MILK CLOTTING ENZYMES RECOVERY

Abstract: A process for isolating milk clotting enzymes from mammal stomachs, comprising a 3-phase centrifugation step.

Description: MILK CLOTTING ENZYMES RECOVERY Field of the invention This invention relates to the recovery of milk clotting enzymes from mammal stomachs. Background of the invention The present invention provides a new process for recovering milk clotting enzymes from animal tissue sources. Milk clotting enzymes are widely used in the cheese industry to produce curd from milk. Curd consists of the major milk proteins. Commercially available milk clotting enzymes include native enzymes derived from their natural microbial or animal tissue source. These enzymes are also derived by recombinant expression. Native milk clotting enzymes are isolated by extraction directly from animal tissues. Such tissues can contain one or more different types of these enzymes. The primary industrial source of native animal milk clotting enzymes is mammal stomach, such as calf stomach, bovine stomach, goat stomach, porcine stomach or sheep stomach. By stomach is meant whole stomachs as well as parts of stomachs as well as stomachs which have been pre-treated physically, chemically and/or mechanically. Chymosin and pepsin (amongst others) can be obtained in this way. Chymosin may occur in prepro, pro, pseudo and mature forms of which pseudo and mature forms are the most active. Prepro and pro-forms of the enzymes are converted into the active forms by removal of pre and pro peptide fragments. The removal of the pre peptide fragment occurs when the prepro enzyme is secreted into the stomach. The pro peptide fragments may be removed by treatments under acidic conditions which also occur in-vivo. In the mammal stomach, pepsin is mainly present as mature enzyme, whereas chymosin may be present as prochymosin, pseudochymosin and mature chymosin. Pseudochymosin is a form of chymosin in which the propeptide fragment has not been completely removed. Pseudochymosin occurs when prochymosin is exposed to a low pH, such as pH 2. At a higher pH pseudochymosin is processed to chymosin. Chymosin is a preferred milk clotting enzyme. Preferably this is obtained from extracts from the fourth stomach of milk fed calves because such stomachs comprise more chymosin than pepsin. The older the calves become the more pepsin they produce. Until finally the calves, (which by this stage are cows) produce more pepsin than chymosin, e.g. newborn sucklings which are less than one month old produce at least 95% chymosin and up to about 5% pepsin, whereas milk fed calves which are 2-3 months old produce 80-90% chymosin and about 1 0-20% pepsin. Heifers which are 1 2-24 months old and fully grown cows produce about 1 5% chymosin and about 85% pepsin. Other mammals also tend to produce more pepsin than chymosin. Extraction of milk clotting enzymes from mammal stomach usually comprises the following steps: - Extracting the enzymes from the stomach tissue by pressing and/or by mixing the stomachs in an aqueous solution of low pH; these steps may optionally be preceded by grinding the stomachs; - Separation of the solid and aqueous phases (containing most of the milk clotting enzyme) from the fat/oil containing phase by centrifugation or flotation; - Separation of the aqueous phase (containing most of the milk clotting enzyme) from the solid phase by filtration, centrifugation or settling. By 'primary liquid extract' is, in this specification, meant, the obtained aqueous phase, after extraction of the milk clotting enzymes from the mammal stomachs, preferably extracted under addition of water, and separation of the fat/oil and solid phases from the aqueous phase comprised in the slurry, which slurry was obtained after extraction. This primary liquid extract may be further purified or concentrated and ultimately made into desired product formulations, such as powders or liquids of different enzymatic activity. A typical process for extracting milk clotting enzymes from mammal stomachs has been described in Nord Europaeisk mejeri-tidsskrift NR 7/78. This process starts with degreasing the mammal stomachs. Then stomachs are cut and/or crushed in one or more steps. The next step is to press the stomachs under high pressure, which may be repeated up to four times. Finally filter aids are added and the slurry is filtered on rotating vacuum filters. This process thus consists of many steps and needs recirculation of stomach material. Due to the unstable character of chymo- sin a considerable amount of the milk clotting activity present in the mammal stomach is lost during this lengthy process. When using this process or other known processes to produce primary liquid extract, the recovery-yield of milk clotting enzymes from the stomach is about 30-70%. So during the recovery at least 30% of the milk clotting enzymes present in the mammal stomachs is lost. Commercially this is a major waste of valuable enzymes. So there is a need for recovery processes which provide higher recovery yields. Summary of the invention The inventors have found that milk clotting enzymes can be isolated from mammal stomach by using a 3-phase centrifugation step, resulting in recovery-yields of at least 85%. The simple process of the present invention, comprises the following steps: a/ extracting milk clotting enzymes from mammal stomachs, to obtain a slurry comprising a fat/oil phase, an aqueous phase and a solid phase; and b/ separating the aqueous phase, which contains most of the milk clotting enzymes, from the fat/oil phase and the solid phase to obtain a primary liquid extract, by centrifuging the slurry using a 3-phase centrifuge. By using a 3-phase centrifugation step, the aqueous phase is separated from the fat/oil phase in the same centrifugation step as separation of the aqueous phase from the solid phase. Thus the aqueous phase is separated in one step from a 3-phase composition, to obtain a single phase composition. In general when producing primary liquid extract according to the process of the invention, the recovery-yield of milk clotting enzymes from the stomach is at least 85%. So the process of the invention allows a primary liquid extract, (an aqueous extract which is separated from the fat/oil- and solid- phase) to be obtained which has a high yield of enzymes. It has further surprisingly been found that using phosphoric acid as the acid in extraction step a/ provides an increased recovery-yield when compared with the use of hydrochloric acid. Detailed description of the invention The process of the invention provides recovery of milk clotting enzymes from mammal stomach. The yield is generally higher than the processes in the prior art. Milk clotting enzymes are isolated from mammal stomach by first extracting the enzymes from the stomach tissue to obtain a slurry, and thereafter the aqueous phase, containing most of the milk clotting activity, is separated from the other phases comprised in the slurry, by 3-phase centrifugation. Milk clotting enzymes are extracted from mammal stomachs by breaking open stomach cells, since most of the milk clotting enzymes can be found inside these cells (intracellular). In order to open stomach cells, stomach tissue is treated e.g.. by acid or mechanically (high pressure, grinding, high shear (e.g. by sand)). Preferably water is added to the stomachs or the stomachs are added to water before rupturing stomach cells, so that the enzymes are solved in an aqueous phase. In a preferred extraction method, mammal stomachs are first mixed in water under acidic conditions in order to extract the milk clotting enzymes from the stomach tissue. During the extraction the stomach turns into a slurry which comprises fat/oil, an aqueous phase and solid particles of meat or other solids. Mammal stomachs which may be processed include for example calf stomach, bovine stomach, goat stomach, porcine stomach or sheep stomach etc. Generally the fourth stomach of calves (abomasum) will provide the highest amount of chymosin. The extraction may be performed using a ratio of stomach/water of from 1 : 1 to 1 : 1 0, preferably the ratio of stomach/water is from 1 :2 to 1 :4. Although higher amounts of water may provide better extraction (and thus yield) of the milk clotting enzymes, the water would have to be removed again if the final product of the extraction process has a milk clotting enzyme activity which is too low to be used in cheese manufacturing. The extraction may be performed at temperatures ranging from 5-55°C, preferably from 20-50°C, more preferably from 35-45°C. Higher temperatures may cause unacceptable enzyme activity losses. Lower temperatures may cause solidification of the fat/oil. During extraction the pH is within from 1 -4, preferably 2-3.5, more preferably 2.5-3.2, most preferably 2.7-3.0. The pH may be adjusted by using any suitable acid, for example hydrochloric acid, sulphuric acid, phosphoric acid, acetic acid or another suitable organic acid. Surprisingly it has been found that extraction using phosphoric acid provides a higher yield of milk clotting enzyme activities from the same stomach tissue. The so-called "extraction output" is highest when phosphoric acid is used as the acid for lowering the pH before extraction. Therefore according to another embodiment of the invention, the pH is kept below 4, by adding acid which on molar basis consists for at least 50%, more preferably at least 75%, most preferably at least 90% of phosphoric acid. This results in high extraction output and in water-layers which are clearer after the 3-phase centrifugation. Clearer water layers are advantageous when using further purification steps like lonexchange or Ultrafiltration. During extraction the low pH causes prochymosin to be converted to chymosin (and in part to pseudochymosin). Chymosin is the most valuable milk clotting enzyme because it has a high milk clotting activity and also has high specificity. Pepsin is able to degrade chymosin. Therefore the conditions and duration of the extraction are optimised so that as little chymosin is degraded by pepsin as possible. Pepsin is most active at a pH of about 2. The extraction (with conditions as described above) is best carried out for a short time period, for example within 1 -8 hours. Thereafter the slurry is optionally freed from course material and is centrifuged in a 3-phase centrifuge. The 3-phase centrifuge separates the aqueous phase from the two other phases: the oil/fat phase and the solid phase in one step. The aqueous phase comprises most of the milk clotting enzyme activity. "3-phase centrifugation" means that in one centrifugation step 3-phases are separated . (e.g. a slurry comprising: ( 1 ) a fat/oil phase (2) an aqueous phase and (3) a solids phase is separated in these three phases in one centrifugation step.) A "3-phase centrifuge" is a centrifuge equipped with (a) a separation ring to separate two liquids of different densities and (b) a solids discharger (to discharge solids (e.g. batchwise) during centrifugation). We have found that with the 3 -phase centrifuges, it is possible to obtain a very sharp separation between the three phases. A separation ring can be installed in the centrifuge by which the fat/oil phase and the aqueous phase are forced to leave the centrifuge on different exits. The size of the separation ring can be determined from the difference between the densities of the fat/oil-phase and the aqueous phase. 3-phase centrifuge manufacturers provide tables, detailing which separation ring is required for particular differences in densities. These centrifuges also have a system of very precise partial discharging of the solids. 3-phase centrifuges which can be used are e.g. Westfalia type SA- and Alfa Laval type CHPX-centrifuges. The slurry can preferably be centrifuged at a pH ranging from 3-4.5 or 5.1 -5.5. Centrifugation at a pH at or around the IEP of chymosin, that is from 4.6-5.0, will cause yield loss of the milk clotting activity. Centrifugation may be performed at temperatures ranging from 5-55°C, preferably from 20-50°C, more preferably from 35-45°C. Higher temperatures may cause unacceptable enzyme activity losses. Lower temperatures may cause solidification of the fat/oil. The process according to the invention may provide a milk clotting enzyme recovery-yield for the primary liquid extract of at least 85%, preferably at least 90%, more preferably at least 95%. The primary liquid extract of milk clotting enzymes may further be concentrated and/or purified using well known techniques such as evaporation, precipitation, ultra- or hyper-filtration or column-chromatography. Powder formulations may be obtained by e.g. (spray) drying. The final products can be used in the manufacturing of many kinds of cheeses. The following examples will further illustrate the invention. Milk clotting enzyme activities were analysed using an official international protocol as described by the International Dairy Federation in the IDF standard 1 1 0A 1 987, appendix A. The applied milk clotting test is a relative test against a standard solution delivered by INRA (Institute National de la Recherche Agronomique). The tests were executed in a Formagraph type 1 1 700 in which 9 samples can be analysed together with the standard. The time needed for coagulation of the milk (26°C) is a measure for the activity. Example 1 To 250 kg of calf stomachs about 750 kg water was added. The mixture was stirred for 5 hours at pH 3 (set by adding HCI) and 40°C. The thus obtained slurry was filtered in line over a rotating sieve (700 micrometer) to remove the meat remains and the course solids, in order not to plug or block the 3-phase centrifuge. The filtered slurry was centrifuged continuously using a 3-phase Westfalia type SA7 centrifuge, to separate the aqueous phase from both the fat/oil phase and the solid phase in one single separation step. The primary liquid extract thus obtained contained up to 93% of the milk clotting enzyme activity present in the original total stomach extract. Example 2 Extractions of milk clotting enzymes from calf stomachs with hydrochloric acid and phosphoric acid were tested. To 500 grams of cut stomachs about 1 250 grams water was added. Thereafter one of the acids was added to provide a pH of 3, which pH was controlled at 3 with the same acid during extraction for several hours at 40°C. During extraction small samples were taken, centrifuged in a Sigma labcentrifuge for 2 minutes and the aqueous phase was analysed for milk clotting activity. Table 1 shows the results of the milk clotting measurements during the extraction with the acids. The 7 hours extraction with hydrochloric acid (HCI) is set on 100%, and the others are calculated in percentages of that activity. Table 1 The relative milk clotting activity during extraction with either hydrochloric acid or phosphoric acid The results given in table 1 show that extraction with phosphoric acid provides higher milk clotting activities then hydrochloric acid. When phosphoric acid was used in the extraction the aqueous phase after centrifugation was the clearest. This resulted in shorter processing times when ionexchange or ultrafiltration was used in further purification steps.

Claims: CLAIMS 1 . A process for isolating an enzyme from a mammal stomach, or from a slurry obtained from mammal stomach, the process comprising a 3-phase centrifugation step. 2. A process for isolating an enzyme from a mammal stomach, comprising: (a) obtaining a slurry from the mammal stomach, the slurry comprising an aqueous phase, a fat/oil phase and a solid phase; and (b) separating the aqueous phase from the fat/oil phase and the solid phase to obtain a primary liquid extract, by 3-phase centrifugation. 3. A process according to claim 1 or 2, which comprises only one centrifugation step. 4. A process according to any of the preceding claims, wherein the slurry is obtained by grinding a mixture of the mammal stomach and water. 5. A process according to any one of the preceding claims, wherein the step to obtain a slurry is done at a pH below 4. 6. A process according to claim 5, wherein the pH is 1 -4, preferably 2-3.5, more preferably 2.5-3.2, most preferably 2.7-3.0. 7. A process according to claim 6, wherein the pH is lowered using an acid selected from hydrochloric acid, sulphuric acid, phosphoric acid, acetic acid or another suitable organic acid, whereby the ' acid comprises preferably on molar basis at least 50%, more preferably at least 75%, most preferably at least 90% of phosphoric acid. 8. A process according to any of the preceding claims, wherein after 3-phase centrifugation the primary liquid extract, is further concentrated and/or purified. 9. Use of phosphoric acid to obtain a pH below 4, preferably a pH within from 1 - 4, in the extraction of enzymes from mammal stomachs. 1 0. A process according to any one of claims 1 to 8 wherein the enzyme is a milk clotting enzyme, preferably chymosin or pepsin.

Inventors: ROBERS HENDRIK JAN, WEBBERS JOSEPHUS JOHANNES PAUL, ROBERS, HENDRIK, JAN, WEBBERS, JOSEPHUS, JOHANNES, PAULUS
Applicants: GIST BROCADES BV, ROBERS HENDRIK JAN, WEBBERS JOSEPHUS JOHANNES PAUL, GIST-BROCADES B.V., ROBERS, HENDRIK, JAN, WEBBERS, JOSEPHUS, JOHANNES, PAULUS
Application year: 1998
Main classification: C12N9
IPC codes: A23C19, C12N9