Patent Application
20070026086
The present invention relates to a process for the separation and isolation of biologically active substances from dairy by-product or secondary waste flows. Many dairy industrial processes, such as in the manufacture of butter and butter concentrates, caseins and caseinates, cheeses, creams, ultra-fresh dairy products, and the like, generate significant amounts of currently unavoidable by-products or waste products. These products must be handled according to legislation that is becoming ever stricter, which often implies a non-negligible cost. One way of recovering that extra cost that the industrialist has to pay, is by attempting to exploit or to find some value added material within the waste or by-product. Unfortunately, such cases are relatively rare on the whole, since the economics of any extra processing steps necessary to retrieve the material are generally unfavorable with respect to value that can be gained from such material. This is especially true of the food and dairy industry, where industrial costs have to be kept as low as possible, in order to maintain economic viability of the general food production processes. Generally speaking, by and waste products of the food industry are handled in the following manner:
In many cases, however, little or no value-added exploitation can occur, either because the by-product or waste product has a too low concentration in dry matter, or on the other hand, because the waste or by-product is extremely unstable and perishable, and thus unsuitable for storage for a time sufficient to retreat it.
An example of a branch of the food industry faced with such difficulties is that relating to cheese product manufacture and treatment. Most dairy products start out from a bulk volume, low cost, starting material, namely milk. This starting material is available in very large quantities and the initial transformation steps, such as protein precipitation, filtration, or heat treatment are considered to form part of the well established economic environment in this field, simply because of the immense volumes involved. However, other products and by-products can also be made starting from milk. One such by-product is whey, which again can be transformed into further value added products. Exemplary value added products obtained from whey would be, for example, whey proteins, whey protein concentrate or hydrolysate, β-lactoglobulin, and α-lactalbumin, phosphopeptides, casein proteins, caseinomacropeptides, immunoglobulins and the like. Unfortunately, these further processing steps are not viable on a large scale, simply because of the high cost of the further technological steps that are currently required to produce these products.
Typically, the processes used in creating value-added products from milk waste or by-products goes through whey, separated from the curds of the milk. Two major techniques are used today:
In these cases, chromatography is generally considered by far the best in terms of pure performance, and is used for the recovery of molecules such as lactoferrin, lactoperoxydase, glycomacropeptides ou simple fragments thereof. In view of the expense of such relatively small scale techniques, most of the applications envisaged are niche markets, such as infant nutrition formulas, sport nutrition formulas, body building formulas, and specialized dietary supplements for the aged.
From the above, one can conclude that there is a long felt need in general to provide a large scale or scalable, cost effective, means of treating industrial dairy waste or by-product flows in order to provide an economic way of dealing with such waste or by-product, whilst at the same time providing a value added substance that has other significant applications.
One object of the present invention is therefore to provide such a low cost, industrially scalable method for the isolation of biologically active substances from by-product or waste flows. Another object of the invention is to provide for biologically active substances obtained via such a method.
Still yet another object of the present invention is to provide for a biologically active complex in which a biologically active substance obtained via the method of the present invention is used.
Other objects may become apparent through the reading of the description and claims.
In the following specification and claims, the following terms have the meaning assigned to them here below:
Particularly preferred solid support materials can be chosen from the group consisting of:
Accordingly, one object of the invention is a method for the isolation of at least one biologically active substance from an industrial dairy by-product or waste flow, comprising:
Preferably, the at least one industrial dairy waste or by-product flow is substantially liquid.
Even more preferably, the at least one industrial dairy waste or by-product flow comprises not more than 30% by weight of dry matter. In accordance with the present invention, the at least one industrial dairy waste or by-product flow is preferably generated or supplied from an industrial process that processes or manufactures a product derived from milk. This means that the method of the present invention can be used for treating waste flows and by-products from a number of dairy industries, and more particularly from the group of industries consisting of butter and butter concentrate manufacture, cream manufacture, ultra fresh dairy products, casein and caseinate manufacture, cheese manufacture, and the like. Such processing plants produce huge volumes of liquid or semi-liquid waste or by-product flows, in which many biologically active substances can be found. Most preferably, however, the at least one industrial dairy waste or by-product flow is milk or derived from milk. The milk industry produces huge amounts of liquid waste or by-products such as whey, that contain very interesting biologically active substances, but which with todays current processing techniques require great efforts to isolate and recover them, at great expense, with the result that the products thereby obtained can only be sold in niche markets with perceived high added value. The method of the present invention manages to solve this problem by producing a workable, industrially scalable alternative, that is also very economic, both in the components used, as in the installation and running requirements. Consequently, in one of the most preferred embodiments, the at least one industrial waste or by-product flow is whey.
In yet another preferred embodiment, the at least one solid insoluble support material and the at least one industrial dairy waste or by-product flow are agitated together after being brought into contact one with the other. The conditions of operation will depend on the type and molecular weight of active substance being recovered from the waste flow. Such conditions can be determined by one skilled in the art, so as to arrive at a system where one can scan a waste product flow for all possible biologically active molecules of interest, and then adapt the adsorption conditions, in a further cycle for example, to retain only those specific molecules that are desired.
The method of the present invention can, for example, be made to function as a small plug-in unit that is connected to the main processing flows of the industrial plant and produces a value added secondary or tertiary material with potential applications in human and animal health and nutrition. In addition, the method of the invention reduces the volumes that need to be dried subsequent to isolation of the biologically active substance, thereby causing further economies of scale to be obtained in heating and energy costs.
The unit can for example comprise a chamber, having a solid support material inlet, and a conduit either for the introduction, or the flow through, of the at least one waste flow or by-product. The chamber can also optionally be provided with agitation means, and temperature regulation means. Naturally, the chamber will also be provided with outlets for both the adsorbed biologically active substance/support material complex, and the remaining waste liquid that is now poorer or depleted in molecules of interest and can either be passed through the chamber once again, or dispatched for disposal. In an industrial plant, one could easily foresee the provision of several cycles through the unit, or the provision of multiple chambers, operating in cascade or parallel, each having a different set of operating conditions to specifically adsorb, and optionally specifically desorb any given biologically active substance from the at least one solid insoluble support material. A final optional step, but one that may be advantageous for the preparation of pharmaceutical, or human or animal health preparations, is the isolation of the at least one biologically active substance from the at least one solid insoluble support material. This can be achieved by the desorption step for example, or could be followed by other refining or purifying techniques that are known to the skilled person such as chromatography.
The invention also therefore provides for a method of screening industrial dairy waste or by-product flows for new biologically active molecules of interest.
Preferably, the at least one biologically active substance is selected from the group consisting of hormones, proteins, peptides, polypeptides, antibodies, glycoproteins, glycosaminoglycans, and enzymes. Even more preferably, the at least one biologically active substance has a biological activity selected from the group consisting of antibiotic, probiotic, anti-microbial, anti-fungal, cell growth regulation, neurogenerative, oestrogenic, anti-thrombotic, anti-viral, free radical and metal ions scavengers, immuno-modulatory, anti atherogenic, anti-inflammatory.
A further object of the invention, is the provision of a complex obtainable from a process as defined previously, wherein the complex comprises at least one solid insoluble support material on which is adsorbed at least one biologically active substance. The complex preferably has a biological activity selected from the group consisting of antibiotic, probiotic, anti-microbial, anti-fungal, cell growth regulation, neurogenerative, oestrogenic, anti-thrombotic, anti-viral, free radical and metal ion scavengers, immuno-modulatory, anti atherogenic, anti-inflammatory.
Still yet another object is an animal feedstock composition comprising a complex as defined previously. In a most preferred embodiment, the complex has antimicrobial activity. In this way, the complex can be used as a substitute for feedstocks that currently contain antibiotics and which will soon be forbidden for use in agricultural animal nutrition. The antimicrobial activity of the feedstock of the present invention will act as a therapeutic solution to minor infections that some agricultural animals tend to suffer from, e.g. poultry, pigs, sheep and cattle, but is also applicable to domestic animals, and aquaculture, i.e. the farming of molluscs, crustaceans, shell animals, and fish.
Finally, it is most preferred that the at least one biologically active substance be a substance naturally present in the at least one industrial waste or by-product, i.e. that it not be a substance that has resulted from the addition by man to the mainstream product or to the animal that created the starting materials for the mainstream product. This of course extends to substances that are introduced by genetic manipulation.
Where the following meaning is given to the symbols in the
In
In
The invention will be further described and explained by the following examples, that are given for the purposes of illustrating some of the preferred embodiments of the present invention, in particular in relation to the recovery of biologically substances of interest from the well known agro-industrial by-product known as whey.
Whey Preparation
The control whey is obtained from fermented milk (Ribot milk™, sold by BRIDEL, France), and simulates whey obtained from cheese manufacturing plants during the manufacture of fresh cheese from cow's milk or certain cow's milk based soft cheeses.
The fermented milk is centrifuged at 6000 g for 20 minutes. The precipitate obtained, that is made up mainly of caseins and caseinates, was eliminated ; the supernatant was collected and made up the control whey sample.
Two types of fermented milk were used
1. low fat type (low in fats);
2. whole fat or creamy (rich in fats).
The pH of each whey obtained was measured and found to be approximately pH 4.6.
For some of the examples, the pH was adjusted to pH 6.8, by the addition of 0.5M aqueous sodium hydroxide NaOH, in order to simulate sweet whey, and in order to limit the effects of dilution of the starting material. Other aqueous hydroxides could also be optionally be used instead of NaOH, but the latter is most often used in the milk industry.
Unless indicated otherwise, 25 millilitres of each whey was taken as the starting material, and the solid support material concentration was 2% (w/v). After addition of the support material to the whey, the mixture was agitated, for example on an agitation table, for an hour.
The mixture was then filtered on a Büchner filter, using a Whatman #4 filter paper. The solid and liquid fractions obtained after filtration were stored at −20° C. for further analysis.
The various different solid support materials used were as follows in Table 1:
Biochemical Analysis
The solid support materials that were tested, either with or without prior treatment, are brought into contact with a solution that simulates an agroindustry effluent or by-product that contains one or more biologically active substances of interest.
Insofar as the experimental parameters are concerned, for example, contact times, type and degree of agitation, etc., these are adjusted to in order to modulate the adsorption and selectivity of the solid insoluble support material for the biologically active substances of interest. After solid-liquid separation, two fractions are obtained
The obtained fractions are analysed using a gel electrophoresis technique known as SDS-PAGE, which involves separating out proteins based on their respective molecular masses, followed by staining. The SDS-PAGE technique enables identification of all of the proteins present in a given sample and provides a visualisation of their distribution in each fraction, when compared to the starting sample analysed. The liquid fraction can be analysed as it is after the solid-liquid separation step, i.e. directly after adsorption. The solid fraction, on the other hand, is put into solution with what is known to the skilled in the art as a “loading” buffer, and brought to the boil. Such drastic conditions enable all of the adsorbed proteins to be desorbed, but are not representative of the enzymatic or temperature conditions prevalent in the normal digestive tract, rumen or stomach. In the figures, the dotted line delimits two areas, with the proteins of interest being located in the area above the dotted line. In the examples given, these proteins are substantially immunoglobulins and lactoferrin. The area below the dotted line is that in which unwanted residual caseins and low molecular weight proteins such as α-lactalbumin and β-lactoglobulin are to be found, which have molecular weights at about 16 to 18 kDa. These latter kind of molecules are not of interest in the present invention.
SDS-PAGE method and analysis conditions.
The following stock solutions were used
Denaturing buffer 5×(5 mL)
Separation buffer, or lower buffer, (50 mL)
Concentration buffer or upper buffer (25 mL)
Migration buffer 10X (200 mL)
APS 10% (1 mL)
The polyacrylamide gels were prepared as follows:
The samples were adjusted to a total protein concentration of approximately 1 mg/mL, by the addition of a volume of loading buffer i.e. sufficient to obtain a concentration that is ready to load of 1 mg/mL. The volume actually loaded onto the gel was comprised between about 25 μl and about 30 μL per well.
Electrophoresis was carried out under the following conditions concentration gel at 60V, then separation gel at 190 V, with migration lasting approximately 45 minutes. After migration was stopped, the gels were stained with Coomassie blue and then dried.
The sample used was a low fat whey, the pH of which had been adjusted to between 4.6 to 6.8 by the addition of 0.5 M NaOH. The results for the solid insoluble support materials 3, 4, 5 and 6 are given in
The four solid insoluble support materials tested all had high adsorptive capacity, cf. A1, but especially A2, A3 and A4. However, desorption, even under drastic conditions, was low, cf. A5 to A8. Support number 4, a clarsol KC1 did show a slight difference in the desorption profile compared to the others, the uppermost band being separating out slightly higher, which indicated the desorption of a protein of higher molecular mass.
The sample tested was a whole fat whey, the pH of which was adjusted to between 4.6 to 6.8 by the addition 0.5M NaOH. The results of solid insoluble supports numbered 3, 4, 5 and 6 are given in
The sample used was a low fat whey, the pH was not adjusted and was naturally close to 4.6. The results of solid support materials 3, 4, 5 and 6 are given in
The sample used was a low fat whey, the pH of which had been adjusted to about 4.6 to 6.8 by the addition of 0.5 M NaOH.
The results of support materials numbered 11, 12 and 13 are presented in
The sample tested was a whole fat whey, the pH of which was adjusted to between 4.6 to 6.8 by addition of 0.5M NaOH. The results for support materials 11, 12 and 13 are given in
The sample used was a low fat whey, the pH was not adjusted and was naturally close to 4.6. The results of support materials numbered 11, 12 and 13 are shown in
Microbiology
Sample Selection
The samples of example 7 were those that showed the most interesting desorption profile, with high desorption of proteins having a molecular weight within the target area being searched for. An in vitro test was carried out on these samples to show that the complex obtained had antimicrobial activity.
Mother Culture Preparation
The bacterial strain used was a strain of E. Coli. This strain was maintained in culture in a Luria-Bertani medium having the following composition: bactopeptone 10 g/L, yeast extract 5 g/L, sodium chloride 5 g/L, prepared in purified water. The culture conditions were as follows: temperature 37° C., agitation 250 rpm on an agitating table, period between reseeding: 24 hours.
At T=0 (where T equals time), an aliquot was thawed. Reseeding was carried out at 2% (v/v) in Luria-Bertani medium. At T=24 hours and 48 hours, a further reseeding at 2% was carried out. The culture at T=48 hours was taken for the experiments, since by this time, the culture had reached the exponential growth phase.
Preliminary Tests
The analytical method used was reading from a spectrophotometer at a wavelength of 565 nm. In a non-inoculated medium, one of either whey n°1 or whey n°2 was added, in order to test whether the endogenous bacteria would not develop in these experimental conditions, and thereby lead to inaccurate results. Under the conditions given, no bacterial growth was observed.
Control Experiments
Several control experiments were carried out in addition to the main experiment involving the complex comprising the solid support material and biologically active substance plus seeded medium (A):
In a glass tube with a diameter of 1.7 mm, 0.1 mL (i.e. 2% v/v) of culture was added to 5 mL of LB media placed within the tube. The culture had reached its exponential growth phase (T=0 h). 10 mg of product was added, whereby the product was the complex, or a whey powder, to give a weight of 0.2% (w/v). A collection of tubes prepared in the same way with different solid insoluble support materials was agitated at 250 rpm and maintained at at temperature of 37° C. Optical density measurements were taken at regular intervals, for example after 1, 3, 5, and 22 hours respectively via spectrophotometry, using a wavelength of 565 nm, in order to check for bacterial growth.
Calculation Method
The results given in Table 2 were obtained after subtraction of the optical density of the supports (D-E) or of complex (A-F), in the LB medium and then compared to the control set up (C) and a whey control (B). Only the values for T=5 hours and T=22 hours are shown in the table. The optical densities read at T=1 hour and T=3 hours did not show any significant differences, since they correspond essentially to the beginning of bacterial growth.
Subtraction of the values is necessary, since bacterial concentration is linked to the turbidity of the seeded medium, and in parallel, the addition of the solid insoluble support material also generates some turbidity that needs to be deducted in order to have the optical density that is directly linked to bacterial growth.
Discussion
Control C shows that in the culture conditions used, a strong rate of bacterial growth was observed.
Control B, obtained by the addition of lyophilized whey powder, shows that, on its own, whey has no inhibiting effect on bacterial growth. On the contrary, the addition of powdered whey seems to have the opposite effect, i.e. stimulate bacterial growth, probably because it is very rich in lactose.
The solid insoluble support materials on their limit microbial growth. At T=5 hours, the results obtained for sialite, metasial C and sokalite powder are respectively 11.8%, 30.1% and 5.4%, compared to the control growth (100%). At T=22 hours, the same measurements gave values of 31.5%, 46.9% and 45.8%. Thus the solid support material on its own has an inhibiting effect on bacterial growth particularly at the beginning of microbial cell growth.
For the complexes formed according to the invention, growth was in general slowed, with an amplified effect. At T=5 hours, the results for the complexes containing sialite and metasial C were respectively 3.2% and 8.6% compared to the growth control (100%). At T=22 hours, these values had reached 6.1%, 55.4% and 8.5% respectively. Although the complex containing metasial C seems to be quite a good inhibitor at the beginning of bacterial growth, the value obtained at T=22 hours would lead one to believe that its inhibitory capacity is in fact similar to that measured for the insoluble solid support alone, and that therefore adsorption of proteins from whey does not lead to increased anti-microbial activity.
However, for sialite and powdered sokalite, strong anti-microbial activity is observed at both T=5 hours and T=22 hours, with an inhibition greater than 90% . Thus the adsorption of proteins from whey, as a waste effluent of the milk industry enables not only amplification of the antimicrobial activity, but also enables this activity to be present for longer, as had been already observed with the solid support materials on their own.
It is thus likely that the complexes formed according to the present invention have bacteriostatic properties.
Further Studies
Selection of the Bacterial Strain
The first set of tests were carried out on a strain known as E. Coli. (gram −). In order to validate the theory behind the invention, other strains were tested. These strains were Staphylococcus Epidermidis (gram +) and Listeria Innocua, the latter often being used to emulate the behaviour of Listeria monocytogenes, but without the precautions necessary to manipulate the monocytogenes strain.
Other tests were carried out to discover the mechanism of action of the complexes according to the present invention, to determine whether the complexes were “concentration dependent” or “time dependent”.
Analytical Method
The results were confirmed through the use of another analytical method, in order to demonstrate that they were not biased by the experimental conditions. Tests were thus carried out on a gelose media, and visible colony counting.
In the same manner, the initial analytical method used was challenged by adopting a different method. The addition of the solid support, or the formation of a particular complex leads to the risk biasing the values obtained. For the concentration of solid support material used and tested, approximately 0.2%, the samples were homogenized by centrifugation at low speed for 5 seconds. For other concentrations, however, for example at 1% concentration, i.e. 50 mg, this simple step was insufficient to obtain a homogeneous sample in which the OD could be said to be representative. The inventors therefore resorted to a visual observation of the turbidity of the samples.
Pilot Scale
As has been mentioned above, the process or method of the present invention is designed to solve the problem of waste, effluent or by-product flows of organic material that are generated in the dairy industry through a industrially economically viable method of recovering biologically active substances from said waste or by-product flows.
Pilot Scale Production
The production of several kilograms of active complex was carried out starting from whey originating from a cheese manufacture. Kaolin, available under the tradename Sialite (Soka, France) at 2% w/v, was added to a total volume of approximately 8m3 whey identical to that mentioned in example 7. Solid-liquid separation was carried out using a press filter. Approximately 160 kg of active complex were recovered for analysis and animal nutrition testing. The whey was tested upon entry into, and exit from, the production process, and the following results obtained:
MAT: total nitrogen content=N*6.38 (where 6.38 is a correction factor specific to milk proteins)
NPN: non-protiein nitrogen (N*6.38*0.97)
NCN non-casein nitrogen (N*6.38*0.98)
MAT−NCN=quantity of casein present, and enables determination of the amount of casein degradation
MAT−NPN=quantity of caseins+serum proteins
NCN−NPN=quantity of serum proteins
An SDS-PAGE analysis of the nature of the proteins retained on the solid support confirmed the result described in example 7. Only high molecular weight serine proteins are adsorbed onto the solid support used.
Animal Studies
A random test on 14 day old Ross breed chickens was carried out for 14 days over a population of 229 individuals, separated into a first group of 114 chickens on a standard dietary regime, and 115 chickens whose standard dietary regime was supplemented with 2% by weight of the active complex obtained above. The diet, which mainly comprised maize (52%), soybean (34%) and wheat (7%), was given to the animals as crumbs ad libitum. Observations and weighings of the chickens were carried out at the start (day 1), and then at days 7 and 14, to determine the general state of health of each group, but also to ascertain the average daily weight gain and a feed efficiency index.
These two parameters are usually considered to be the most important parameters for an industrial evaluation of an animal feed test. The average daily weight gain indicates how much weight the animal puts on and the feed efficiency index indicates how much the animal has put on weight compared to the quantity of food ingested. For a farmer, the idea is to have a minimum quantity of feed to distribute for a maximal weight gain. The product described and produced according to the present invention does just that. Physico-chemical and microbiological analyses of the excreta were also undertaken.
In comparison to the standard dietary regime test group, the tests on the supplemented group of animals showed that during the first week, known as the growth stage, an average daily weight gain increase of +4.2% and a 5.3% reduction of the feed efficiency index were observed. No statistically significant difference was observed for mortality and morbidity rates. The analysis of the excreta showed a reduction in coliform bacteria of −0.5Log CFU/batch of standard food, an increase in dry matter of +12.9%, as well as a reduction in organic matter of −7.9%.
A random test in an industrial pork farm on piglets that were 28 days old was carried out over 14 days and on 74 specimens, split into two groups of 36 and 38 piglets respectively. The group of 36 piglets was put on a standard dietary regime, and the group containing 38 piglets were fed with an active complex 1% by weight supplement to the standard diet. The standard diet for piglets post-weaning was given as granules ad libitum. Observations and weighings were carried out at day 1 and day 14 to determine the general state of health of each group, but also the average daily weight gain and feed efficiency index. Physico-chemical and microbiological analyses of the excreta were also carried out. In comparison to the chickens on the standard diet, the tests showed that over a period of 14 days, an increase in the average daily weight gain of +23.2% and a reduction of the feed efficiency index of −4.0% was observed. No significant statistical difference was observed for the mortality and morbidity rates. Analysis of the excreta showed an increase in dry matter of +53.9%, a reduction in organic matter of −12.9%, as well as a reduction in the total quantity of phosphorus of −33.3%.
| Number | Date | Country | Kind |
|---|---|---|---|
| 03292605.7 | Oct 2003 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB04/03420 | 10/19/2004 | WO | 4/18/2006 |
