, maximum lysine production occurred with the addition of 0.1% yeast extract. Increasing the yeast extract level to 0.2% or higher did not increase the amount of lysine produced. Among the selected nutrients added to AHWP, yeast extract had significant stimulatory effects on the production of lysine. Therefore, the effect of yeast extract concentration on lysine production was investigated in detail. The relationship of yeast extract concentration to lysine production, cell mass, glucose utilization, and final ph is shown in Table 1 (treatments 3 through 6). Increasing yeast extract levels up to 0.2% stimulated lysine production with 3.3 g of lysine per liter being produced at this level. However, further addition of yeast extract (0.5%) reduced lysine production markedly and only stimulated cell mass production. The final ph and residual glucose tended to drop with increasing yeast extract levels. (iii) Effect of enzyme hydrolysis of lactose in whey permeate. Lactose in whey permeate was hydrolyzed by E. coli,3-galactosidase. The degree of hydrolysis was 92%. EHWP was fortified with (NH4)2SO4 (Kubota et al., U.S. patent 3,527,672, 1970; Nakayama et al., U.S. patent 3,684,654, 1972) and yeast extract or peptone and sterilized by membrane filtration. Table 1 (treatments 11 through 14) shows the effects of enzyme hydrolysis and additives on lysine production. Generally speaking, EHWP supported high amounts of cell mass production. Maximum lysine production (2 g/liter) was in EHWP containing 1% (NH4)2SO4 and 0.5% peptone. (iv) Effect of amino acid supplementation. B. lactofermentum ATCC is an auxotrophic mutant. Threonine, isoleucine, and valine must be present in the culture medium to support growth, and the addition of methionine is necessary to stimulate lysine production (Kubota et al., U.S. patent 3,527,672, 1970). The effect of amino acid concentration in culture medium was investigated in detail by the addition of amino acids at levels ranging from to 1 times the previously reported levels (Kubota et al., U.S. patent 3,527,672, 1970). The

4 VOL. 45, 1983 AMINO ACID PRODUCTION FROM WHEY PERMEATE 613 TABLE 3. Threonine production from whey permeate and AHWP by E. coli ATCC Treatment Klett Final Residual lactose no. Medium' units ph or glucose Threonine (gfliter) 1 Whey permeate (5% lactose)' Whey permeate, 0.1% yeast extract Whey permeate, 0.5% peptone Whey permeate, 0.5% tryptone Whey permeate, diaminopimelic acid (5 mg/ ml), isoleucine (2.5 mg/100 ml) 6 Whey permeate, 0.5% soytone Whey permeate, 0.1% yeast extract, 0.1% Na2CO3 8 Whey permeate, 0.1% yeast extract, 0.3% Na2CO3 9 Whey permeate, 0.1% yeast extract, 0.5% Na2CO3 10 AHWP (5.4% glucose)b (50 ml), 0.1% yeast ex tract 11 AHWP (50 ml), 0.5% peptone AHWP (50 ml), 0.5% tryptone AHWP (50 ml), diaminopimelic acid (5 mg/ ml), isoleucine (2.5 mg/100 ml) 14 AHWP (50 ml), 0.5% soytone AHWP (50 ml), 0.1% yeast extract, 0.1% Na2CO amedia of treatments 1 through 9 contained 1.4% (NH4)2SO4, and media of treatments no. 10 through 15 contained 50 ml of demineralized, double-distilled water. b Initial concentration of lactose or glucose. relationships of amino acid concentration to lysine production, cell mass, final ph, and residual glucose are shown in Table 2. Cell mass, glucose utilization, and ph reduction were in proportion to the amino acid concentration. However, lysine production decreased at amino acid concentrations above 0.25 times the previously reported levels. Threonine production from whey permeate and AHWP by E. coli ATCC E. coli ATCC was used as the test organism. Although most of the amino acid-producing bacteria cannot utilize lactose or galactose, E. coli ATCC can utilize both (verified by inoculation of phenol red broth base plus carbohydrate; data not shown). This organism is an auxotrophic mutant that requires diaminopimelic acid and isoleucine for its growth (Nakayama et al., U.S. patent 3,684,654, 1972). (i) Threonine production from whey permeate. Whey permeate (containing 5% lactose) with 1.4% (NH4)2SO4 added (as a nitrogen source) was the basic fermentation substrate. This base was fortified with one or more of the following nutrients: yeast extract, peptone, tryptone, diaminopimelic acid plus isoleucine, and soytone. Among the tested substrates, the highest amount of threonine (1.1 g/liter) was produced from whey permeate containing (NH4)2SO4 and diaminopimelic acid plus isoleucine (Table 3, treatment 5). Fortification with yeast extract was almost as effective, resulting in the production of 1.0 g of threonine per liter (Table 3, treatment 2). (ii) Effect of Na2CO3 on the production of threonine. During the preceding fermentations (Table 3), the ph of the medium dropped to 4.5 to 4.7. Because the optimum ph during fermentation is about 5.0 to 8.5 (Nakayama et al., U.S. patent 3,684,654, 1972), Na2CO3 was tested as a buffering agent. The results in Table 3 (treatments 7 through 9) show that the addition of Na2CO3 to a yeast extract-fortified base medium caused a significant improvement in the production of threonine. The highest amount of threonine (3.6 g/liter) was produced with 0.3% added Na2CO3. Further addition of Na2CO3 (0.5%) resulted in a higher final ph and reduced cell mass and threonine production. (iii) Threonine production from AHWP. AHWP was fortified with one or more of the following nutrients or buffering agents: yeast extract, peptone, tryptone, diaminopimelic acid plus isoleucine, soytone, and Na2CO3. Among the tested substrates, the highest amount of threonine (1.0 g/liter) was produced from a mixture of AHWP and diaminopimelic acid plus isoleucine (Table 3, treatment 13). The addition of Na2CO3 did not improve the production of threonine. Stimulation of threonine production by nutrient addition was not due to the presence of

5 614 KO AND CHIPLEY threonine in the above nutrients since contribution of threonine by these nutrients could account for less than 1% of the observed production. DISCUSSION Unhydrolyzed whey permeate was not a good substrate for the growth of B. lactofermentum and production of lysine because this organism cannot utilize lactose. However, the organism did grow and produced small amounts of lysine probably by use offree amino acids and peptides present in the permeate. AHWP was also a poor substrate for the growth of this organism and production of lysine. Substantial quantities of free amino acids and peptides were destroyed during the acid hydrolysis of whey permeate (data not shown). The organism is an auxotrophic mutant and requires certain amino acids for growth. Therefore, it became apparent that AHWP must be fortified with other nutrients to be of value as a substrate for industrial production of lysine. Among the nutrients tested, yeast extract had a remarkably stimulatory effect on the production of lysine. It appears that yeast extract contains certain components that stimulate lysine production. Yeast extract is an excellent source of B-complex vitamins, free amino acids, and peptides. These nutrients in yeast extract apparently stimulated lysine production. The effect of yeast extract deserves further comment because of its stimulatory effect on lysine production. Cell mass and lysine production were stimulated by increasing the yeast extract level up to 0.2%. However, further addition of yeast extract (0.5%) reduced lysine production markedly, only stimulating cell mass production. It is well known that a suboptimal concentration of a nutrient (in this case, amino acids) required for cell growth is necessary for the accumulation of lysine (10). The inhibitory effect of excess nutrients (amino acids) on lysine production would be the result of feedback inhibition of enzymes in the lysine biosynthetic pathway (such as aspartate kinase, a key enzyme for the synthesis of lysine, threonine, and methionine). This was supported by the observation that when high levels of amino acids were added to AHWP, lysine production was decreased. It is apparent that only suboptimal concentrations of the amino acids required for cell growth are conducive for the accumulation of lysine. EHWP would be a good substrate for the APPL. ENVIRON. MICROBIOL. production of lysine because it contains lower amounts of salts and higher amounts of amino acids and peptides as compared with the amounts in AHWP. However, enzyme hydrolysis of whey permeate did not result in stimulation of lysine production in comparison with acid hydrolysis. It is likely that the enzyme hydrolysis procedure (batch type) employed was not properly suited to this study. After hydrolysis, EHWP contained sufficient amounts of P-galactosidase to cause partial clogging of the membrane that was used for sterilization of the substrate. Partial clogging of the membrane would interrupt the passage of nutrients in the prepared substrate, and as a result, the sterilized substrate would be somewhat different from the original substrate. From this point of view, other enzyme hydrolysis systems, such as immobilized enzymes, may be more desirable for whey hydrolysis. E. coli ATCC 21151, which was used for threonine production, can utilize lactose. The use of unhydrolyzed whey permeate or whey permeate fortified with other nutrients did not result in production of high amounts of threonine. During fermentation, the ph of the medium dropped rapidly, which may have inhibited the growth of this organism and production of threonine. When unhydrolyzed whey permeate was buffered with Na2CO3 (up to 0.3%), threonine production was markedly improved. This is vivid evidence that ph is a critical factor in amino acid production. At a higher level (0.5%), Na2CO3 was inhibitory to both the growth of the organism and production of threonine. It is likely that the high level of Na2CO3 increased the osmotic pressure of the medium and inhibited growth of the organism. When AHWP or AHWP fortified with nutrients or buffering agents was used as a fermentation substrate, threonine production was relatively low. AHWP probably contained too much salts for active growth of the organism. In summary, the highest amount of lysine (3.3 g/liter) was produced from a mixture of AHWP and yeast extract (0.2%) by B. lactofermentum ATCC 21086, and the highest amount of threonine (3.6 g/liter) was produced from a mixture of whey permeate, (NH4)2SO4 (1.4%), yeast extract (0.1%), and Na2CO3 (0.3%) by E. coli ATCC LITERATURE CITED 1. Ashwell, G Colorimetric analysis of sugars. Methods Enzymol. 3: Bahl, S., S. Naqvi, and T. A. Venklitasubramanian Simple, rapid quantitative determination of lysine and arginine by thin-layer chromatography. J. Agric. Food Chem. 24: Charles, M., and M. K. Radjal Xanthan gum from acid whey, p In Extracellular microbial polysaccharides, American Chemical Society Symposium Series 45. American Chemical Society, Washington, D.C. 4. Clark, W. S., Jr Major whey products markets- 1975, p Proceedings of Whey Products Conference,

6 VOL. 45, 1983 Atlantic City, N.J. 5. Clark, W. S., Jr Major whey products markets J. Dairy Sci. 62: Coughli, J. R., and T. A. Nickerson Acid-catalyzed hydrolysis of lactose in whey and aqueous solutions. J. Dairy Sci. 58: Gawel, J., and F. V. Kosikowskl Improving alcohol fermentation in concentrated ultrafiltration permeates of cottage cheese whey. J. Food Sci. 43: Hargrove, R. E., F. E. McDonough, J. A. Alford, and P. G. Lynch Conversion of deproteinized whey to solid animal feeds. J. Dairy Sci. 57: Hargrove, R. E., F. E. McDonough, D. E. Lacroix, and J. A. Alford Production and properties of deproteinized whey powders. J. Dairy Sci. 59: Hhrose, Y., K. Sano, and H. Shibai Amino acids. Ann. Rep. Ferm. Proc. 2: Huang, H. T Production of L-threonine by auxotrophic mutants of Escherichia coli. Appl. Microbiol. 9: Lynch, G. P., M. I. Poos, and R. E. Hargrove Nutritional value of whey (permeate) blocks for calves. J. Dairy Sci. 57:652. AMINO ACID PRODUCTION FROM WHEY PERMEATE Mallette, M. F Evaluation of growth by physical and chemical means. Methods Microbiol. 1: Moon, N. J., E. G. Hammond, and B. Glatz Conversion of cheese whey and whey permeate to oil and single-cell protein. J. Dairy Sci. 61: O'Leary, V. S., C. Sutton, M. Bencivengo, B. Sullivan, and V. H. Holsinger Influence of lactose hydrolysis and solids concentration on alcohol production by yeast in acid whey ultrafiltrate. Biotechnol. Bioeng. 19: Patakl, G Thin-layer chromatography of amino acids, p In Techniques of thin-layer chromatography in amino acid and peptide chemistry. Ann Arbor Science Publishers, Ann Arbor, Mich. 17. Saylock, M. J., M. A. Uebersax, and R. C. Chandan Utilization of cheese whey permeate in canned beans. J. Dairy Sci. 62(Suppl. 1): Tosaka, O., and K. Takinaml Pathway and regulation of lysine biosynthesis in Brevibacterium lactofermentum. Agric. Biol. Chem. 42: Wierzbicki, L. E., and F. V. Kosikowski Kinetics of lactose hydrolysis in acid whey by B-galactosidase from Aspergillus niger. J. Dairy Sci. 56: