PROCESS FOR HECOGENIN PRODUCTION FROM ANAEROBIC FERMENTATION OF SISAL JUICE

Abstract: Anaerobic fermentation caused by inoculation, in the purified sisal juice, of microorganisms of rumen mixed with a salt solution. The process demands a pH control, an anaerobic environment, a temperature around 37<o>C, a 50-70 r.p.m. agitation for one week period. The sapogenins (hecogenin, tigogenin) are purified by crystallization using a solvent made of ethyl acetate and cyclohexane (1:1), the process also produces useful secondary compounds as ethanol, acetic acid, cells, CO2, H2, and methane.

Description: Report describing a patent of invention for "PROCESS FOR HECOGENIN PRODUCTION FROM ANAEROBIC FERMENTATION OF SISAL JUICE" . The process herein described points out the transformations, by autolysis and fermentation, on the sisal juice (resulting from the decortication operation of sisal leaves), aiming at obtaining hecogenina, by an anaerobic cultivation using a 0.9% saline solution of rumen. Usually, when sisal juice is used to produce hecogenina and tigogenina, the sisal precipitate is submitted to an acid hycrolysis; this precipitate is obtained through an aerobic fermentation of sisal juice. This kind of process has many problems with no satisfactory solutions. Among those one can point out the following ones: 1 ) Difficulty to maintain autolysis for extended period at constant rate in the sisal juice, under normal conditions, without deterioration (of secondary metabolites), including saponina and genina caused by contamination of bacteria and fungi from air. 2) The contamination precipitates the secondary metabolites forming an insoluble solid; decantation will cause loss of hecogenina glicosides, forming a precipitate which will not suffer further hydrolysis. 3) Autolytic enzimes from the sisal juice are strongly affected by competition or/and inibition, due " SUBSTITUTE SHEET' to presence of sintases and fructosyl or glycosyl transferases in the sisal juice. 4) Low amount of carbohydrates (including glucose) affecting the activity of -glucosidase and -galactosidase, which are used in the autolysis process. 5) Necessity of performing a further acid hydrolysis on the solid precipitate, to obtain a limited autolysis. 6) Low hecogenina yield due, among other factors, to the formation of secondary products which are related to the amount of acid and heating applied in the precipitate hydrolysis. 7) High cost involved in the acid hydrolysis. Aiming at solving the problems above mentioned, studies were conducted with the objective of obtaining high yields of hecogenina, using microbial biochemichal hydrolysis; in this way Harssal and Smith have obtained hecogenina from extracted saponina incorporated to an agar medium (pH 7.2) of Czape Dox, inoculated with strains of Alternaria sp. or Corynespora. But the aerobic microorganismus took long time to reach maximum enzimatic activity and revealed to be inadequate for the sisal juice process, for several reasons: 1) high degree of mixing in the sisal juice; 2) carbohydrate inhibition of -glicosidases; 3) high costs of aeration; 4) contamination by microorganism from air. S UBSTITUTE SHEEi The aerobic microorganisms above mentioned (Alternaria sp. and Corynespora) and other ones, have an important role in the decomposition of -glycosidic linkage (like saponina from sisal) . Like the anaerobic microorganisms found, especially, in the rumen; which do not need neither oxygen nor strong agitation. The development of the process proposed in this report, since it needs only carbohydrates and saponinas from sisal, shows an inherent degree of simplicity and o economy, which are: 1 ) The preservation of anaerobic conditions ,demande by the process, is assured through gas formation of C0 2 ,H 2 from anaerobic metabolism of the rumen microorganisms. 2) The ethanol and organic acids, especially acetic acid, produced by anaerobic fermentation together with the saline solution of rumen and the saponina from sisal, sterilize the sisal juice preventing it from being contaminated by airborne microorganisms. 3) The autolytic digestion of saponinas in the sisal juice is performed by -glicosidase with the help of anaerobic microorganisms of the rumen, producing the sapogeninas (hecogenina, tipogenina, etc.) . 4) The ethanol produced by anaerobic microorganisms during the fermentation produced by the 0.9% rumen suspension, have an important role in the autolysis of the saponina and its derivatives, as a solvent. 5) The ethanol, acetic acid, H„ and methane SUBSTITUTE H produced could be recovered and used properly while the residue could be utilized as a source of single cell protein. 6) The proposed process used thyl acetate and ciclohexane (1:1) for hecogenina isolation from a mixture of tigogenina and hecogenina. 7) A clean sisal juice is prepared for the fermentation using the Yang's automatic press centrifuge developed by author. To reach the anaerobic condition demanded by the process any consuming 0- microorganisms can be used. The rumen Ercherichia coli can reach the anaerobic condition in shorter time than the Candida utilis IFO 4277, besides it does not affect the hydrolysis being alive or not because of its limited growth. To keep anaerobic microorganisms at high activity for long period of time it is important to obtain a high production of ethanol, acetic acid, H„, methane, sapogeninas (hecogenina e tigogenina) and yeast. Consequently it is recommended to cultivate the anaerobic microorganisms with high concentration of saponins. Identic recommendation is not valid for an aerobic fermentation where the high saponins concentration could lead to unfavourable results due to the high viscosity of the medium. This is one reason for the use of anaerobic microorganisms to perform the glycosidic decomposition. " SUBSTITUTE SHEET' The microorganisms Ruminococcus albus and Ruminococcus flavepaciens from rumen are excelent for the glycosidic decomposition of sapogenin and utilization of C0 2 - Ethanol and acetic acid, main products of fermentation from sugars of the sisal juice or resulting from saponin' s hydrolysis by anaerobic microorganisms do not inhibit growth and hydrolysis as far as a 40 g per liter of saponin is maintained in the sisal juice. The specific average rate of saponin digestion is 0.25 g of hydrolised saponin/g dry of dry cells. The digestion rate of saponin is related to the consumption of carbohydrates. The amount of ethanol and acetic acid are respectively 7.5 g and 6 g per liter, during the fermentation. The hecogenine produced in a 7 days fermentation batch reached a level of 1:2% (w/w) related to sisal juice while tigogenin reached 0.4%, these results are superior to those usually got by any known process . Two known process for hecogenin production are described in the following pages and later the new process. The two selected known processes are Spensley's and Marker' s ones. 1 st process: A hecogenina source P.C. Spensley, 1956 Extraction of sisal juice Shrededded left over dry Maves were pressed and the juice left to ferment and rest for about one week. "SUBSTITUTE SH T The supernatant was separated and boiled for 4 hours with H SO. enough to reach a 0.75 - 1.0 N solution. The solid residue was filtered, whashed and dried. 50 g of that residue was boiled in reflux for 4 hours with 500 ml alcoholic HC1 2N or H 2 SO.. When the alcohol was stripped off, 10-20 g of active carbon was added. The suspended material was separated, washed with water, treated with 200 ml of NaOH 1N, separated, washed with water, dried and extracted for 72 hours with isopropylic ether. The extract was concentrated to 50-100 ml and left at room temperature. After a few hours raw hecogenin was precipitated, which was dissolved with careful boiling using 2 times greater volume of acetic anhydride for 5 minutes, after, it was cooled. In the cooling process, a precipitate of hecogenin acetate was formed, with boiling point p.e. 242-278°C. To separate the hecogenin acetate the girard's reagent was used. The hecogenin yield was 0.18% related to the pressed juice from leftover sisal leaves. _ J 2 process: Isolation of new sapogenins R.E., Marker et al. 1947 The fresh and shreded sisal leaves were processed in about 25 kg batches. Each batch was extracted with 32 liters of ethanol 95% at low temperature in steam bath for 12 hours. The aqueous extract was filtered with cotton mesh. The residue was washed with 2 portions of 7 liters of hot ethanol and after pressed, dried using "SUBSTITUTE SHEET" a press for wine manufacture. The washed extract kept in 22 liters containers or in tin cans of 30 gallons were evaporated under low vacuum until a concentrated sirup was obtained, a flux of air was also blown on the liquid surface. When alcohol was difficult to store and handle, leaves were treated with hot water. This process was considered as satisfactory, although it was lengthy, since the aqueous extract had to be evaporated for the following step. For this reason the aqueous extract kept in 5 gallons containers was concentrated by heating in a water bath with air blowing on the liquid surface. The concentrated liquid was boiled with reflux for two hours with 8 liters of ethanolic HC1 2N. The resulting solution was cooled and fitered. When a significat amount of tar appeared, it was ground and digested several times with an equal volume of hot ethanol. The filtered solutions were combined and diluted with 20 liters of ether and the solutions were washed consecutively with water, NaOH at 5%, water and later, it was evaporated and concentrated. The fatty esters were hydrolised with reflux using 3 times the volume of alcoholic sodium hidroxide at 10% for 30 minutes. The cold hydrolised solution was treated with ether and the resulting solution was washed with water and concentrated to a small fraction. The raw sapogenin was separated, dissolved with acetone and purified with morita. Usually it was converted to acetate using TϋTE boiling acetic anhydride for a better purification. Proposed process: "Process for Hecogenin Production from Anaerobic Fermentation of Sisal Juice". The process for hecogenin production from anaerobic fermentation of sisal juice, starts with the production of sisal juice using the Yang's automatic press centrifuge which removes fibers and impurities, yielding a clear sisal juice plenty of original enzymes. It is recomended the previous esterilization of the equipment which contacts the juice with sodium hypochloride at 20-30 p.p.m. The diagram presented in figure 1 shows the operations which are part of the whole process; the numbers indicate the yield over a 1000 kg of processed sisal jigice. After the extraction of raw juice by the automatic press, 2 liters of 0.9% saline of rumen solution with 400 g of rumen (suplemented with cysteine, HC1 0.25 g/1 to complete the juice and Na-S. 9H 2 0 0.25 g/1 the same), the solution was thoroughly mixed. Afterwards the solution was adjusted to pH = 6.8 with NaOH 10% (w/w) and 0- was stripped off with a C0„ bubbling operation and incubated at 37°C, under agitation at 50-70 r.p.m. for one week. During the fermentation, pH was ajusted to 6.8 with a 10% (w/w) NaOH solution and anaerobic condition was tested by the resazurin teste. After one week solids were recovered by filtration using a press filter and SUBSTITUTE SHEET' dried. The dried solids which contains sapogenins (hecogenin, tipogenin, etc.) and cells (monocelular protein) were extracted with a mixture of acetone and chloroformium (1:1) using and extractor (basket tipe or similar) . The extracte was evaporated and the sapogenins were obtained as a solid residue. The solvent used was recycled. The raw sapogenins were dissolved with ethyl acetate and cyclohexane (1:1) , free from impurities. Pure hecogenin was obtained from the mixture of sapogenin/tigogenin by cristalization with the above described solvent (ethyl acetate and cyclohexane (1:1)) , finally the tigogenin was obtained in the sane operation. The hecogenin yield was 1.2%, boiling point 265-267 C, 12 kg/metric ton of sisal juice. The tigogenin yield was 0.4%, boiling point of 182-183 C; 4 kg/metric ton of sisal juice. From the filtrate ethanol and acetic acid were recovered, the ethanol yield was 7.5 ml/1 of sisal juice, the acetic acid yield was 6 ml/1 of sisal juice. The cells obtained could be used as single cell protein for runinants feeding. The CO- obtained was 9 1/1 of the juice and the mixture of C0 2 + H was 10 1/1 of juice, at normal pressure. The "Process for Hecogenin Production from Anaerobic Fermentation of Sisal Juice", above described, points out an economic dimension for its clear viability when ' U T E S iPFT'' employed at large scale, since it is compatible with the whole process of fiber extraction adopted by most of the industries dealing with sisal fiber. "SUBSTITUTE SHEET"

Claims: - ~ CLAIMS 1. "PROCESS FOR HECOGENIN PRODUCTION FROM ANAEROBIC FERMENTATION OF SISAL JUICE" , which could avoid the usual acid hydrolysis on the solid residue; this process is σaracterized by promoting an anaerobic autolysis of the sisal juice by Ruminococcus albus, Ruminococcus flavefaciens and Erσherichia coli, found in the rumen. 2. "PROCESS FOR HECOGENIN PRODUCTION FROM ANAEROBIC FERMENTATION OF SISAL JUICE", according to claim 1 , is caracterized by the fact that it can supply the surrounding air with fermentation gases (C0 2 ,H 2 ,CH.) , resulting from the microbial metabolism inherente to the process, which could have a profitable use. 3. "PROCESS FOR HECOGENIN PRODUCTION FROM ANAEROBIC FERMENTATION OF SISAL JUICE", according to claim 1, is σaracterized by adopting an anaerobic digestion of saponin in the sisal juice, promoted by the -glucosidase formed by the anaerobic microorganisms of the rumen (producing hecogenin, tigogenin, etc.). 4. "PROCESS FOR HECOGENIN PRODUCTION FROM ANAEROBIC FERMENTATION OF SISAL JUICE" , according to claim 1 , caracterized by promoting an assepsys of the medium relatively to aerobic microorganisms, such as, Aespergillus sp, penicillum sp, Aureobasidium sp, and Palcilomyces sp, by the combined action of ethanol, acetic acid, sodium chloryde and saponins present in the medium culture. 5. "PROCESS FOR HECOGENIN PRODUCTION FROM ANAEROBIC "SUBSTITUTE SHEET -it FERMENTATION OF SISAL JUICE", according to claim 1 or 3, caracterized by the utilization, for hecogenin isolation, a mixed solvent made of ethyl acetate and cyclohexane (1:1) for the crystalization method. 6. "PROCESS FOR HECOGENIN PRODUCTION FROM ANAEROBIC FERMENTATION OF SISAL JUICE", according to claims 3, caracterized by the utilization the Yang's automatic press centrifuge to obtain a fresh sisal juice. " SUBSTITUTE SHEEP

Inventors: YANG CHANG IL, YANG, CHANG, IL
Applicants: UNIV FEDERAL DA PARAIBA, UNIVERSIDADE FEDERAL DA PARAIBA
Application year: 1985
Main classification: C12P33
IPC codes: C12P33