Stabilized milkproteins-containing compositions
The invention relates to a process for the preparation of homogeneous, liquid compositions. In particular the invention relates to a process for the preparation of edible, aqueous suspensions comprising particles which display a tendency to settle or to precipitate and which contain, for instance, isoluble, often denatured proteins, insoluble carbohydrates, and/or particles which display a tendency to migrate to the surface of the liquid, such as fats or other components, the specific weight of which is appreciably smaller than the specific weight of the liquid in which the particles are suspended or emulsified. The occurrence of these undesirable phenomena results in suspensions being obtained which have no homogeneous composition, which is visually--and in the case where edible compositions are involved nutritionally--unacceptable. 
     These frequently occurring problems will in particular arise in the preparation of liquid animal feed compositions, such as calf milk replacers, which have a relatively high content of insoluble carbohydrates and fats. 
     Attempts have been made to solve these problems by incorporating a stabilizer into the composition. 
     As stabilizers hydrocolloids have been proposed. However, these stabilizers are unattractive, chiefly by reason of cost price. Moreover, to achieve optimum activity of these stabilizers the water used as diluent for the composition should be brought to a temperature above 37° C., which is in some cases objectionable. 
     Applicants have now found a process which alleviates the above disadvantages. 
     The present invention provides a process for the preparation of homogeneous compositions starting from (a) a solvent, (b) a blend containing components which, when mixed with said solvent, tend to precipitate or to migrate to the surface of the solvent, and (c) a stabilizer which keeps said components in suspension, which process comprises using a stabilizer consisting of a casein which has been hydrolysed by a proteolytic enzyme under conditions such that the K-casein moiety is hydrolysed whereby no peptides having a molecular weight below 10 4  are formed. 
     As stated above, the components which should be kept in suspension can comprise all kinds of materials whose specific weights differ to an appreciable extent from the specific weight of the diluent. 
     Where the stabilization of, for instance, calf milk replacers is involved, these components consist of denatured proteins and high molecular weight carbohydrates, having a specific weight ranging between 1 and about 1.5, and fats having a specific weight of below 1. As diluent for these calf milk replacers water is used. 
     The casein which is treated with the proteolytic enzyme can be an acid casein or a casein salt such as sodium caseinate. 
     The proteolytic enzymes preferably applied for this purpose are those which are capable of partially hydrolysing casein and particularly hydrolysing K-casein, whereby no substantial amount of peptides with a molecular weight of less than 10 4  is formed. In other words, preferably those enzymes are used which mainly achieve curdling or clotting and thus mimic rennet. 
     In the literature these enzymes are in general specified by the term &#34;rennet substitutes&#34;. These rennet substitutes are products commercially available under the trade names Noury Liquid, Noury Granules, Meito Rennet, Emporase (derived from the organism Mucor Pusillus Lindt), Marzyme, Fromase, Rennilase, Hannilase (derived from the organism Mucor Miehei). It was further found that proteolytic enzymes of microbiological origin, especially subtilisin, were particularly advantageous. In a preferred embodiment of the process according to the invention for the preparation of calf milk replacers, it is recommended to use subtilisin originating from Bacillus licheniformis- or subtilis strains. These subtilisins, marketed by the firm of NOVO under the tradename Alcalase (R), the principal component of which is subtilisin Carlsberg, and Neutrase (R), were found to give excellent results in stabilizing calf milk replacers based on soyameal concentrate, milk proteins and fats. 
     These enzymes are especially suitable because at a pH within the range of 5.0 to 7.5, prevailing in calf milk replacers, they ensure the in situ formation of the required, partially hydrolysed casein, as defined above, without it being necessary that the hydrolysis be carried out under strictly controlled time and temperature conditions. 
     In the process according to the invention it is possible either to incorporate into the composition an effective amount of enzymatically treated casein, or to incorporate into the starting mixture--which is to be diluted to a liquid composition--untreated casein together with the enzyme, after which the hydrolysis is allowed to take place in situ subsequent to dilution of the mixture with the solvent. 
     Preferably the enzyme is applied onto a carrier. The carrier may comprise a water-soluble or water-dispersible salt. Preferably the salt is a substance binding calcium and magnesium ions, such as an alkali metal polyphosphate, e.g. sodium pyrophosphate, sodium tripolyphosphate and/or sodium hexametaphosphate, or an alkali metal citrate or ethylenediamine tetraacetate (EDTA). 
     These substances promote keeping the proteins in suspension. Moreover, it is believed that the use of these substances has an emulsifying effect on fat globules. 
     Especially when the composition to be stabilized contains whey proteins, it is recommended to use these substances in an amount of 0.1-10% by weight calculated on the amount of protein, whereby these substances can be partly or wholly used as a carrier for the enzyme. 
     In a preferred embodiment of the process of the present invention the enzyme applied onto a carrier is coated with a water-soluble or water-dispersible polymer, e.g. a cellulose derivative or polyethylene glycol. 
     A particular application of the present invention is concerned with the preparation of calf milk replacers. According to the invention these calf milk replacers are obtained by 
     (a) preparing a mixture, comprising proteins, fats, carbohydrates, at least 0.5% casein calculated on dry matter, and an effective amount of the proteolytic enzyme; 
     (b) diluting this mixture with enough water to obtain a suspension having a solids content of 5-20% by weight; 
     (c) if necessary, adjusting the pH to a value ranging from 5.0 to 7.5. 
     Preferably 1-10% by weight of sodium caseinate is incorporated into the mixture. Preferably 10-30% by weight of fat, 10-30% by weight of protein and 10-50% by weight of carbohydrates are incorporated into the mixture. 
     The enzyme preferably used is a subtilisin, which has as its principal enzyme component subtilisin Carlsberg. This enzyme is preferably used in an amount of from 0.01-3% by weight, calculated on the amount of casein when an enzyme is used having an activity of 0.5-0.6 Anson units/g, measured at 25° C. and at pH 7.5. 
     The protein source preferably comprises whey protein and/or soya proteins. It should be understood that other proteins, such as potato protein or single cell protein, can be used as well. It was found convenient to introduce as a further adjuvant to the mixture an amount of 0.1-10% by weight of the above-mentioned substances capable of binding calcium and magnesium ions. 
     The invention also relates to dry animal feed compositions comprising besides the usual ingredients such as fats, carbohydrates, proteins, vitamins and minerals, also casein and a proteolytic enzyme capable of hydrolysing only the K-casein moiety of the casein molecule. 
     The animal feed compositions according to the invention preferably contain 0.5-10% by weight of sodium caseinate, 10-30% by weight of fat, 10-30% by weight of proteins and 10-50% by weight of carbohydrates. The enzymes and adjuvants such as substances capable of binding calcium and magnesium ions to be introduced into these animal feed compositions, and of the manner of processing these compositions have been mentioned elsewhere in the specification and will therefore not be repeated. 
     It will be understood that animal feed compositions according to the present invention can be provided which besides the usual ingredients also comprises a casein, the K-casein moiety of which has already been hydrolysed by means of a proteolytic enzyme. For the majority of animal feed compositions an amount of 0.5-10% by weight of enzymatically treated sodium caseinate will suffice. 
     In the specification the application of the invention for the preparation of animal feed compositions has been illustrated. It will be understood, however, that the gist of the invention can also be applied for the preparation of other liquid compositions adapted for human purposes. 
    
    
     The invention will now be illustrated with reference to the following Examples. 
     EXAMPLE 1 
     A mixture was prepared comprising 
     
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                % by weight                                               
______________________________________                                    
fat               20                                                      
soya protein concentrate                                                  
                  15                                                      
whey powder       55                                                      
sodium caseinate   9                                                      
vitamins/minerals  1                                                      
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     To this mixture was added an amount of 0.05% by weight of subtilisin from Bacillus subtilis (Neutrase ex Novo, activity 0.5 Anson units/g at 25° C. and pH 7.5). A suspension (pH 6.6) having a solids content of about 10% by weight was prepared by adding tap water to the mixture in a vessel and stirring it for 5 minutes. By the action of the enzyme the suspension gradually resembled buttermilk. After the suspension was allowed to stand for one hour, no precipitate was observed on the bottom of the vessel. 
     No fatty layer was observed at the surface of the liquid. The liquid was of a completely homogeneous composition. SDS-polyacrylamide gel electroforese showed that the enzyme that had been used had hydrolysed K-casein, whereas α- and β-caseins had not been affected even after four hours. The electroforese patterns showed that casein was broken down into proteins the molecular weight of which ranged between 10 and 20×10 3 , whereas no peptides had been formed of a molecular weight ranging between 5 and 10×10 3 . 
     When a composition was prepared starting from the same ingredients but without enzyme, almost immediately a precipitate was formed which mainly consisted of insoluble carbohydrates and proteins, and gradually creaming of fat could be observed. 
     EXAMPLE 2 
     Example 1 was repeated, except that the enzyme used this time was subtilisin from Bacillus licheniformis (Alcalase ex Novo, activity 0.6 Anson units/g at 25° C., pH 7.5). The results were as satisfactory as in Example 1. 
     EXAMPLE 3 
     Example 1 was repeated, except that the enzyme used this time was commercially available rennet. The results were as satisfactory as in Example 1. 
     EXAMPLE 4 
     Examples 1-3 were repeated, except that the amount of sodium caseinate was reduced to 5% by weight, while the amount of soya concentrate was proportionally increased. Also in this case a satisfactory stabilizing effect was achieved. 
     EXAMPLE 5 
     A mixture was prepared comprising: 
     
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                % by weight                                               
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fat               22                                                      
whey powder       55                                                      
sodium caseinate   1                                                      
soya protein concentrate                                                  
                  21                                                      
vitamins and minerals                                                     
                   1                                                      
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     To this mixture were added 0.05% by weight of the enzyme used in Example 2 and 0.4% by weight of sodium tripolyphosphate. From this mixture an aqueous suspension was prepared having a pH of 6.6 and a solids content of 7% by weight. Also this suspension displayed an excellent stability. 
     Example 6 
     Example 5 was repeated, except that 0.8% by weight of acetic acid was used instead of sodiumtripolyphosphate. The resulting aqueous suspension also displayed an excellent stability. 
     EXAMPLE 7 
     The formulation of Example 1 was repeated, except that the enzyme was added in the form of granules. The enzyme granules comprised 12.5% by weight of enzyme, 77.5% by weight of sodiumtripolyphosphate, 4% by weight of magnesiumoxide and 6% by weight of dextrin/H 2  O. 
     Granulation was effected by spraying a solution of the enzyme and dextrin onto polyphosphate particles. Powdered magnesium oxide was used to prevent the formation of aggregates. Dosing of the necessary amount of enzyme granules was very readily effected by adding them to the dry starting mixture of the ingredients or to the fat. 
     EXAMPLE 8 
     To the mixtures of Example 1 and Example 5 were added 9% by weight and 1% by weight respectively of the product of the enzymatic reaction of rennet and sodium caseinate. 
     The mixtures so obtained were used for the preparation of aqueous suspensions having a solids content of 10% by weight. Also these suspensions displayed an excellent stability.