NZ619868B2

NZ619868B2 - Dairy based compositions with low lps

Info

Publication number: NZ619868B2
Application number: NZ619868A
Authority: NZ
Inventors: Vries Ynte Piet De; Anouk Leonie Feitsma; Paula Maria Leandro Garcia; Andries Dirk Siemensma; Der Padt Albert Van
Original assignee: Friesland Brands Bv
Priority date: 2011-07-13
Filing date: 2012-07-13
Publication date: 2015-05-28

Abstract

method to produce a dairy based food composition with low lipopolysaccharide (LPS) comprises the steps (a) Providing a milk with a storage time of less than 264 hours (11 days old), (b) Treating the milk such that at least 98wt% of the Gram negative bacteria is removed and (c) Heating the milk wherein at least 98wt% of the Gram negative bacteria is removed to 60-90 °C. The method can further comprises (d) Treating the milk with a micro filter of pore size of from 0.05-1.2 ?m such that at least a casein rich fraction and a serum protein rich fraction are obtained. The treatment to remove at least 98wt% of the bacteria is selected from the group consisting of Bacterial filtration with a poresize of 0.5-2.5 micron; Centrifugation; or Use of antibody to remove Gram negative bacteria. Dairy based food composition obtained by a method is further disclosed. Also disclosed is a dairy based food composition where the amount of lipopolysaccharide (LPS) is less than 4000E3 endotoxin units (EU) per liter ready to use food composition or where the amount of LPS is less than 30E3 endotoxin units (EU) per gram dry product. rein at least 98wt% of the Gram negative bacteria is removed to 60-90 °C. The method can further comprises (d) Treating the milk with a micro filter of pore size of from 0.05-1.2 ?m such that at least a casein rich fraction and a serum protein rich fraction are obtained. The treatment to remove at least 98wt% of the bacteria is selected from the group consisting of Bacterial filtration with a poresize of 0.5-2.5 micron; Centrifugation; or Use of antibody to remove Gram negative bacteria. Dairy based food composition obtained by a method is further disclosed. Also disclosed is a dairy based food composition where the amount of lipopolysaccharide (LPS) is less than 4000E3 endotoxin units (EU) per liter ready to use food composition or where the amount of LPS is less than 30E3 endotoxin units (EU) per gram dry product.

Description

W0 20132‘009182 Title: DAIRY BASED ITIONS WITH LOW LPS Field of the Invention The invention relates to a method of providing milk proteins. Particularly, the invention relates to a method of making a milk protein composition with a low lysaccharide (LPS) concentration. More specifically the invention relates to a method of making milk protein compositions with low LPS concentration for infant and toddler formula.

Background The epithelial integrity in the gut is of pivotal importance for an optimal lial barrier, e.g. in mammals. The outer membrane of Gram negative bacteria, in contrast to Gram positive bacteria, contains lipopolysaccharides (LPS), which are xines. A single bacterial cell contains imately 3.5 x 106 LPS molecules.

LPS is a complex, negatively charged molecule composed of a distal polysaccharide chain called the O-specific chain, a core polysaccharide and a lipid moiety referred to as lipid A. LPS acts as endotoxin resulting in induction of strong inﬂammatory responses in s and human beings and is involved in the development of several diseases, e.g., sepsis. The toxic part of LPS is the lipid A moiety, which consists of two phosphorylated glucosamine residues and at least 6 fatty acids. These two phosphate groups are essential for the bioactivity of LPS. Incorporated in the outer leaﬂet of the Gram-negative bacterial cell ne, the LPS molecule is relatively non-toxic as the lipid A moiety is more or less stown away. However, when a dividing or dying bacterium spontaneously releases LPS into the mammalian ation, it can interact with several proteins such as albumin, lactoferrin, high-density lipoproteins (HDL), low-density lipoprotein (LDL) and bacterial permeability- increasing protein (BPI).

The LPS-binding protein(LBP) is the most important protein in this respect. The LBP-LPS complex binds to CD14 (‘Cluster of differentiation 14’) after being released from the membrane of the cells of the d e (macrophages, monocytes and polymorphonuclear leukocytes). The CD14 require an accessory or complex, the toll-like receptor 4/ myeloid differentiation-2 complex MD-2) to initiate a cell signal transduction e in the cell. This e promotes nuclear translocation of NF~KB and transcription of pro- inﬂammatory cytokines such as Tumor Necrosis Factor alpha (TNFOL). TNFoc and other pro-inﬂammatory cytokines induce vascular permeability, enhanced blood flow, and phil recruitment to the LPS source as well as systemic responses such as fever. At high concentrations, LPS s toxic by overstimulation of TLR4 signalling, leading to an excessive inﬂammatory response that results in adverse reactions such as septic shock. An aberrant immune response to LPS or other bacterial antigens (eg. in or T cell epitopes) has been linked to inﬂammatory bowel disease (IBD) and necrotizing enterocolitis (NEC). NEC predominantly affects premature infants and is assumed to be caused by an immature immune response to commensal organisms that colonize the gut after birth.

Premature infants probably have impaired LPS sensing since they miss an essential molecule from the TLR4- induced signalling pathway and therefore es are predisposed to NEG upon microbial colonization of the immature intestine since one of the most abundant sources of LPS encountered by vertebrates is their resident gut microbiota. These gut microbiota do not elicit pathological inﬂammation in adult hosts. There are also indications that LPS plays an important role in the ion of systemic inﬂammation. Since newborn babies have a higher permeability of their gut epithelium during the first months or their lives, pathogenic substances have a higher chance to cross the lial r and induce inﬂammation in the child.

Raw milk can contain significant numbers of Gram negative ia. For health safety reasons, in food, ia, including the Gram negative bacteria are killed by pasteurisation and sterilisation techniques. Unfortunately killing the Gram negative bacteria does not result in complete degradation of the membrane of Gram negative bacteria and the LPS molecules stay largely intact and are therefore ed into the composition. In addition, during the processing of a food composition, LPS is released from the bacterial wall due to the shearing forces caused by the food processing techniques. LPS is heat stable at 100°C and can survive during the processing of food products.

Often the proteins in infant food and other dairy based products come from a whey fraction that stems from the cheese making process. The whey fraction contains many ional whey proteins such as a-lactalbumin and B-lactoglobulin.

During the process of cheese making, the milk is first often heat treated to kill bacteria including Gram negative bacteria and to inactivate unwanted enzymes. As explained above these treatments will release LPS in the milk. During the cheese making process the LPS may thus end up in the whey fraction. In addition, during the next processing steps of the whey ﬁ'action in the making of the infant formula or other dairy based food, contamination of Gram negative bacteria may again occur and therefore additional heat treatments may be carried out. Although the bacteria count may be under control this way, this can lead to an increasing amount of LPS that ends up in the formula. As is explained above, a high LPS load is not desirable in infant a.

Another method to provide a protein fraction that may be used to e infant formula or other dairy based food is a method that uses microﬁltration. A background nce on providing protein fraction from milk with iltration is US 5,169,666. Herein bovine milk is subjected to low temperature ultraﬁltration or iltration.

Another background reference is EP 1 138 238. Herein a protein composition, derived from whey, is manufactured by subjecting milk that has not been heat-treated, or at most has undergone a moderate heat treatment, to iltration at elevated temperature (typically 50°C).

A further background reference is . This concerns a serum protein product suitable as an ingredient for e.g. babyfoods, which is ed by micro-filtration of bovine milk at a temperature of MPG-20°C utilizing a membrane having a pore size of between 0.3 and 0.5 pm.

The prior art methods including the ones mentioned above may e protein fractions that are suitable for producing infant formula and other dairy based products, but they are not directed to minimize the LPS content, and thus the LPS content in the n fractions, and thus in the final dairy product, may be quite high. For instance the LPS is not removed by the microfiltration method and may end up in the permeate fraction.

EP 1 859 924 B1 discloses that lactic acid bacteria and biﬁdobacteria, particularly those with hydrophobic surface, have the ability to bind endotoxins. The hydrophobic lactic acid ia or bifidobacteria should have a percent hydrophobicity (%H) of at least 80%H. As these bacteria grow they may bind up to 95% of the LPS molecules present. However, the LPS molecules are still present in the composition and may thus become detached from the hydrophobic ia by eg. heat treatment or shear forces during the sing of the composition. In addition, if one does not want to add the hydrophobic bacteria to the composition, there is still no satisﬁable method to produce a composition with a low level of LPS.

Summary of the invention It is ore desirable to have a composition that is microbiologically safe but that also has a low LPS load. The t invention provides a solution.

In a ﬁrst aspect the present invention is directed to a method to produce a dairy based food composition with low LPS comprising the steps (a) Providing a milk with a storage time of less than 264 hours (b) Treating the milk with a microfilter of poresize of from 0.01-2 am such that at least a casein rich fraction and a serum protein rich on is obtained In a second aspect the present invention is directed to a method to produce a dairy based food composition with low LPS comprising the steps (a) Providing a milk with a storage time of less than 264 hours (b) Treating the milk such that at least 98% of the Gram negative bacteria is removed (0) Heating the milk wherein at least 98% of the Gram negative bacteria is removed to °C.

In a r aspect the present invention is d to a method to produce a dairy based food composition with low LPS comprising the steps 80 (a) Providing a milk with a storage time of less than 264 hours (b) Treating the milk such that at least 98wt% of the Gram negative bacteria is removed WO 2013009182 (0) Heating the milk wherein at least 98wt% of the Gram negative bacteria is removed to 60-90°C. (d) Treating the milk with a microﬁlter of poresize of from 0.01-2 um such that at least a casein rich fraction and a serum protein rich fraction is C31 obtained In yet r aspect the present invention is directed to a dairy based food composition comprising less than 4000E3 EU LPS per liter ready to use composition or comprises less than 80E3 EU LPS per gram dry product.

Detailed description The invention is directed to a dairy based composition with low LPS load.

In raw milk Gram negative bacteria are present as well as Gram positive bacteria and enzymes that may spoil the milk. For these s the milk is usually pasteurized or sterilized to kill bacteria and vate s. During heat treatment and other processes of milk treatment such as ng and homogenization, the present bacteria can be ruptured. When the Gram negative bacteria are ed the LPS is released in the milk. Unfortunately LPS is heat stable and thus after these treatment the milk is loaded with LPS that may still cause harm, especially in vulnerable groups such as s. The amount ofLPS in the milk is dependent on the amount of Gram negative ia in the milk. The more Gram negative bacteria in the milk the more LPS will be released in the milk. The older the milk is the more Gram negative bacteria are present in the milk when the milk is not pasteurized or sterilized. as these bacteria will grow and multiply during storage at low temperature.

At the farm the milk that comes from the cow is ﬁrst stored in a cold tank to keep the milk until the milk is collected and transported to the milk processing factory. In normal circumstances, the milk is collected generally every 2-3 days. The milk of all the cows in these 2-3 days is collected in the cold tank. The milk that is transported to the milk factory is thus a mixture of milk of different ages. It means that the time between the milking of the cow and the arrival of the milk at the factory may be some 3-4 days for the milk that was collected first in the tank and may be only 0.5-1 day for the milk that was last collected.

W0 2013/‘009182 The storage time of the milk in the present invention is deﬁned as the storage time of the oldest milk, i.e. the milk that is the first collected in the storage tank at the farm. The storage time is the time between the time of milking the cow and the time the milk or products derived therefrom is sed into a dry product or UK finished concentrate product. The storage time thus comprises the time in the tank on the farm, the transportation time, the storage time in the factory and the processing time in the factory. Normally the e time may be up to about 2 weeks. For many dairy based products such as infant formulas, part of the protein on is often ed from whey from a cheesemaking process. For such products with a whey fraction the total time between the milking of the cow and the ﬁnished product such as a dry infant a may be up to about 3 weeks.

In the present invention the maximum e time is 264 hours, or 11 days. This is the maximum storage time of the oldest milk. As said , the milk that arrives at the milk factory is a mixture of milk of different ages, the oldest may have been stored in the tank on the farm for up to 3-4 days while the youngest milk may be only half a day old. Thus the maximum time between the milking of the cow and the milk being orated into a dry product or finished concentrate is 264 hours or 11 days.

Preferably the storage time is from 250 to 40 hours, more preferably from 220 to 60 hours, even more preferably from 200 to 80 hours, more preferably from 180 to 100 hours, more preferably from 160 to 110 hours, even more preferably from 150 to 130 hours, and most ably from 145 to 185 hours. Suitable storage times may also be from 10.5 to 1.5 days, more suitably from 10 to 2~days, more suitably from 9.5 to 2.5 days, more suitably from 9 to 3 days, even more suitably from 8.5 to 3.5 days, more suitably from 8 to 4 days, even more suitably from 7.5 to 4.5 days, more suitably from 7 to 5 days, even more suitably from 6.5 to 5.5 days, and most suitably from 6 to 5 days.

The time that it takes from milking the cow and l at the milk factory, the arrival time, is also important, and preferably should be as short as possible. Again it should be understood that the arrival time is the time of the oldest milk present in the mixture of milkings. In a preferred embodiment, the arrival time is less than 8 days or less than 70 hours. Preferably the arrival time is less than 2.5 days, more preferably less than 2 days, even more preferably less than 1.5 days, more preferably less than 1 day, and most preferably less than 0.5 day. Suitably the arrival time is less than 60 hours, more preferably less than 50 hours, even more preferably less than 35 hours more preferably less than 25 hours, and most preferably less than hours. As it is important to keep the contamination of the milk of Gram ve bacteria to a minimum, the milking, storing and transfer of the milk, as well as the Cl handling in the milk factory is done in a hygienic or aseptic way.

The milk provided to the process of the invention can, in principle, be from any dairy animal. This is mostly cattle, and particularly cow (adult female cattle), but in addition to cattle, the following animals provide milk used by humans for dairy products: Camels, Donkeys, Goats, Horses, Reindeer, Sheep, Water buffalo, Yaks, and moose. Most preferably, the milk used in the invention is cow’s milk.

The microfiltration is generally ted using a lter having a pore size in the range of from 0.01 to 2 , preferably from 0.1 - 1.2 micron, more preferably from 0.2 - 0.5 micron and most preferably from 0.15 to 0.45 .

Suitable microfilters are known in the art and include, eg. spiral wounded polymer or ceramic based systems For the microﬁltration, any conventional apparatus for ow microﬁltration can be used. Thus, for instance, use can be made of a spiral-wound microﬁltration membrane, for instance as described in ERA-1873975. Preferably, a process system with multiple spiral-wound modules is used. it has been found that it is helpful that in the crossﬂow microfiltration process measures are taken for reducing the transmembrane pressure across the membrane, in such a manner that the embrane pressure is 2.5 bar at a maximum. For that reason, preferably, the embrane pressure during microfiltration in-a method according to the invention is kept relatively low, that is, 2.5 bar at a maximum. Good s as regards the protein composition of the permeate have for instance been obtained at a maximum transmembrane pressure of 2 bars. The average transmembrane pressure may vary, and is for instance 0.1 to 1.8 bar. In a specific embodiment, the maximum transmembrane pressure is from 0.2 to 1.5 bar, more preferably from 0.8 to 1.2 bar, more preferably from 0.5 to 1 bar and most preferably from 0.6 to 0.8 bar.

Instead of reducing the transmembrane re, a different on may be the use of microﬁltration membranes having a gradient in the porosity or thickness of the membrane layer.

In a method according to the invention, standard microfiltration membranes having a pore size of between 0.1 and 1.2 pm may be used. As is known in general. pore size inﬂuences the eventual protein composition of the permeate and the retentate. In the light of the present invention, the pore size proves to have an inﬂuence inter alia on both the serum protein to casein ratio and the proportion of beta casein in the casein fraction. In an embodiment, use is made of a membrane, for instance a spiral-wound membrane, having a pore size of between 0.2 and 0.5 tun, ably between 0.15 and 0.45 pm.

The microﬁltration steps are conducted starting from milk that comprises non- red milk protein and with ient microbiological quality. This may refer to raw (untreated) milk, or to milk that has undergone a mild heat treatment, but has not been subjected to a temperature higher than 90 °C. The milk may be whole milk or milk which has been skimmed to a greater or lesser degree, raw milk, bactofuged milk or bactoﬁltered milk or milk pasteurized under mild conditions or reconstituted from powdered milk dried at low temperature. Preferably, non heat-treated, skimmed raw milk is used. If reated, this is done at a ature below the ature where Gram negative bacteria are broken down, preferably below 80 OC. Suitably the milk and ts obtained from the milk during the process are not subjected to a heat treatment at a temperature above 75°C, more preferably not above 70°C, even more preferably not above 65°C, also more preferably not above 60°C, more preferably not above 55°C, most preferably not above 50 °C.

The microfiltration step may be performed at a temperature between 0 and 65°C. Preferably the microfiltration is performed at a temperature of between 25 and 65°C or between 0 and 25°C. More preferably the microfiltration step is performed at a temperature of from 0 to 25°C, more preferably of from 5 to 20°C and most preferably from 10 to 15°C. In another red embodiment, the microfiltration step is performed at a temperature of from 25 to 65°C, more preferably of from 35 to 60°C and most preferably from 45 to 55°C.

The microﬁltration tes the milk into a permeate and a retentate.

The retentate is a casein rich fraction and the permeate a serum n rich fraction.

In the casein rich fraction the amount of casein on total protein is more than the amount of casein on total protein in milk that has not been subjected to iltration. Preferably, the casein rich fraction comprises 1wt% more casein on total protein than non-microﬁltered milk, more preferably 5wt% and most preferably 10wt% more. In the serum protein rich fraction the amount of serum protein on total 01 protein is more than the amount of serum protein on total protein in milk that has not been ted to microfiItration. Preferably, the serum protein rich fraction comprises 20wt% more serum on total protein than croﬁltered milk, more preferably 40wt% and most preferably 60wt% more.

In a red embodiment the heating step is done at a temperature of between (SO-85°C, more preferably between 65 to 80°C, and most preferably between 70 and 75°C. Suitable heating regimes are at 60-65 °C for 1-2 minutes or at a ature of 70-72 °C’ for 5—30 seconds.

In a preferred embodiment the heating step is done at a temperature of 60- 65 °C for 1-10 minutes or at a temperature of 65-85 °C for 5-180 seconds, preferably at a temperature of 65-76°C for 10- 120 seconds, most preferably at a temperature of 66-71°C for 5 to 180 seconds.

In another aspect the invention is d to a method to produce a dairy based food composition with low LPS comprising the steps (a) Providing a milk with a storage time of less than 264- hours (b) Treating the milk such that at least 98% of the Gram negative ia is removed (c) Heating the milk wherein at least 98% of the Gram negative bacteria is removed to (SO—90°C.

For microbial safety the milk is treated such that at least 98% of the Gram negative bacteria is removed. Preferably the Gram negative bacteria are removed as intact cells. In a preferred ment, the Gram negative bacteria are removed such that the bacterial cells remain intact and that preferably as little as possible and most ably no additional LPS is released in the treated milk. Bacterial removal techniques are known such as a bacterial ﬁltration with a poresize of 5 micron, centrifugation, or use of antibodies to remove Gram negative bacteria. It is to be understood that there may be other methods that remove Gram negative bacteria.

Any method is suitable as long as it removes at least 98% of the Gram negative bacteria and is safe for a food product. With removal is meant that the Gram negative bacteria are taken out of the product, in contrast to being killed but still present in the product, such as e.g. pasteurization and sterilization methods do.

O! Bacterial ﬁltration with a filter with a pore size of 0.5-2.5 micron s Gram negative bacteria and spores that are larger than about 0.5-2.5 microns. ly the poresize of the bacterial filter is between 0.7 and 2 micron and more preferably between 1 and 1.5 micron. A suitable example of such a bacterial filtration is bactocatch. In a preferred embodiment the filtration to remove Gram negative bacteria and spores is conducted at a temperature of from 0 to 65 °C, more preferably of from 35 to 60 °C and most preferably of from 45 to 55 °C.

Gram negative ia may also be removed by centrifugation. The milk is centrifuged at high speed, e.g. from 5000 rpm to 8000 rpm to remove the Gram negative bacteria. Suitable centrifuge speeds are from 5500 rpm to 7500 rpm, more suitably from 6000 rpm to 7000 rpm. Suitably the Gram ve ia are removed by a uge (ex Tetrapack). r suitable method to remove Gram negative bacteria is the use of dies. Antibodies may be designed to recognize specific Gram ve bacteria or a wide range of Gram negative bacteria. Preferably the antibodies are immobilized to a column or beads so that they can be easily removed.

In a red embodiment at least 98.5% of the Gram negative ia are removed, more preferably at least 99% of the Gram negative bacteria are removed, and more preferably at least 99.5% of the Gram negative bacteria are removed. Most preferably at least 99.9% or even 100% of the Gram negative bacteria are removed.

After the removal of at least 98% of the Gram negative bacteria, a heating step is performed to inactivate unwanted enzymes and other pathogens that may not have been removed by the bacteria removal step. The heating gives another effect to the microbial safety. e most of the Gram negative bacteria are removed, the 3O milk may be subjected to a heat treatment up to 90°C, as the disruption of the Gram negative bacteria that are left may release their LPS in the milk, however due to the very low amount of Gram negative bacteria left, if at all, the amount of LPS in the milk is still very low. Preferably, during the process to produce the food product, the W0 2013i009182 milk and t obtained therefrom are not subjected to a heat treatment above 85°C, more preferably not above 80°C and most preferably not above 70°C. In a red embodiment the heating step is done at a temperature of 60-65 °C for 1-10 minutes or at a temperature of 65-85 °C for 5-180 seconds, preferably at a temperature of 65-76°C for 10- 120 seconds, most preferably at a temperature of 66- 71°C for 5 to 180 seconds.

A le mild pasteurization technique is at a temperature of 60-65 °C for 1—2 minutes or at a temperature of 70-74°C, preferably 70-72 °C’ for 5-30 seconds.

In a preferred aspect of the invention a method is provided comprising the steps (a) Providing a milk with a storage time of less than 264 hours (b) Treating the milk such that at least 98% of the Gram negative bacteria is removed (0) Heating the milk wherein at least 98% of the Gram negative bacteria is removed to 60-90°C (d) Treating the milk with a microfilter of ze of from 0.01-2 1.1m such that at least a casein rich fraction and a serum protein rich on is The bacteria removal step and the microfiltration step may be performed in any order, however it is preferred to have the heating step to follow after the bacteria removal step. In a preferred embodiment, the bacterial l step is performed before the microﬁltration step and most preferably the heating step is performed before the microﬁltration step.

Suitably, the microﬁltration and/or Gram negative bacteria removal step is performed on milk that has been subjected to a decreaming ent. Decreaming may be performed with any suitable method known to the skilled person. A suitable method is centrifugation, wherein the r proteins and carbohydrates are separated from the less heavy fat particles. Preferably the milk is decreamed to a fat content that is about 70wt% of the original fat content, more preferably to about 50wt% of the original fat content, more preferably to about 25wt% of the fat content and most preferably to about 10wt% of the original fat content.

In order to make the food composition, the casein rich ﬂaction and/0r serum protein rich fraction are used. In a preferred embodiment the serum n rich fraction is combined with the casein rich fraction or the serum protein rich fraction is combined to another milk product with a storage time of less than 264 hours. Suitably the milk with a storage time of less than 264 hours has been subjected to a treatment wherein at least 98t% of the Gram ve bacteria have been removed and has been subjected to a heat treatment above temperature (SO-90°C. ably the serum rich on and/or casein rich fraction, or milk is combined to obtain a casein: serum protein ratio of from 0.1 to 2.5 in the dairy based composition, ably 0.2-2, more preferably 0.8—1 most preferably 0.4-0.7.

In another preferred embodiment fat is added to the composition. The fat may be any fat but is preferably a vegetable fat. Suitable fats comprise sunﬂower oil, soy oil, safﬂour oil, rape seed oil, palm oil, palm kernel oil, ricebran oil, olive oil, arachis oil, and t oil. Milk fat, butter oil and other animal fat such as lard are also suitable. Fish oil and algae oil are also very suitable. The fat may be a combination of different fats. Suitably the fat is a mixture of ble oils and milk fat, cream, butter milk or butter oil. Preferably at least at least 25wt% of the fat ses milk fat or butteroil, more preferably at least 40wt% 0f the fat comprises milk fat or butter oil.

In addition, other ingredients may be added to the food ition such as vitamins, minerals, polyunsaturated fatty acids, prebiotics, probiotics, protein, antibodies, nucleotides, idants, polar lipids including phospholipids. Eg. it is conventional to add to the food compositions carbohydrates, such as lactose and oligosaccharides, lipids and ingredients such as vitamins, amino acids, minerals, taurine, carnitine, nucleotides and polyamines, and idants such as BHT, ascorbyl palmitate, vitamin E, Ot- and B-carotene, lutein, zeaxanthin, lycopene and lecithin. In addition, the food composition may be enriched with polyunsaturated fatty acids, such as gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid and docosapentaenoic acid. With a View to a proper development of the intestinal ﬂora, probiotics may be added, such as lactobacilli and/or biﬁdobacteria, as well as tics. A preferred combination of probiotics is for instance Bifidobacterium lactis and/or WO 2013009182 Bjﬁdobaczferjum amMans with L. rhamnosis, L. casei, L. paracasei, L. salivarius or L. reuteri. Examples of prebiotics e fuco-, fructo- and/0r galacto-oligosaccharides, both short- and hain, (fuco)sialyloligosaccharides, branched (olig0)saccharides, sialic acid-rich milk products or derivatives thereof, inulin, carob bean flour, gums, which may or may not be hydrolyzed, fibers, etc.

It should be understood that it may be necessary to concentrate the food product. If such a concentration method is employed it is desirable to use a mild concentration method such that less than 25wt% of the protein is denatured in the trated product. Suitable concentration methods are forward osmosis, reverse osmosis, membrane distiliation, freeze concentration,thin-film spinning cone evaporator, and scraped film ators. Concentration techniques may be optimised by reduced residence time distribution, and/or improved heat transfer to minimise denaturation.

Dry products have the advantage that they have a longer shelf life due to the reduced level or even lack of water. In addition, dry products are less heavy, and have a smaller volume so that transportation is . However, conventional drying techniques will denature a considerable amount of the proteins present. Therefore, 2O the drying is preferably a mild drying step, such that less than 25wt% of the protein is denatured in the dried product. Suitable drying steps are spray drying, drying in the presence of surface active ents, gas injection, drying with super critical CO2, freeze . In the present invention a dry product is a product that contains at least 70wt% dry , preferably at least 75wt% dry matter, more preferably at least 80 wt% dry matter, more preferably at least 85 wt%, more preferably at least 90 wt%, more preferably at least 95 wt% dry matter and most preferably at leat 98 wt% dry matter.

It is to be tood that the food product of the present ion may be 3O any food product that is dairy based and comprises protein, such as yoghurt, desert, dairy drink, cream, creme fraiche, sour cream, ice cream, cheese, dairy spreads.

However because of the low LPS load due to the method of the present invention it is W0 2013(009182 especially suitable for a food product for infants and toddlers, medicinal food, food for elderly people and neutraceutical food The present invention is also directed to dairy based food composition obtainable by a method according the invention.

The method of the present ion es food compositions that n very little LPS. As mentioned , from EP 1 359 924 Bl it is known that lactic acid bacteria and biﬁdobacteria, particularly those with hydrophobic e, have the ability to bind endotoxins. The present invention provides methods that reduce the amount of LPS without the addition of lactic acid bacteria and biﬁdobacteria.

Therefore a preferred embodiment comprises a food composition comprising less than 4000E3 EU LPS per liter ready to use composition in a composition more preferably less than 3500E8 EU LPS, preferably less than 8000E8 EU LPS, more preferably les than 2000E3 EU LPS, more preferably less than 1800E3 EU LPS, even more preferably less than 700E3 EU LPS, and most preferably less than 200E8 EU LPS per liter ready to use composition. Suitably for dry product, the food composition comprises less than 30E8 EU LPS per gram dry product, more suitably less than 20E3 EU LPS, more suitably less than 15E8 EU LPS, even more suitably less than 10E3 EU LPS, more suitably less than 7E8 EU LPS, even more preferably less than 5E3 EU LPS and most suitably less than 1.5E3 EU LPS per gram dry product, Preferably the food ition does not comprise lactic acid bacteria and bifidobacteria with a percent hydrophobicity of at least 80%H, r, the food composition of the present invention may comprise lactic acid bacteria and biﬁdobacteria and this will even further lower the amount of LPS in the composition. Suitably the food composition of the present ion ses less than 5100 EU LPS per liter ready to use composition, more ably less than 5000 EU LPS, preferably less than 4500 EU LPS, more preferably les than 4000 EU LPS, more preferably less than 3000 EU LPS, even more preferably less than 2500 EU LPS, more preferably less than 2000 EU LPS, more preferably less than 1500 EU LPS, more preferably less than 1000 EU LPS, even more preferably less than 750 EU LPS, more preferably less than 500 EU LPS, more preferably less than 250 EU LPS, more preferably less than 150 EU LPS, and most preferably less than 100EU LPS per WO 2013009182 2012/050504 liter ready to use composition. Suitably for dry product, the food composition comprises less than 39 EU LPS per gram dry product, more suitably less than 85 EU LPS, more suitably less than 30 EU LPS, even more suitably less than 25 EU LPS, more suitably less than 20 EU LPS, more suitably less than 15 EU LPS, even more cm ably less than 10 EU LPS, more ly less than 5 EU LPS, more suitably less than 2 EU LPS and most suitably less than 1 EU LPS per gram dry product. ably the food composition comprises lactic acid bacteria and bifidobacteria, ably with a percent hydrophobicity of at least 80%H, EU stands for endotoxin units. 10 endotoxin units (EU) are approximately equal to 1 ng of endotoxin. It is to be understood that the E notation as used in the present description stands for “times ten raised to the power of”, thus replacing the X 10 in the Scientiﬁc notation, also known as standard form or as exponential notation, with a superscript indicating the power.

Suitably the amount of LPS is ed according to a LAL gel-clot assay or a c chromogenic LAL assay (Gehring et a1, Environmental Int 2008; 34:1182- 1186). Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the horseshoe crab, Limulus polyphemus. LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane component of Gram negative bacteria. LAL contains enzymes that are activated in a series of reactions in the presence of LPS into the Limulus coagulation cascade. This reaction is the basis of the LAL test, which is used for the detection and quantiﬁcation of bacterial endotoxins. The LAL containing enzymes can split the chromophore, paranitro aniline (pNA), from the chromogenic substrate, producing a yellow color in the c genic LAL assay.

However, LPS enbedded in lipid complexes such as phospholipids in milk derived from milkfat globules, or bound to lactic acid bacteria and bifidobacteria mask the Lipid A moiety from the LPS-binding protein (LPB) and therefore escape ion.

Therefore, in such cases the amount of LPS t in the food composition can be detected by combining an aqueous suspension of the lipid complexes with a suitable detergent such as Lubrol-PXTM as described in US 6,015,716. 2012/050504 The method according to the invention yields a casein rich fraction and a serum protein rich fraction. Preferably the casein rich fraction ses more than 81wt% casein on total protein, more preferably more than 85 wt% casein on total protein, even more preferably more than 90wt% of casein on total protein, and most preferably more than 95wt% of casein on total protein .

Also preferred is a serum protein rich fraction comprising more than 20wt% serum n on total protein, more preferably more than 30 wt% serum protein on total protein, even more preferably more than 40wt% serum protein on total protein, more preferably more than 45wt% serum n on total n, more preferably more than 50wt% serum protein on total n, even more preferably more than 55wt% serum protein on total protein, and most preferably more than 60wt% serum protein on total protein.

In another aspect, the present invention is directed to a dairy based food ition wherein the ratio of casein: serum protein is 0.1-4.0. Preferably the composition according to the present invention comprises 1 to 40 wt% protein for a ready to use product, and 10 to 80 wt% protein in a dry product, more preferably 20 to wt% of n for a ready use product, or 20 to 60 wt% of a dry product, most preferably 3 to 25 wt% protein for a ready to use product, or 30 to 50 wt% for a dry product. For infant forumulas suitably the ratio of casein: serum protein is from 0.1 to 4, preferably 0.2-2.5, more preferably 0.3-1 most preferably 0.4-0.7. For medical nutrition or nutrition for y a suitable ratio of caseinzserum protein is from 3-15, more preferably from 4-12, more preferably from 5-11, even more preferably from 6— , and most preferably from 7-9.

The dairy based food composition may also comprise fat in an amount of between 0.5 and 15 wt% fat for a ready to use product and 2 to 40wt% fat in a dry product, more preferably between 1 and 8 wt% fat for a ready to use product or 3 to 30 wt% in a dry product, most preferably 2 to 5 wt% fat in a ready to use product or 5 to 20 wt% in a dry product . The fat may be any fat but is preferably a vegetable fat.

Suitable fats se sunﬂower oil, soy oil, safﬂour oil, rape seed oil, palm oil, palm kernel oil, an oil, olive oil, arachis oil, and coconut oil. Milk fat, cream, butter milk or butter oil and other animal fat such as lard are also suitable. Fish oil and algae oil are also very suitable. The fat may be a combination of different fats.

Suitably the fat is a mixture of vegetable oils and butter oil. Preferably at least 25wt% of the fat comprises butteroil, more preferably at least 40wt% of the fat comprises butter oil.

In a preferred embodiment the composition ing to the invention comprises an amount of betaccasein of from 2 to 4.5 g/L of a ready to use product, preferably from 2.5 to 4 g/L ready to use product and most preferably from 3 to 3.5 g/L ready to use product. Suitably a dry product contains 10-50 mg beta—casein, more ly 15-40 mg beta casein and most preferably from 20-30 mg beta casein per gram dry product.

In another preferred embodiment the composition according to the ion comprises an amount of alpha lactalbumin from 2 to 4.5 g/L of a ready to use product, preferably from 2.5 to 4 g/L ready to use t and most preferably from 3 to 3.5 g/L ready to use t. Suitably a dry product contains 10-50 mg alpha lactalbumin, more suitably 15-40 mg alpha lactalbumin and most preferably from 20-30 mg alpha lactalbumin per gram dry t.

In r preferred embodiment, the composition according to the invention comprises less than 2 g/L alpha casein in a ready to use product, more preferably les than 1 g/L, even more preferably less than 100 mg/L and most preferably less than 10 mg/L in a ready to use product. Even less than 1 mg/L alpha casein in a ready to use product is very suitable. In a dry product, preferably less than 15mg alpha casein per gram dry product is present, more preferably less than 1 mg alpha casein per gram dry product is t, more preferably less than 500 mcg/g and most preferably less than 100 mcg/g alpha casein in a dry product.

In another preferred embodiment, the composition according to the invention comprises less than 2 g/L beta lactoglogulin in a ready to use product, more preferably les than 1 g/L, even more preferably less than 100 mg/L and most preferably less than 10 mg/L in a ready to use product. Even less than 1 mg/L beta logulin in a ready to use product is very suitable. In a dry product, preferably less than 15mg beta lactoglogulin per gram dry product is present, more preferably less than 1 mg beta lactoglogulin per gram dry product is present, more preferably less than 500 meg/g and most ably less than 100 meg/g beta lactoglogulin in a dry product.

Infant (baby) formula is generally for use, in addition to or in lieu of human breast milk, with infants up to 18 months old. Toddler formula generally refers to follow-on formula for children of 18-48 months. Obviously, it is not excluded in accordance with the invention to use the milk proteins and milk protein compositions obtained, also for other purposes such as enteral food, medical nutrition for children and for the elderly.

It will be tood that any nutritional compositions, such as infant or toddler formula, provided in accordance with the invention, may se any further conventional ingredients. E.g. it is conventional to add to baby and infant food and therapeutic compositions carbohydrates, such as lactose and oligosaccharides, lipids and ingredients such as ns, amino acids, minerals, taurine, carnitine, tides and polyamines, and antioxidants such as BHT, ascorbyl palmitate, vitamin E, (1- and B—carotene, lutein, zeaxanthin, lycopene and lecithin. The lipids are mostly of vegetable origin. In addition, the food or the therapeutic composition may be ed with polyunsaturated fatty acids, such as gamma-linolenic acid, dihomo- gamma-linolenic acid, arachidonic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid and docosapentaenoic acid. With a View to a proper development of the intestinal ﬂora, probiotics may be added, such as lactobacilli and/or bifidobacteria, as well as prebiotics. A preferred combination of probiotics is for instance Bifidobacterium lactis and/or Bjﬁ'dobacterjum zls with L. rhamnosis, L. casei, L. paracasei, L. salivarius or L. reuteri. Examples of prebiotics e fuco—, fructo- and/or galacto-oligosaccharides, both short- and hain, (fuco)sialyloligosaccharides, branched (oligo)saccharides, sialic acid-rich milk ts or derivatives thereof, inulin, carob bean ﬂour, gums, which may or may not be hydrolyzed, ﬁbers, etc.

The t ion is also directed to the use of a casein rich fraction and a serum protein rich fraction according to the invention to produce a food composition, preferably an infant formula. It is also directed to the use of a serum rich fraction as described herein and a milk wherein at least 98% of the gram negative W0 2013/‘009182 bacteria are removed and which has been subjected to a heat ent at a temperature between 60 and 90°C and/or a milk protein concentrate wherein at least 98% of the gram negative bacteria are removed and which has been subjected to a heat treatment n temperature 60-90°C, to produce a food composition, C1“! ably an infant formula.

Preferably the use of the serum rich fraction and/or casein rich fraction to produce a food composition is wherein the ratio of casein: serum protein is from 0.1- 2.5 or from is from 8-15. For infant formulas suitably the ratio of casein: serum protein is from 0.1 to 2.5, preferably 0.2-2, more preferably 0.3-1 most ably 0.4- 0.7. For medical nutrition or nutrition for elderly a suitable ratio of caseinzserum protein is from 3-15, more ably from 4-12, more preferably from 5-11, even more preferably from 6- 10, and most preferably from 7-9.

The ion is illustrated in the following, non-limiting examples.

Example 1 Preparation of Low LPS/endotoxin milk protein products with microfiltration Raw milk with a storage time of 72 hours is centrifuged (temp: 50-55°C) in order to separate the cream and to obtain the skimmed milk.

In the next step milk is ﬁltered on a ceramic microfilter (poresize 1.5 pm, temp 50- 55°C) and then subsequently pasteurized (725°C, 20 s) and cooled down to 6°C and then stored in a tank at 6°C. The max storage time in this tank is 24 hours.

The incoming skimmed milk and the resulting ltrated milk is sampled for determination of total microbial counts (CFU/ml at 30°C) and xin (EU/g DM).

The results are shown in Table l.

Table 1 _Endotoxin (EU/g DM) CFUfml (30°C) Skimmed milk 1155:10 ~9500 Skimmed milk (microfiltrated) The microﬁltrated d milk (storage time 108 hours) is stored in a tank at a temperature of 6°C. In the following step, milk is microfiltrated with a spiralwound membrane with a poresize of 0.15 pm (DSS). Filtration is carried out at a temperature of 10-12°C. The transmembrane pressure is max 1.8 bar, preferably 1.8 bar. The preset volume reduction factor was in this example 8.3. In this setting casein micelles are retained in the concentrate, while the majority of the serum proteins will pass the ﬁlter in the ﬁltrate.

The obtained casein concentrate is stored in a tank at <6°C. The storage time was 150 xin (EU/g DM) CFUfml (30°C) Milk casein concentrate 108i 13 30 The obtained permeate with serum ns is collected in a tank and then concentrated on an ultraﬁlter system (spiralwound ne, cut off IOkD) at a temperature of 10-12°C. Concentration is carried out until the protein content of the retentate has reached a value of 14-15%. The obtained serumprotein concentrate is stored in a tank at 6°C. The storage time was 150 h.

Endotoxin (EU/g DM) CFU/ml (30°C) Example 2 Infant formula compositions with low LPS t In Table 2, an e is given of an infant formula composition.

Table 2 Component Per 100 g Proteins g 10.6 Serum or whey protein g 6.4 Casein ; 4.2 Fat g 27 Linoleic acid g 3.4 A-Linolenic acid :, 0.48 DHA mg 53 AA mg 58 Carbohydrates __g_ 55 Lactose :, 53 Dietary Fiber 1 1.9 Galacto-oliosaccharides ; 1.9 Minerals, Vitamins, nucleotides g 1.9 The infant formula compositions are prepared by using either the serum protein C11 concentrate or the milk casein concentrate or both, which are ed according to Example 1, as main protein . These can be combined with (minor amounts of) tional protein sources, such as milk powder and/or demineralized whey protein powder.

Claims

1. Method to produce a dairy based food composition with low lipopolysaccharide (LPS) comprising the steps (a) ing a milk with a e time of less than 264 hours (b) Treating the milk such that at least 98wt% of the Gram negative bacteria is removed (c) Heating the milk wherein at least 98wt% of the Gram negative bacteria is removed to 60-90 °C.

2. Method according to claim 1 further comprising the step of (d) Treating the milk with a micro filter of pore size of from .2 um such that at least a casein rich fraction and a serum protein rich fraction are obtained.

3. Method according to claim 1 or 2 wherein the treatment to remove at least 98wt% of the bacteria is selected from the group consisting of ial filtration with a poresize of 0.5-2.5 micron; fugation; Use of antibody to remove Gram negative bacteria.

4. Method according to claim 3 wherein the bacterial filtration with a pore size of 0.5-2.5 micron is carried out at a temperature of from 25 to 65 °C.

5. Method according to any one of the preceding claims wherein during the process to produce the dairy based food composition the milk and product obtained therefrom are not subjected to a heat treatment above 90°C.

6. Method according to any one of claims 2 to 5 wherein the microfiltration is performed at a temperature of .

7. Method according to any one of claims 2 to 6 wherein the pore size of the microfiltration is from 0.1 to 0.8 micron.

8. Method according to any one of claims 2 to 6 n the pore size of the microfiltration is from 0.15 to 0.5 micron.

9. Method ing to any one of claims 2 to 8 wherein the transmembrane pressure during microfiltration is less than 2.5 bar.

10. Method according to any one of claims 2 to 9 wherein the microfiltration is performed at a temperature of 25-65°C, or O-25°C.

11. Method according to any one of the preceding claims wherein the milk is heated at a temperature of 60—65 °C for 1—10 minutes or at a temperature of 65—85 °C for 5-180 seconds.

12. Method according to any one of the preceding claims wherein the milk is heated at a temperature of 65-76°C for 10—120 seconds.

13. Method according to any one of the preceding claims wherein the milk is heated at a 66-71°C for 5 to 180 seconds.

14. Method according to any one of the preceding claims wherein the storage time of milk is from 200 to 80 hours.

15. Method according to any one of the preceding claims wherein the milk is subjected to a decreaming treatment before the microﬁltration step and/or removal step of the Gram negative bacteria.

16. Method according to any one of claims 2 to 15 wherein the serum n rich fraction is combined with the casein rich fraction or with a milk protein product with a storage time of less than 264 hours to obtain a casein: serum protein ratio of from 0.1 to 4.0 in the dairy based composition.

17. Method according to any one of claims 2-16 n the serum protein rich on is combined with the casein rich fraction or with a milk protein product with a storage time of less than 264 hours to obtain a casein: serum protein ratio of from 0.2- 2 in the dairy based composition.

18. Method according to any one of claims 217 wherein the serum protein rich fraction is combined with the casein rich on or with a milk protein product with a storage time of less than 264 hours to obtain a casein: serum protein ratio of from 0.8- 1 in the dairy based composition.

19. Method according to any one of claims 2-18 n the serum protein rich fraction is combined with the casein rich fraction or with a milk protein product with a storage time of less than 264 hours to obtain a casein: serum protein ratio of from 0.4— 0.7 in the dairy based ition.

20. Method according to any one of the preceding claims wherein a fat is added to the composition.

21. Method according to claim 20 wherein at least 25wt% of the fat comprises butter oil.

22. Method according to any one of the preceding claims wherein ingredients ed from the group consisting of vitamins, minerals, saturated fatty acids, prebiotics, probiotics, protein, antibodies, nucleotides, idants and phospholipids are added to the ition.

23. Method according to any one of the preceding claims wherein a drying step is present.

24. Method according to any one of the preceding claims wherein the food composition is selected from the group consisting of infant formula, medicinal food, nutraceutical food composition, yoghurt, drink, spread, or cream.

25. Method according to any one of the preceding claims wherein the milk is a bovine milk.

26. Method according to claim 25 wherein the milk is cow’s milk.

27. Dairy based food composition ed by a method according to any one of the preceding claims.

28. Dairy based food composition wherein the amount of lipopolysaccharide (LPS) is less than 4000E3 endotoxin units (EU) per liter ready to use food ition or wherein the amount of LPS is less than 30E3 endotoxin units (EU) per gram dry product.

29. Dairy based food composition according to claim 27 n the amount of LPS is less than 5100 EU per liter ready to use food composition or wherein the amount of LPS is less than 39 EU per gram dry product.

30. Dairy based food composition according to any one of claims 27 to 29 comprising lactic acid bacteria and bifidobacteria.

31. Dairy based food composition according to any one of claims 28 to 30 comprising lactic acid bacteria and bifidobacteria with a hydrophobicity of at least 80%H.

32. Composition according to any one of the claims 27 to 31 wherein the composition is a casein rich fraction wherein more than 80wt% of the protein is casein.

33. Composition according to any one of claims 27 to 31 wherein the composition is a serum protein rich fraction wherein more than 30wt% of the n is serum protein.

34. Composition according to any one of claims 27 to 33 wherein the amount of beta-casein on total casein is at least 37%.

35. Composition according to any one of claims 27 to 34 wherein the amount of beta-casein on total casein is at least 39%.

36. Composition according to any one of claims 27 to 35 wherein the amount of beta-casein on total casein is at least 41%.

37. Composition according to any one of claims 27 to 36 wherein the ratio of casein: serum protein is from 0.1 to 4.0.

38. Composition ing to any one of claims 27 to 37 wherein ratio of casein: serum protein is from 0.2-2.5.

39. Composition according to any one of claims 27 to 38 wherein ratio of casein: serum protein is from 0.3-1.

40. Composition according to any one of claims 27 to 39 wherein ratio of casein: serum protein is from 0.4-0.7.

41. Composition according to any one of claims 27 to 40 comprising fat wherein the fat comprises at least 25 wt% of butter oil.

42. Composition according to any one of claims 27 to 41 wherein the food composition is selected from the group ting of infant formula, medicinal food, nutraceutical food composition, yoghurt, drink, spread or cream.

43. Use of a casein rich fraction as claimed in claim 32 and a serum protein rich fraction as claimed in claim 33 to produce a food composition.

44. Use of a casein rich fraction as claimed in claim 32 and a serum n rich fraction as claimed in claim 33 to produce an infant formula.

45. Use of a serum rich on as claimed in claim 33 and a milk n at least 98wt% of the gram negative bacteria are removed and which has been subjected to a heat treatment at a temperature between 60 and 90°C and/or a milk protein concentrate wherein at least 98wt% of the gram negative ia are removed and which has been subjected to a heat treatment between temperature 60—90°C, to produce a food composition.

46. Use of a serum rich fraction as claimed in claim 83 and a milk n at least 98wt% of the gram ve bacteria are removed and which has been subjected to a heat treatment at a temperature between 60 and 90°C and/or a milk protein concentrate wherein at least 98wt% of the gram negative bacteria are removed and which has been subjected to a heat treatment n temperature 60-90°C, to produce an infant formula.

47. Composition according to any one of claims 28 to 46 n the amount of LPS is measured according to a LAL assay or a kinetic chromogenic LAL assay.

48. Composition according to any one of claims 28 to 47 wherein the amount of LPS is measured according to a LAL assay or a kinetic chromogenic LAL assay in the presence of a detergent.

49. Method according to claim 1, substantially as herein described with reference to any one of the Examples thereof.

50. Method according to any one of claims 1 to 26, substantially as herein described.

51. Composition according to claim 28, ntially as herein described with nce to any one of the Examples thereof.

52. Composition according to any one of claims 28 to 42, 47 or 48, substantially as herein described.

53. Use according to any one of claims 43 to 46, substantially as herein described.