FR2487642A2 - Sepn. of protein fractions esp. from milk prods. - using membrane ultrafiltration, exclusion chromatography and ion exchange resins - Google Patents

Abstract

In the prodn. of protein-contg. fractions using exclusion chromatography and ion-exchange resins, as described in the parent patent (79/08555), the new feature is that before or after processing the starting material is subjected to membrane ultrafiltration. Pref. the starting material is skimmed milk, sweet or acid whey, buttermilk, colostrum, etc. Before or during the ultrafiltration, the material can be subjected to enzymatic, chemical or thermal proteolysis to increase the amt. of proteins of mol.wt. below 15000; particularly the proteolytic enzymes are attached to the membrane. The sepd. fractions are variously useful in human or animal nutrition; human or veterinary medicine (e.g. recovered immunoglobulins can be used to protect new-born animals against septicaemia) and cosmetics. Particularly they have foaming, gelling, emulsifying and sequestering effects so can be used in baking, cheese-making, etc.

Description

 The present invention relates to changes, additions and improvements made to French patent No. 79/08555 taken on April 4, 1979 for "Process for the preparation of animal or vegetable proteins, particularly lactoproteins, and new products thus obtained.

 The main patent describes a process for the preparation of animal or vegetable proteins, in particular lactoproteins, using exclusion and ion exchange chromatography. Before this chromatographic treatment, the raw materials are subjected to judiciously chosen physicochemical pretreatments, leading to the isolation and fractionation of the proteins present in an original state. These protein isolates are then treated by operations which give them particularly interesting and rewarding properties in the food industry. Thus, proteins are transformed to give them original properties and better economic interest, by stabilizing, exacerbating or revealing some of these properties.

 The raw materials used in the main patent process are chosen from the group consisting of milk, skim milk, sweet or acid whey, buttermilk, colostrum, any other protein material obtained from mammalian milk and any other product resulting from the lactose fermentation.

 Regarding the physico-chemical treatments of the raw material, either physical treatments are used, such as the action of temperature, pH, passage through resins, concentration by means acceptable in the food industry (evaporation , ultrafiltration, ultracentrifugation, etc.), electrophoresis, melting and extrusion, either chemical treatments such as the action of chemical agents (for example, acids, salts, bases) and action of enzymes (for example rennet).

 Among the physico-chemical treatments, it appears that one of the most promising treatments is the passage over an ultrafiltration membrane.

 An object of the present invention is to provide a process for the preparation of protein fractions by combining, as appropriate, ultrafiltration techniques and exclusion chromatography and ion exchange techniques.

 Another object of the present invention is to combine ultrafiltration techniques and techniques of exclusion chromatography and ion exchange to increase the yield of these different techniques, taken in isolation, and to improve the quality of products obtained.

 Other objects will become apparent from the following description of the present invention.

 These objects are now achieved, according to the present invention, by a process for the preparation of protein fractions using exclusion chromatography and ion exchange techniques, according to French patent No. 79/08555, in which, before or after passing through an exclusion chromatography system and ion exchange on resin, the raw material is subjected to a membrane ultrafiltration process.

 The raw material used in the process of the present invention is chosen from the group consisting of at least one of the products consisting of skimmed milk, sweet whey, acid whey, buttermilk, colostrum and any other protein material animal or vegetable.

 From a quantitative point of view, the combination of ultrafiltration techniques and exclusion and ion exchange chromatography makes it possible to increase the ultrafiltration yields, by recovering the protein fractions of the ultrafiltration permeate on the exclusion chromatography apparatus and to increase the yield of this apparatus by treating an ultrafiltered product therein, since an advantage of the pretreatment of the raw material by passage over an ultrafiltration device, before chromatography, is to reduce the cycles of raw material passages on this latter device. In addition, ultrafiltration techniques are used as means of concentrating the eluate from the passage through the exclusion and ion exchange chromatography apparatus. The advantage of this concentration is that it does not denature thermally proteins as is the case with other concentration processes, in particular vacuum evaporation.

 From a qualitative point of view, thanks to the combination of ultrafiltration techniques and exclusion and ion exchange chromatography, ultrafiltration can be used as a means of concentrating the effluent obtained after passage through a column chromatography, which does not denature the proteins and, moreover, which selectively isolates certain protein fractions, such as a-lac talbumin. In addition, thanks to this association, it is possible to selectively recover the protein fractions of the ultrafiltration permeate passed through an exclusion and ion exchange chromatography system, and it is possible to separate the constituents of the proteins, in particular dairy proteins having particular applications, these constituents (such as peptides, lactotransferins, serum albumin and all other fractions present in minimal quantities) which cannot be recovered by other processes. This technique can be further improved by subjecting the raw material, before or during the ultrafiltration treatment, to proteolysis by enzymatic, chemical or thermal means, in order to cut the starting material into constituents with lower molecular weights, which increases thus the content of low molecular weight nitrogen fractions (less than about 15,000) of the permeate. This latter process, in a preferred embodiment, is such that ultrafiltration and proteolysis treatment are combined by fixing the enzyme on the ultrafiltration membrane, which results in a marked improvement in productivity.

 By combining the techniques of ultrafiltration and exclusion and ion exchange chromatography, protein fractions are obtained in native form (not denatured), thus retaining their original properties. If these protein fractions are to be given specific properties, they are added with specific substances as defined in the main patent, or they are subjected to physico-chemical treatments as indicated in the main patent.

 The products obtained from a lactic raw material are mainly: peptides, protein-peptones, amino acids, lactotransferins, ss-lactoglobulins, α-lactalbumin, serum albumin, immunglobulin -, or any other product present in small quantities in the raw material.

 The protein fractions obtained can be used in the food, chemical or pharmaceutical industries for their functional, therapeutic or nutritive properties. In particular, owing to their foaming, gelling, emulsifying and sequestering properties, protein fractions can be used in the food industries (bakery, pastry, confectionery, cold meats, cheese, ice cream products, etc. ...) and in cosmetic products.

 Due to their nutritive and therapeutic properties, protein fractions can be advantageously used in industries for animal nutrition (animal feed supplements), in industries for human nutrition (diet products, infant formula and for children) and in the pharmaceutical and veterinary industries (use of immunglobulins in medicines).

 Tests indicate that immunglobulins could be used in preventive treatments of newborn babies who are at birth completely devoid of antibodies. Indeed, these immunglobulins could provide protection against sepsis during the first twenty-four hours of life of the newborn (period during which the intestinal barrier of calves, like that of lambs, is completely permeable). After this period, the immunglobulins "line" the inner lining of the intestine and can therefore prevent or even treat diarrhea.

 The present invention will now be described with the aid of the following examples which are given only by way of illustration and not by way of limitation of the present invention.

EXAMPLE 1
The permeate (2,600 liters) from the ultrafiltration of milk or serum (8,000 liters of whey) is recovered, the average composition of which is as follows
Dry extract 37.9 g / l
Nitrogenous matter N x 6.39 1.8 g / l
This permeate can be concentrated or not by conventional techniques, and then cooled to 50C. It is percolated directly on two columns of series chromatography an anionic column, followed by a cationic column.

 A variant may consist in modifying the order of passage, the number and the quality of the columns involved.

 After passage of 1N hydrochloric acid (50 liters), an eluate is isolated from the first column whose protein content is close to 70% and whose composition indicates the presence of a large amount of serum albumin (500 g of proteins recovered of which approximately 5% of rumum albumin.

EXAMPLE 2
A variant can be made by adding, during or before the ultrafiltration operation, a protein "- which is made to act on milk or whey (1,200 liters) by adjusting the hydrolysis parameters. can thus choose an alkalase (2 g per 30 liters of milk), which is left to act at a pH of 5 to 8.5, preferably 7 to 8 for several hours. Be permeate which results from the ultrafiltration of the product proteolysed, is rich in peptides. The implementation of ion exchange and exclusion chromatography allows, in the same way as in Example 1, to recover a concentrated protein material (about 4 kg of proteins) , more or less charged with peptide fractions according to the degree of progress of the hydrolysis (80% of peptides under the preceding conditions).

 Depending on the quality and action of the proteases involved, we thus isolate fractions whose technological properties (foaming, emulsifying ...) make them usable in the food industry, in cosmetology or in the fields of therapies human or veterinary.

EXAMPLE 3
A whey (150 liters) is concentrated by means of ultrafiltration to a concentration factor close to 5 so as to obtain a product (30 liters of retentate) whose protein content is close to that of a milk.

The composition of this retentate is approximately as follows
Dry extract 97.5%
Nitrogenous matter 36.3%
Ash 8.25%
It is cooled to about 50C. One percolates successively on the anionic and cationic columns, as in the examples of the main patent.

 However, compared to the usual functioning developed on whey, one can decrease the quantity of liquid injected in proportion to the concentration factor, and thus decrease the duration of the work cycles. This can therefore increase the overall productivity of the installation. In addition, by adjusting the concentration factor so as to provide a sufficient amount of protein to saturate the binding sites, it is possible to work in a fixed bed.

 Similarly, a gain in productivity can be observed due to the increase in the protein / ash ratio. Indeed, during ion exchange, all of the anions compete with proteins at the binding sites. The value of the concentration factor for working in a fixed bed is 10 and the productivity gain is 15%.

EXAMPLE 4
A quantity of whey (240 liters) in excess relative to the fixing capacity (10 kg) of the resins is successively percolated on anionic, then cationic columns. Since p-lactoglobulins preferentially bind to o-lactalbumin, it is possible to obtain an effluent containing only the latter fraction. As this effluent is very diluted, ultrafiltration makes it possible to selectively recover and concentrate the α-lactalbumin fraction
- either to place oneself in the composition of a team
are worth serum free of p-laetoglobulin
12 or 13% protein,
- either to obtain a protein concentrate of
a-lactalbumin up to purities
protein higher than 60%.

 In both cases, this type of product is particularly suitable for use in infant formula.

Thus, under the preceding conditions, the last 80 liters of effluent are collected and, the resin fixing capacity being 90 mg / g, we recover
- 0.9 kg of p-lactoglobulin per elution
- 0.4 kg of a-lactalbumin in the effluent.

 The same type of treatment and application can be carried out on an effluent which has not undergone percolation on a cationic column, therefore containing the mixture of α-lactalbumin and immunglobulins, or else on an effluent of cationic column not containing more than im munglobul ines alone.

 The present invention is not limited to the exemplary embodiments which have just been described, it is on the contrary liable to variants and modifications which will appear to those skilled in the art.

Claims (10)

 1 - Process for the preparation of protein fractions using exclusion chromatography and ion exchange techniques on resins, according to French patent No. 79/08555, characterized in that before or after passage through a chromatography system exclusion and ion exchange on resin, the raw material is subjected to a membrane ultrafiltration process.  2 - Process according to claim 1, characterized in that the raw material is chosen from the group consisting of at least one of the products consisting of skimmed milk, sweet whey, acid whey, buttermilk, colostrum and any other animal or plant protein matter.  3 - Process according to claim 1 or claim 2, characterized in that the protein fractions of the ultrafiltration permeate are not selectively recovered on an exclusion chromatography and ion exchange system, and separates the constituents of proteins with particular applications, present in minimal quantity in the raw material.  4 - Method according to claim 3, characterized in that before or during the ultrafiltration treatment, the raw material is subjected to proteolysis by enzymatic, chemical or thermal, in order to increase, in the permeate, the content of protein fractions with molecular weights less than about 15,000.  5 - Process according to claim 4, characterized in that the enzyme is fixed on the ultrafiltration membrane.  6 - Process according to any one of claims 1 to 5, characterized in that, in the case where a lactic raw material is used, the protein fractions obtained are chosen from peptides, protein-peptones, amino acids , lactotransferins, (3-lac-toglobulins, α-lactalbumin, serum albumin, immunglobulin and other products present in minimal quantities in the raw material.  7 - As new industrial products, protein fractions obtained by the process according to any one of Claims 1 to 6.  8 - Products according to claim 7, characterized in that they are obtained in native form (not dSna-tubed).  9 - Products according to claim 7, characterized in that the products are given specific properties by subjecting them to physical-chemical treatments or by adding specific compounds to them, as defined in French patent No. 79/08555 .  10 - Use of the products indicated in any one of claims 7 to 9, characterized in that these products are used in human or animal food, in pharmacy or in cosmetology, because of their functional, therapeutic or nutritive properties.