NL2012795B1

NL2012795B1 - Novel hydrolysate.

Info

Publication number: NL2012795B1
Application number: NL2012795A
Authority: NL
Inventors: Jan Leonhard Ackerman Koert; Anna Van Vuure Catherina; Maria Henrica Doremalen-Van Der Steen Johanna; Beekmans Frederik; Adrianus Hage Johannes
Original assignee: Sonac B V
Priority date: 2014-05-09
Filing date: 2014-05-09
Publication date: 2016-02-24
Also published as: WO2015170988A2; WO2015170988A3

Abstract

Described is a novel method for the preparation of a hydrolysate comprising phospholipids and at least 6 w/w% cholesterol, the weight ratio between cholesterol and phospholipids being between 1 : 0.5 and 1 : 3, comprising the steps of providing animal brain tissue, animal spine tissue, or a combination thereof, and optionally bile, subjecting the tissue to hydrolysis to obtain a hydrolysate, antimicrobial heat treatment of the hydrolysate and drying the antimicrobially treated hydrolysate.

Description

Title: Novel hydrolysate

The invention relates to a method for the preparation of a hydrolysate of animal origin comprising phospholipids and at least 6 w/w% cholesterol, to such a hydrolysate and to uses of such a hydrolysate.

In the art, cholesterol is an important ingredient for aquafeed, in particular shrimp feed, and in the pharmaceutical industry e.g. for the preparation of vitamin D3 (cholecalciferol). Although egg yolk has a high dietary cholesterol content, wool fat of sheep is used as a main source for cholesterol in industry. Whereas egg yolk comprises significant amounts of phospholipids, wool fat does not. Phospholipids, such as phosphatidylcholine, are known to be emulsifiers and are also used for the preparation of liposomes as medicinal transport vesicles. Phospholipids such as phosphatidylserine are known in brain functions to improve memory and reduce stress.

The present invention now for the first time provides a method for the preparation of a hydrolysate comprising phospholipids and at least 6 w/w% cholesterol, the weight ratio between cholesterol and phospholipids being between 1 : 0.5 and 1 : 3, comprising the steps of a. providing animal brain tissue, animal spine tissue, or a combination thereof, b. subjecting the tissue of step a. to hydrolysis to obtain a hydrolysate, c. antimicrobial heat treatment of the hydrolysate of step b., d. drying the heat treated hydrolysate of step c.

The term ‘hydrolysate’ herein means a protein hydrolysate, comprising partial or completely hydrolysed protein. It has surprisingly found that animal brain tissue, animal spine tissue, or a combination thereof, is an optimal source for cholesterol, and also for phospholipids. For example, mother milk and egg yolk contain less cholesterol compared to brain and spine tissue and are much less suitable as a source for cholesterol and phospholipids. Lecitine is a known source for phospholipids. Brain tissue of e.g. porcine origin comprises about 10 w/w% cholesterol and about 20-24 w/w% phospholipids, such as phophatidylcholine, phophatidylserine, phophatidylinositol and phophatidyl-ethanolamine, whereas porcine spine tissue comprises about 7-8 w/w% cholesterol and 7-15 w/w% phospholipids. The dry weight content of brain is about 20% and spine tissue is about 45-55 w/w%, based on the total tissue weight.

In the art, methods are known as how to hydrolyse animal tissue, e.g. by enzymatic treatment in an aqueous medium using a hydrolytic enzyme such as pepsin, chymotrypsin or trypsin and/or others, or by chemical means at elevated temperature and acid or basic pH.

In order to remove micro-organisms and to provide a food grade hydrolysate, the hydrolysate is heat treated. The skilled person is aware of suitable heat treatments, such as pasteurisation or sterilisation. Attractively, the antimicrobial heat treatment is combined with the hydrolysis, or, when the hydrolysis step is performed in the presence of a hydrolysing enzyme, the heat treatment may be performed such, that also the enzyme is inactivated by this treatment.

The heat treated hydrolysate is then dried by known techniques in the art, such as e.g. by spray drying, fluidised bed drying or drum drying.

The animal tissue can be of any vertebrate, and is preferably from avian or mammalian origin. Attractive examples are domestic animals such as livestock and cattle, such as e.g. chicken, bovine, porcine, goat etc. In a preferred embodiment, the animal tissue comprises porcine tissue. It is also possible for the animal tissue to comprise a mixture of tissue of different animal origin, e.g. a mixture of porcine and bovine tissue.

The animal tissue in step a. may consist of brain tissue, but it has also been found that spine tissue is a very attractive source for the cholesterol and phospholipids comprising hydrolysate, although the absolute content may be somewhat lower. In a very attractive embodiment, the animal tissue comprises a mixture of brain and spine tissue, but can also consist of spine tissue.

It can be chosen to use any relative amount of spine tissue as compared to brain tissue, depending on the content of cholesterol and phospholipids in the starting material (spine and brain tissue) and in the envisaged hydrolysate. In case a relatively high cholesterol or phospholipid content is wished, it can be chosen to incorporate more brain tissue, whereas more spine tissue will be preferred when a lower content of phospholipids is desired. It has been shown that spine tissue can at least partly or for the majority provide for the required cholesterol and phospolipids in the hydrolysate. In view of the relatively low costs for animal spine tissue, it may be preferred to use as much spine as possible. Because of the tendency of fat segregation from the spine tissue, in other attractive embodiments, the animal tissue may comprise relatively less spine tissue. Therefore the weight ratio, based on wet tissue, of brain tissue : spine tissue is between 1.00 : 0.00 and 0.00 : 1.00, preferably between 0.95 : 0.05 and 0.05 : 0.95, more preferably between 0.80 : 0.20 and 0.20 : 0.80, even more preferably between 0.60 : .40 and 0.40 : 0.60, and most preferably between 0.55 : 0.45 and 0.45 : 0.55.

Cholesterol is also known as precursor of ecdysteroids, such as ecdysone, the molting hormone for crustaceae like shrimps. Bile comprises about 1 w/w% cholesterol, and 65 w/w% bile acids, based on dry weight content. The dry weight content of mammalian bile is about 11 w/w%, based on the liquid bile.

Bile acids are known to stimulate fat digestion. Further, bile acids are like cholesterol, in chemical structure (cholate) precursor for ecdysteroids. Therefore, in an attractive embodiment, step a. of the method of the present invention further comprises adding animal bile to the animal tissue, resulting in a hydrolysate that also comprises bile acids, e.g. as a source for ecdysteroid production in the animal consuming the hydrolysate. Preferably, the bile used to add to the animal brain and/or spine tissue originates from the same animal species as the said tissue. However, also bile of one or more different animal species can be chosen. As bile has a cholesterol content of about 1 w/w%, the skilled person will be aware of the proper amount of bile to add to the animal tissue to arrive at the envisaged cholesterol content of the hydrolysate.

The weight ratio, based on wet tissue, of brain and/or spine tissue : bile is preferably between 1 : 0 - 1.5. In case of the maximum bile content of 60 w/w%, based on the wet weight of brain tissue and bile, the cholesterol content equals about 6 w/w% (based on dry weight of the hydrolysate). More preferably, the weight ratio of brain and/or spine tissue : bile is between 1 : 0.8 - 1.2, even more preferably between 1 : 0.9 - 1.1. In case the said ratio is 1 : 1, the cholesterol content of the hydrolysate is about 7 w/w% (based on dry weight of the hydrolysate). The animal tissue in a bile containing hydrolysate preferably comprises brain tissue and particularly consists thereof. In case it is less important for the hydrolysate to comprise a high phospholipid content, a bile containing hydrolysate can be produced by using spine tissue as animal tissue. Again, any combination of brain and spine tissue can be combined with the bile, in particular in the above preferred ratios.

Very attractively, at least a portion, but preferably all, of the spine tissue, if present, is at least partially defatted during or before step a. By defatting the spine tissue, i.e. by removing 10 - 50%, preferably 15 - 25% w/w% of the spine fat, based on the total weight of the spine tissue, by common defatting methods such as heat or enzymatic treatment, the problem of fat segregation during the preparation method of the invention, in particular during drying, is minimized. It has surprisingly been found, that the cholesterol and phospholipid content and ratio may slightly change after defatting. It has been found that most of the cholesterol and phospholipids remain in the remaining protein-fat matrix of the defatted tissue. The cholesterol content of defatted spine tissue may e.g. increase to 10 w/w%, whereas the removed fat portion is can still contain cholesterol and phospholipids, in particular 1 - 4 w/w %, preferably 2-3 w/w % cholesterol. This means that also the spine fat fraction, obtained after defatting the spine tissue, can be regarded as a valuable cholesterol source.

Attractively, hydrolysis step b. comprises enzymatic hydrolysis. Chemical hydrolysis requires rather harsh conditions such as high or low pH and high temperatures, conditions at which the envisaged components, cholesterol and the phospholipids tend to be degraded. Therefore, enzymatic hydrolysis by a hydrolysing enzyme such as proteinases and peptidases are preferred. Any such enzyme capable of cleaving proteins can be used, such as the enzyme mentioned above, but in a preferred embodiment, step b. comprises treatment with alcalase. Alcalase can be used at conditions (pH, temperature) that do not have negative effects on cholesterol and phospholipids. To this end, step b. is preferably performed at 6 - 65°C, more preferably at 45 - 65°C.

The heating step c. is preferably performed for 10 - 20 minutes at 65 -90°C, preferably for 13 - 17 minutes at 75 - 85°C, sufficient to impair or take away the viability of any micro-organisms present. The conditions are preferably chosen such, that the hydrolytic enzymes used in step b. will readily degrade, resulting in inactivation of the enzymes free in the hydrolysate. In a preferred embodiment, 0.01-0.1 w/w%, preferably 0.02 - 0.05 w/w% (based on the animal tissue weight) alcalase is added to the animal tissue, and the tissue is heated from the temperature at which the tissue is kept, e.g. 8°C to 80°C, in e.g. 10 - 30 min. Alcalase is preferably be added before the heating, but can also be added in an early stage of the heating step, i.e. before the hydrolysate reaches a temperature of 30°C - 40°C .

During the heating, the tissue is hydrolysed and antimicrobially treated, while at 80°C, the alcalase is denatured.

The drying step d. preferably comprises drum-drying. Although other drying techniques, known to the skilled person may be used, it has been shown that for hydrolysates of the invention, having a rather high fat content, drum drying results in an attractive dry particulate.

The hydrolysate preferably comprises at least 6 w/w%, preferably at least 7, more preferably at least 8 w/w%, even more preferably at least 9 w/w% cholesterol and most preferably at least 10 w/w% cholesterol, based on dry weight of the hydrolysate. By changing the ratio of brain tissue relative to spine tissue and optionally relative to bile, the cholesterol content can be varied. As defatting of the spine tissue also results in a relative elevation of cholesterol (and phospholipid) content, also the level of defatting has an impact on the cholesterol (and phospholipid) content of the hydrolysate to be obtained.

The weight ratio between cholesterol and phospholipids in brain and spine tissues may vary, and also among different animals. The starting materials are chosen such, both in relative amount, and animal origin, that the weight ratio between cholesterol and phospholipids of the hydrolysate is between 1 : 0.5 and 1 : 3, preferably between 1 : 0.7 and 1 : 2.7, more preferably between 1 : 0.8 and 1: 2.5, even more preferably between 1 : 0.9 and 1 : 2.4. By using a 1:1 ratio of porcine brain tissue as related to whole (i.e. non-defatted) porcine spine tissue, the said ratio cholesterol : phospholipid is about 1 : 1.7- 2.3, whereas said ratio using only porcine brain tissue is about 1 : 2.0 - 2.4. From porcine spine tissue alone, said ratio is about 1 : 1.8 - 1.9.

Defatting the spine tissue preferably comprises the steps of: i. heating the spine tissue in an aqueous medium at 70°C - 90°C to melt at least a portion of the fat from the spine tissue, ii. separating the heated spine tissue in a spine tissue fraction and a molten spine fat fraction.

By this method, cholesterol and phospholipids are not damaged and about 15-20 w/w% relative to the weight of the spine tissue, fat is removed from the spine tissue, resulting in a relative increase of the cholesterol content in the remaining spine tissue fraction, and in a spine fat fraction that comprises 1-4 w/w%, preferably 2-3 w/w% cholesterol, rendering both defatted spine fraction as the spine fat fraction as valuable starting materials, the defatted fraction as starting material for the hydrolysate as decribed above, and the spine fat fraction as an alternative cholesterol source e.g. for aquafeed, cosmetics of pharmaceuticals.

In another embodiment, defatting the spine tissue comprises the steps of: i. heating the spine tissue in an aqueous medium at 100°C - 150°C in the presence of acid and/or lipase to remove at least a portion of the fat from the spine tissue, ii. separating the heated spine tissue in a spine tissue fraction and a spine fat fraction.

Using the above more harsh conditions, more fat can be removed from the spine tissue, but some of the phospholipids may be degraded during the process. In case however, a higher ratio cholesterol to phospholipids is envisaged, such a more harsh treatment may be preferred. A more defatted spine tissue also results in improved drying, such as drum drying and may therewith allow for a relative higher spine content.

The invention further relates to a protein hydrolysate and a spine fat fraction. The protein hydrolysate comprises phospholipids and, on dry weight basis of the hydrolysate, at least 6 w/w% cholesterol, preferably at least 7 w/w% cholesterol, the weight ratio between cholesterol and phospholipids being between 1 : 0.5 and 1 : 3.

If present, the protein hydrolysate preferably comprises up to 35 w/w% bile acids, based on dry weight of the hydrolysate. Such a high bile acid content can be achieved when using brain/spine tissue and bile in a wet weight ratio of 1 : 1.5, as discussed above. The protein hydrolysate preferably comprises 15-30 w/w%, more preferably 20-25 w/w% bile acids. The protein hydrolysate of the invention, preferably comprises 9 w/w% or less cholesterol, on dry weight basis of the hydrolysate,. As outlined above, such hydrolysates can be obtained by the method of the invention. By using more brain tissue relative to spine tissue or, if present, bile, a higher cholesterol content can be obtained. The same is true when using partially defatted spine tissue. The ratio cholesterol : phospholipids can also be varied, e.g. by subjecting any of the tissue materials before or during the method of the invention to harsh conditions, that degrade or remove either cholesterol or phospholipids. The phospholipids of the protein hydrolysate of the invention or obtained by the method of the invention may comprise one or more of e.g. phosphatidylcholine, phosphatidylserine, phosphatidylinositol and/or phosphatidylethanolamine, e.g. in a relative weight ratio of 7 - 8 : 3 - 4 : 0.7 - 1 : 7 - 8. As indicated above, the weight ratio between cholesterol and phospholipids of the hydrolysate is preferably between 1 : 0.5 and 1 : 3, preferably between 1 : 0.7 and 1 : 2.7, more preferably between 1 : 0.8 and 1: 2.5, even more preferably between 1 : 0.9 and 1 : 2.4.The invention also relates to a spine fat fraction, as obtainable by the method of the invention, comprising, based on dry weight of the spine fat, 1 - 4 w/w%, preferably 2 - 3 w/w % cholesterol, and 0.1 - 0.7, preferably 0.1 - 0.5 w/w% phospholipids.

The invention further relates to the use of the hydrolysate of the invention, or as obtained by the method of the invention, in aqua feed, in the preparation of pharmaceuticals or as food additive, in particular feed stock food additive. The hydrolysate is very suitable to be used in aqua feed for feeding e.g. fish or crustaceae like shrimps because the hydrolysate comprises a) cholesterol and optionally also bile acids as a precursor for ecdysteroids such as ecdysone, the molting hormone, b) docosahexaenoic acid (DHA) and taurin, which are essential for shrimps, c) phospholipids as a source of phosphorus, inositol and choline and d) hydrolysed proteins which makes the hydrolysate a good attractant. This hydrolysate has advantages above addition of pure compounds like choline and cholesterol in shrimp feed, because of 1) less leakage of choline and inositol in the surrounding aqua due to the form of phosphatidylcholine and phosphatidylinositol, 2) better distribution of small cholesterol particles which should make cholesterol more available for shrimp digestion and 3) better homogenisation of hydrolysate powder compared to liquid lecithin during shrimp feed production. Therefore, in a very attractive embodiment, the hydrolysate is used as source of cholesterol and other ecdysteroid precursors in aquafeed.

Also in the preparation of pharmaceuticals, as cholesterol is an important precursor of Vitamin D3, both the hydrolysate as well as the spine fat of the invention are very suitable, for this purpose.

The enriched content of phospholipids makes the hydrolysate, but also the spine fat very suitable as food additive for food, pet food or food for feed stock, such as for animals of bovine, avian, or porcine origin. The food additive is particularly useful for juvenile feed stock, in particular piglets. It has surprisingly been shown that piglets become more relaxed when consuming the hydrolysate of the invention, resulting in a significant stress reduction. From literature is known that phospholipids can also improve memory. Therefore, the hydrolysate is also very useful in the preparation of a medicament for stress reduction, in particular for juvenile feed stock, like piglets or broilers, sport animals like racing horses and sled dogs, show animals and elderly animals like dogs. The hydrolysate can be used as an active ingredient or precursor thereof in the said medicament, or be used to further isolate one or more components, in particular one or more phospholipids, such as phosphatidylserine, to be included in such a medicament as active ingredient.

As indicated above, both the hydrolysate and the spine fat fraction, alone or in combination, are also very useful for the extraction of cholesterol and preparation of vitamin D3.

The invention will now be further illustrated by way of the following figures and examples, wherein

Figure 1 shows the effect of heat load on cholesterol and phospholipids content in free fat fraction, and

Figure 2 shows the effect of ratio brain - (defatted) spine tissue on fat, cholesterol and phospholipids content.

Example 1

Time and temperature treatment effects defatting spine tissue.

Spine tissue (collected at the slaughterhouse) was treated with different heat loads on lab scale. Thereafter, the tissue was centrifuged (10 minutes at 4500 g) and the free fat, sludge (water + fine protein particles) and protein (large, solid protein particles) were isolated, weighted and freeze dried for dry matter (DM) determination. In table 1 the different heat treatments are written. In table 2, the % free fat, sludge, protein and evaporated water are written including the % dry matter of these fractions.

Table 1.

Different heat treatments to 1 kg spine tissue.

Table 2. % free fat, sludge, protein and evaporation and the dry matter (DM) of these fractions.

Example 2

Defatting spine effects content cholesterol and phospholipids in free fat fraction.

Spine tissue (collected at the slaughterhouse) was treated with different heat loads on lab scale as written in table 1. Thereafter, the tissue was centrifuged (10 minutes at 4500 g) and the free fat, sludge (water + fine protein particles) and protein (large, solid protein particles) were isolated. The free fat fraction was analyzed on % cholesterol (using HPLC-MS) and % phospholipids (using enzymatic method Instruchemie [RuiterJ., de Graaf, A.F., Analytics BV,

Delfzijl, The Netherlands, 2011]). In figure 1, the effect of the heat load on the % cholesterol and phospholipids in the free fat fraction is shown.

Example 3

Ratio brain - (defatted) spine tissue effects content fat, cholesterol and phospholipids.

Spine and brain tissue were collected at the slaughterhouse. Defatted spine tissue was prepared on lab scale by heating spine tissue 30 minutes at 95°C in a water bath during mixing with a spoon. Thereafter the solid protein fraction was collecting by decanting and leak out the liquid fat and water with fine protein particles fraction. Brain, spine and defatted spine were mixed on lab scale in different ratio’s together and hydrolysed with 0.04% Alcalase 4L (Novozymes) during 5 minutes at 60°C in a water bath. The enzyme was inactivated by treating the hydrolysates at 80°C in a water bath during 15 minutes. The hydrolysates were cooled on ice and freeze dried before analyzing the % fat (using acid hydrolysis before collecting fat fraction of the sample in petroleum ether), cholesterol (using HPLC-MS) and phospholipids (using enzymatic analysis method Instruchemie). In figure 2, the analysis results are shown.

Example 4

Shrimp feeding trail 100% brain hydrolysate versus pure cholesterol. 100 kg brain tissue was collected in the slaughterhouse and transported into a tank at 8°C. 0.04% Alcalase 4L was added and the tissue was heated using direct steam during mixing to 80°C during 15 minutes. Thereafter, the hydrolysate was dried using a drum dryer (GMF) at 7 bar. The dried product was sieved and used in a shrimp feeding trial. In this shrimp feeding trial, the effect on growth, mortality and feed conversion ratio were studied using 100% brain hydrolysate powder versus pure cholesterol as feed ingredients in such way that the amount cholesterol in both feed was 0.08-0.09%. The rest of the feed components were the same (5% corn gluten, 26% fishmeal, 2% squid meal, 16.9% wheat, 25% wheat flour, 2% soya lecithin, 2% fish oil, 4% wheat gluten, 2% premix and the rest adjusted to 100% with soybean meal). The shrimp trail starts with 4 x 40 shrimps (.Litopenaeus vannamei) of 1.0-1.4 gram. The shrimps were feed during 6 weeks and each week the shrimps were counted and weight. In table 3 the average % growth, mortality and feed conversion ratio (FCR) of the shrimps are written.

Table 3. % growth, mortality and FCR of shrimps using pure cholesterol versus 100% brain hydrolysate as feed ingredient.

Example 5

Composition and performance on shrimp growth of hydrolysates porcine brain -spine marrow and porcine brain - bile and combinations thereof.

Brain tissue, spinal marrow and bile were collected in the slaughterhouse and transported into a tank at 8°C. Defatted spine tissue was prepared on lab scale by heating spine tissue 30 minutes at 95°C in a water bath during mixing with a spoon. Thereafter the solid protein fraction was collecting by decanting and leak out the liquid fat and water with fine protein particles fraction. Brain, defatted spinal marrow, bile and combinations thereof (brain - defatted spinal marrow (1:1), brain - bile (1:1) defatted spinal marrow - bile (1:1) and brain - defatted spinal marrow - bile (1:1:1)) were prepared. 0.04% Alcalase 4L was added and the tissues were heated in a water bath during mixing to 80°C during 15 minutes. Thereafter, the hydrolysates were dried using a drum dryer (GMF) at 7 bar. In the shrimp feeding trial, the effect on growth was studied using 100% brain hydrolysate powder versus brain - bile (1:1) versus brain - defatted spinal marrow (1:1) versus defatted spinal marrow - bile (1:1) versus brain - defatted spinal marrow - bile (1:1:1) as feed ingredients in such way that the amount hydrolysate in all feed was 0.9 %. The rest of the feed components were the same (5% corn gluten, 26% fishmeal, 2% squid meal, 16.9% wheat, 25% wheat flour, 2% soya lecithin, 2% fish oil, 4% wheat gluten, 2% premix and 14.2% soybean meal). The shrimp trail starts with 4 x 40 shrimps (Litopenaeus vannamei) of 1.0-1.4 gram. The shrimps were feed during 6 weeks and each week the shrimps were observed on growth (on scale 1-10). In table 4, the composition and total observation on shrimp growth of the hydrolysates is written.

Table 4.

Composition and observation on shrimp growth of dried hydrolysates brain, defatted spinal marrow, bile and combinations thereof.

Observation shrimp growth expressed on scale 1-10; 1=poor; 10=excellent.

Example 6

Stress reduction piglets trail 100% brain hydrolysate versus placebo. 16 weaned piglets (ca 12 kg) were random divided into 2 groups. 1 group was fed with control weaner diet and the other group was fed with weaner diet + 0.2% 100% brain hydrolysate. The brain hydrolysate was produced by collecting brain tissue in the slaughterhouse and transported into a tank at 8°C. 0.04% Alcalase 4L was added and the tissue was heated using direct steam during mixing to 80°C during 15 minutes. Thereafter, the hydrolysate was dried using a drum dryer (GMF) at 7 bar and sieved. The piglets were fed 200 - 1000 grams a day till 25 kg. To indicate the amount of stress of the piglets, the piglets were visual observed and the amount of cortisol and growth were measured. More cortisol is present when the body is stressed to restore the energy (glucose) balance. In table 5 the results are written.

Table 5.

Stress observation of weaned piglets using control weaner diet versus weaner diet with 100% brain hydrolysate as feed ingredient.

The visual stress observation is expressed on scale 1-10; 1=no stress; 10=very stressed.

The % cortisol response and growth are compared to the control group. Similar results were obtain when a 1:1 ratio of brain : spinal tissue was used for the preparation of the hydrolysate.

Claims

A method for the preparation of a hydrolyzate comprising phospholipids and, based on the dry weight of the hydrolyzate, at least 6% by weight of cholesterol, the weight ratio between cholesterol and the phospholipids being between 1: 0.5 and 1: 3, comprising the steps of: a. providing animal brain tissue, animal spinal cord tissue or a combination thereof, b. subjecting the tissue of step a. to hydrolysis to obtain a hydrolyzate, c. antimicrobial heat treatment of the hydrolyzate of step b., d. drying the heat-treated hydrolyzate of step c.

The method of claim 1, wherein the animal tissue comprises bovine tissue or pig tissue or a mixture thereof.

The method of claim 2, wherein the animal tissue comprises pig tissue.

A method according to any of the preceding claims, wherein the animal tissue in step a. Comprises a mixture of brain tissue and spinal cord tissue.

A method according to claim 4, wherein the weight ratio based on wet tissue of brain tissue: spinal cord tissue is between 0.95: 0.05 and 0.05: 0.95, preferably between 0.80: 0.20 and 0 , 20: 0.80, more preferably between 0.60: 0.40 and 0.40: 0.60, most preferably between 0.55: 0.45 and 0.45: 0.55.

The method of any one of the preceding claims, wherein step a. Further comprises adding animal bile to the animal tissue.

A method according to claim 6, wherein the weight ratio based on wet tissue of animal brain and / or spinal cord tissue: bile is between 1: 0-1.5, preferably between 1: 0.8 - 1.2, with more preferably between 1: 0.9 - 1.1.

A method according to any of the preceding claims, wherein if present, at least a portion of, but preferably all of, the spinal cord tissue is at least partially degreased during or prior to step a.

A method according to any of the preceding claims, wherein step b. enzymatic hydrolysis.

The method of claim 9, wherein step b. treatment with alkalase.

The method of claim 10, wherein step b. is carried out at 6 - 65 ° C, preferably at 45 - 65 ° C.

A method according to any of the preceding claims, wherein the heating step c. is carried out at 65-90 ° C for 10-20 minutes, preferably at 75-85 ° C for 13-17 minutes.

A method according to any of the preceding claims, wherein the drying step d. drum drying.

A method according to any of the preceding claims, wherein the hydrolyzate is at least 6% by weight, preferably at least 7% by weight and more preferably at least 8% by weight, even more preferably at least 9% by weight and most preferably comprises at least 10% by weight of cholesterol based on the dry weight of the hydrolyzate.

The method according to any of the preceding claims, wherein the weight ratio between cholesterol and phospholipids of the hydrolyzate is between 1: 0.7 and 1: 2.7, preferably between 1: 0.8 and 1: 2.5, more preferably between 1: 0.9 and 1: 2.4.

The method of any one of claims 8-15, wherein degreasing the spinal cord tissue comprises the steps of: i. heating the spinal cord tissue in an aqueous medium at 70 ° C - 90 ° C to melt at least a portion of the spinal cord fat from the spinal cord tissue, ii. separating the heated spinal cord tissue into a spinal cord tissue fraction and a molten spinal cord fat fraction.

The method of any one of claims 8-15, wherein degreasing the spinal cord tissue comprises the steps of: i. heating the spinal cord tissue in an aqueous medium at 100 ° C - 150 ° C in the presence of acid and / or lipase to remove at least a portion of the spinal cord fat from the spinal cord tissue, ii. separating the heated spinal cord tissue into a spinal cord tissue fraction and a spinal cord fat fraction.

A protein hydrolyzate comprising phospholipids and at least 6% by weight of cholesterol based on the dry weight of the hydrolyzate, the weight ratio between cholesterol and phospholipids being between 1: 0.5 and 1: 3.

The protein hydrolyzate according to claim 18, further comprising up to 35% by weight of bile acids based on the dry weight of the hydrolyzate.

A protein hydrolyzate according to claim 19, which comprises bile acids from 15-30% by weight, preferably 20-25% by weight, based on the dry weight of the hydrolyzate.

The protein hydrolyzate of any one of claims 18 to 20, comprising, based on dry weight of the hydrolyzate, 9% by weight or less of cholesterol.

The protein hydrolyzate according to any of claims 18 to 21, wherein the weight ratio between cholesterol and phospholipids of the hydrolyzate is between 1: 0.5 and 1: 3, preferably between 1: 0.7 and 1: 2.7, with more preferably between 1: 0.8 and 1: 2.5, even more preferably between 1: 0.9 and 1: 2.4.

Spinal cord fat obtainable by the method according to claim 16 or 17, comprising, based on the dry weight of the spinal cord fat, 1 to 4% by weight, preferably 2 to 3% by weight of cholesterol and 0.1 to 0.7% by weight. %, preferably 0.1 -0.5% by weight of phospholipids.

Use of a hydrolyzate according to any of claims 18 to 22 or obtainable according to a method according to any of claims 1-17, as a food additive, animal feed or pet food or as an active ingredient or precursor thereof in the preparation of a medicament.

Use of a hydrolyzate according to claim 24 as a food additive in animal feed or pet food, in particular for young cattle or older animals, sport animals and show animals.

Use of a hydrolyzate according to claim 24 in the preparation of a medicament for stress reduction or memory improvement, in particular for young cattle or for older animals, sport animals and show animals.

The use of a hydrolyzate according to claim 24 as a source of cholesterol and other acdysteroid precursors in aquafeed.

Use of spinal cord fat according to claim 24 or available as a spinal cord fat fraction according to claim 16 or 17 as a source of cholesterol in aqua feed or as an active ingredient or precursor thereof in the preparation of a medicament, in particular preparation of vitamin D3.