Biotechnol. Prog.

1991, 7 , 67-69

67

NOTES
Fermentative Production of Injectable-Grade Calcium Gluconate
Deepak N. Shah? and Raman M. Kothari'
Industrial Fermentation Division, Sarabhai Research Centre, Baroda 390 007, India

Optimized economical procedures for the submerged batchwise (250-L scale) production and recovery of injectable-grade calcium gluconate are devised. While the optimal parameters of production envisaged the use of Aspergillus niger, salts-fortified dextroserich production medium, pH 6.5 f 0.1, 29 f 1 "C, 250 f 10 rpm, 1.0-1.5 vol of air (vol of medium)-' min-I aeration rate over 24 f 2 h duration, and intermittent neutralization with calcium carbonate, t h e optimal recovery procedure consisted of charcoal decolorization, vacuum filtration, methanol-aided precipitation, centrifugation, vacuum drying, % recovery of calcium gluconate, passing Indian/British a n d pulverization t o provide 85 5 pharmacopeial tests for the injectable-grade preparation.

Introduction The production of injectable-grade calcium gluconate by using an electrolytic process has been a closely guarded trade secret. Moreover, the process being energy and capital intensive, and electricity being costly in India, calcium gluconate injections are exorbitantly costly, in turn questioning the continuation of this process. Its preparation through alternate modes of production always posed a formidable problem of solubility. Most of the batches of calcium gluconate turned turbid either immediately after dissolution or subsequently upon storage, thus disqualifying them as injectable grade, solely on solubility characteristics. They met with pharmacopeial approval to be useful as tablet grade, in turn, causing a tremendous difference in profitability. To alleviate these problems, we have devised a simple and reproducible procedure for its fermentative production, an account of which is given here. Materials and Methods Chemicals. Corn steep liquor (CSL), dextrose monohydrate (Maize Products, Ahmedabad, India), diammonium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, urea, calcium carbonate, hydrochloric acid, and activated charcoal (Sarabhai M. Chemicals, Baroda, India), caustic lye (Gujarat Alkalies & Chemicals, Baroda, India), antifoam H-601 (Hico Products, Bombay, India), methanol (GNFC Ltd., Bharauch, India), dicamol (Amol Dicalite, Kadi, India), metal-distilled (DM) water, and analytical-grade chemicals were used. Culture. Aspergillus niger was subjected to UV mutagenesis and picked up on PDA slants (Shah et al., 1987)

* To whom correspondence should be addressed at the Biotechnology Division, Thapar Corporate Research and Development Centre, Patiala 147 001, India. t Present address: Biotechnology Department, Research Centre, Gujarat State Fertilizer Co. Ltd, Fertilizernagar, Baroda 391 750, India.
8756-7938/91/3007-0067$02.50/0

for its high glucose oxidase activity, incubated for 72-96 h at 29 f 1 "C, and upon sporulation preserved a t 4 "C. Analytical Methods. Glucose was estimated as percent reducing sugars as per the method of Bernfeld (1955). Calcium gluconate was estimated by the hydroxamate method as per Lien (1959). Chromatography of organic acids was carried out on Whatman No. 1 paper by using n-butanol/formic acid/water (5:1:4 v/v/v) as a solvent phase, spraying with 0.2% (w/v) bromocresol purple in 8% (w/v) formalin in ethanol, and drying; yellow spots against a blue background were developed by exposing the paper to ammonia vapors. Calcium gluconate preparations were further tested as per Indian Pharmacopeia (IP)/British Pharmacopeia (BP). Suitability tests for injectable-grade calcium gluconate were carried out as per the procedures of May & Baker Co. and Sarabhai M. Chemicals Ltd. The May & Baker test composed of suspending 50 g of calcium gluconate in 50 mL of water to which a filtered solution of 10 g of boric acid in 170 mL of water was added. The volume was made up to 250 mL with hot DM water. The solution must be colorless and completely free from opalescence. The SMC suitability test composed of suspending 50 g of calcium gluconate in 50 mL of boric acid solution (made up to 10 g of boric acid in 50 mL of glycerin) and adding 150 mL of hot water to make up the final volume to 200 mL. The resultant solution should be colorless and completely free from opalescence. For judging total solubility, in both these tests, the final solution was read for optical density at 590 nm. Optical density of 0.005 was considered the maximum allowable to pass the material as injectable grade. Media. Table I gives the compositions (w/v) of an optimized inoculation medium (IM) and a production medium (PM), adjusted to pH 5.5 and 6.5, respectively. While the IM was sterilized at 121 "C for 20 min, the P M was sterilized at 100 "C for 10 min only. Inoculum. A 10-mL suspension containing 1 X l o 7 spores, prepared in 0.19 ; aqueous Tween-80, incubated in a 500-mL Erlenmeyer flask containing 100 mL of IM on a rotary shaker (220 rpm, 29 f 1 "C, 24 h) served as an

0 199 1 American Chemical Society and American Institute of Chemical Engineers

60 Table I. Composition of Inoculation and Production Media

ingredient dextrose monohydrate diammonium hydrogen phosphate CSLC potassium dihydrogen phosphate magnesium sulfate urea H-601

Biotechnol. Prog., 1991, Vol. 7, No. 1

IWng/L
5 0.03 0.2 0.02 0.02 0.01 0.05

PMVb g/L 20 0.02

0.04 0.01 0.01 0.04 0.05

Table 11. Profiles of Calcium Gluconate Recovery as a Function of Processing Steps

E/C

volume, step spent liquor filtration (inclusive of washings) charcoal treatment vacuum concentration precipitation, washings, and centrifugation

L
30 36

calcium total, % gluconate kg recovery

20 16
14 18

6 5.8 5.5 5.4 5.1

100 96 92 90 85

40 30

Initial pH was 5.5. Initial pH was 6.5. Presterilized at 121 "C for 30 min.

inoculum. A 10% inoculum was transferred to 100 mL of P M in a 500-mL Erlenmeyer flask and, during scaling-up studies, to 250 L of P M in a 600-L pilot bioreactor. Fermentation. Calcium gluconate production was standardized to 250 L by undertaking 25 batches with 10 c( inocula and production medium with the above given composition. The optimal rate of agitation was 240 f 20 rpm, and the optimal rate of aeration was 1 volume of sterile air per volume of PM per minute (vvm) for the first 12 h and then 1.5 vvm until harvesting (24 f 2 h). Air pressure and temperature during fermentation were 10 psi and 29 f 1 "C, respectively. A 270 (wjv) calcium carbonate slurry upon sterilization (121 "C, 90 min) was added in three installments such that 6 kg was added in the first 3 h, 3 kg was added during 13-16 h, and a just adequate amount was added upon harvesting (residual reducing sugar 0.2 70, 22-26 h) to adjust the pH, which was 3.4 f 0.1, to 5.4. Postfermentation Processing. The spent liquor was heated (75 "C, 10 min), filtered hot through a preformed dicamol bed in a filter press (60 X 60 cm plate, 3 plates) at a rate of 40 L/h to remove mycelial cake. The free gluconic acid content of clearate was determined (Lien, 1959) and accordingly it was neutralized with a calcium carbonate slurry (60 "C, 1 h, pH 5.4 f 0.1) by constant stirring. Subsequently, the calcium gluconate slurry was decolorized (ZCL charcoal, 60 "C, 1h, continuous stirring) and filtered hot through the above-mentioned filter press. Calcium gluconate (18 f 2 % , w/w) was optimally precipitated (15 "C, 8 f 2 h) by addition of 1 5 T (v/v) methanol, leaving minimal material in the mother liquor, without affecting solubility characteristics. The precipitated matter was spun in a basket centrifuge (1700 rpm, 27 "C) and spray-washed with methanol to obtain a snowwhite, fluffy, tasteless, and odorless cake of calcium gluconate. Finally, it was vacuum-dried at 45 "C and pulverized to the desired mesh size. All the operations after decolorization were performed in a fiber- and dustfree area. Calcium gluconate, thus prepared, was subjected to the following tests as per Indian and British Pharmacopeia (IP, BP): description, identity, solubility, clarity, acidity, arsenic and heavy metal content, chloride and sulfate content, magnesium and alkali salts, boric acid test, dextrose content, bacterial and mold count, and assay.

Results and Discussion Effect of Fermentation Parameters. The above protocol provides a simple and cost-effective batchwise mode of fermentative production of injectable-grade calcium gluconate. A 10% (v/v) inoculum has promoted a faster rate of conversion of glucose to gluconic acid, leaving the least amount (0.2%, w/v) of residual dextrose a t harvest period. This observation is significant since dextrose concentrations exceeding 0.5 % at harvest period gave a slightly yellowish calcium gluconate preparation.

Higher rates of aeration in the later period of fermentation (after 12 h) expedited the harvesting period by 4-6 h. Crucial Recovery Steps. The profiles of recovery as a function of processing steps are summarized in Table 11. Due to nonavailability of appropriate equipment, only 30 L of spent liquor was processed out of a total of 250 L. Most of the losses up to the vacuum concentration step are due to handling of large volumes of almost concentrated solution. The overall 85 % recovery is quite satisfactory and the mother liquor could be processed in the next batch of calcium gluconate preparation for marginal improvement in recovery. In our opinion, the first crucial step in the recovery process is neutralization of the spent liquor after the removal of the mycelial cake, since lower pH (4.8-5.2) led to failure in the acidity test and higher pH (above 5.5) led to opalescence, disqualifying it from being injectable grade. The quality and quantity of charcoal used for decolorization also played an important role in causing minimal changes in pH after neutralization, besides giving a colorless preparation. Addition of 15% (v/v) methanol not only promoted optimal recovery; a methanol wash also expedited complete and rapid drying by alleviating foul smell formation during drying and by prohibiting fungal growth during storage. Temperatures higher than 45 "C for drying led to opalescent preparations for reasons so far not clearly understood. Specifications of Calcium Gluconate. Calcium gluconate preparations, thus obtained, passed all the tests given above as per IP/BP to qualify as an injectable grade with 99.9% purity. Difficulties in Upgradation. It seems that upgradation of tablet-grade calcium gluconate to injectable grade is not feasible for a variety of reasons: (i) Redissolution and recrystallization, although the simplest classical method of purification (upgradation), did not selectively remove the insoluble impurities, presumably due to similar solubility and precipitation characteristics of marginal extra calcium carbonate in calcium gluconate. While only a few batches passed as injectable grade (due to solubility), the majority of the batches failed (due to opalescence), for reasons not quite clear. (ii) Selective conversion and subsequent removal of extra calcium carbonate by acidification using HCl/HZS04 as soluble (CaC12) or insoluble (CaS04) salt did not materialize, since acidification led to less recovery. (iii) It was, however, found that the use of technical-grade calcium carbonate for neutralization of gluconic acid led to turbidity due to presence of marginal impurities in it (such as calcium hydroxide, calcium sulfate, Ba2+,etc.). Their presence at more than 10 ppm led to opalescence/turbidity. The possible presence of citrate and oxalate salts of calcium (which are intrinsically insoluble) as byproducts of fermentation causing opalescence was investigated. However, neither citrate nor oxalate salts could be detected. (iv) The spore count (1 X lO5/g), molds, specks, and fibers arising out of fermentation process/atmosphere/ filtration process/ postfermentation operations until pulverization and packing contributed to

Biotechnol. Prog. 1991, 7 ,69-71

69

opalescence to some extent. These could, however, be removed by optimally autoclaving (121 C, 90 min) the desired solution of calcium gluconate and passing it through successive steripads (White Clouds Co., Pune, India). Three passages were adequate for the removal of such a high spore count. In a nutshell, we have a reproducible and cost-effective protocol for the preparation of injectable-grade calcium gluconate meeting desired pharmacopeia1 specifications. Its success depends upon crucial recovery steps discussed above. Any attempt at upgradation of tablet-grade calcium gluconate is not a commercially feasible proposition.

Indian Pharmacopeia, 3rd ed.; Controller of Publications, Government of India: New Delhi, India, 1985; pp 85-86. Lien, 0. G., Jr. Determination of gluconolactone, galactonolactone, and their free acids by the hydroxamate method. Anal. Chem. 1959,31, 1363-1366. Shah, D. N.; Shah, N. K.; Kothari, R. M. Isolation of transglucosidase nonproducing mutant of Aspergillus awamorii yielding improved quality and production of amyloglucosidase. J.
Ind. Microbiol. 1987, 2, 175-180.

Literature Cited Bernfeld, P. Enzymes of carbohydrate metabolism. Methods Enzymol. 1955, 1, 149-158.

Accepted October 25, 1990.

Registry No. Calcium gluconate, 299-28-5.

Determination of Antigenic Characteristics and Stabilities of Mycoplasma hyopneumoniae

C. N. Weng,? S. Y. Lin,i Y. L. Tzan,s and C. J. Lee*9
Department of Chemical Engineering and Center of Bioengineering, National Tsing Hua University, Hsinchu, Taiwan, ROC, Department of Medical Research, Veterans General Hospital, Taipei, Taiwan, ROC, and Department of Pathology, Pig Research Institute, Chunan, Taiwan, ROC

T h e aim of this study was t o find a titering method that could rapidly and accurately determine the antigenic activities of Mycoplasma hyopneumoniae under specific conditions. By using the enzyme-linked immunosorbent assay (ELISA) method, the activity of M . hyopneumoniae was found t o be affected by acidic environment, temperature, and the existence of trypsins.

Introduction Mycoplasmal pneumonia of swine (MPS) infected by Mycoplasma hyopneumoniae has been recognized internationally as a very serious disease of pigs (Huhn, 1970; Mori et al., 1987). Such disease caused a big economic loss in pig farms due to deduction in the meat gain rate, which also reflected in a low return on investment of feed (Huhn, 1970). Survey reports from different countries have shown that 30-80% of slaughtered pigs revealed positive mycoplasmal pneumonia lesions (Mori, 1987). Although certain drugs and antibiotics have been tested in vitro against M . hyopneumoniae and observed to be capable of reducing the clinical symptoms of the disease (Hannan et al., 1982),they are not yet proven effective in vivo to eliminate the pathologic organism sufficiently. The concept of a common mucosal immune system has been advocated and supported by several researchers (Mestecky et al., 1979; Bergmann et al., 1986; Husband, 1987)for oral immunization of flockand herd in the animal farms. The oral administration of antigens can effectively induce antibodies through the lymphoid collection of distal small intestines, which are known as Peyers patches (Bergmann et al., 1986). Weng (1985)was able to inoculate intramuscularly with formalin-inactivated M . hyopneumoniae vaccine (MHV) in incomplete Freunds adjuvant

and boost intraintestinally. The absorption of MHV via Peyers patches would be able to induce the common mucosal immune system not only in the GI tract but also in the respiratory tract, in order to obtain the efficacy of immunofunctions. However, practical application of such an immunization route was hampered by antigen degradation through gastric acidity and proteolytic enzymes of the GI tract. Therefore, the antigenic activities of M. hyopneumoniae must be protected and persisted through the course of oral feeding and stomach digestion and not be released until it reaches the Peyers patches. In order to obtain efficacy of oral immunization, the enteric coating of MHV seems to be necessary. The purpose of this work is to determine the antigenicity of MHV under various conditions and to establish the criteria of stability for the specific vaccine. The preparation of enteric coated MHV and the correct method of oral administration of such vaccination will be further studied and reported subsequently.

* To whom correspondence
+ Pig Research Institute.
f

should be addressed.

* Veterans General Hospital.

National Tsing Hua

University.

Materials and Methods M. hyopneumoniae Antigen. M . hyopneumoniae (strain PRIT-5),isolated and supplied by the Pig Research Institute, was cultured for at least 48 h in Friis mycoplasma broth. Friis broth was prepared by following the standard procedures (Friis, 1979). The incubation was carried out until the pH of the incubator shifted from 7.4 to approximately 6.5. The broth was then inactivated with 0.5% formalin and the antigens were harvested by centrifugation a t 15000g for 50 min. The collected antigens

8756-7938/9 1/3007-0069$02.50/0 0 199 1 American Chemical Society and American Institute of Chemical Engineers