Process for producing hydrolysed protein

Abstract

The present disclosure relates to a novel enzymatic-free process for the production of hydrolysed protein derived from animal raw materials, as animal by-products. Moreover, the present disclosure also relates to the hydrolysed protein obtained through such process and to products comprising such hydrolysed protein.

Description

D E S C R I P T I O NPROCESS FOR PRODUCI NG HYDROLYSED PROTEI NTECHNICAL FIELD

[0001] The present disclosure relates to a novel enzymatic-free process to produce hydrolysed protein derived from animal raw materials, wherein the digestibility of the hydrolysed protein obtained is superior to 90%. More particularly, the present disclosure pertains to a method that avoids the use of enzymes during the production process, thereby providing a cost-effective and environmentally friendly approach to obtain improved, protein-rich products and powders, to be applicable in various industries, including food and cosmetic industry.BACKGROUND

[0002] The treatment and referral of animal by-products is subject to tight European regulation arising from the BSE (bovine spongiform encephalopathy) or mad cow disease crisis. Since that time, around the year 2000, this industry, rendering, has adapted in the best possible way to the requirements created by the authorities. The prion, a protein with a size of about 30 kDa, has been identified as responsible for the transmission and induction of the disease, bovine spongiform encephalopathy, and ruminant animal species are the vehicle of generation of this pathology.

[0003] Protein hydrolysis is a method developed several years ago, and there are several ways to be performed, from enzymatic, acid / basic or physical hydrolysis. In the field of rendering, the development of hydrolysed proteins has become a legislated form to circumvent the problem of prions in proteins derived from animal by-products, ensuring food safety.

[0004] Currently, the standard method to perform protein hydrolysis is enzymatic hydrolysis.

[0005] Document EP3518686A4 describes a process for producing an animal protein hydrolysate, including the steps of subjecting raw material to the pasteurization process and performing an enzymatic digestion, among other steps.

[0006] Document EP3052641B1 also provides a manufacturing method of a hydrolysate of animal protein, wherein one of the steps is an enzymatic hydrolysis through the addition of specific proteases.

[0007] Nonetheless, enzymatic hydrolysis presents some disadvantages. On one hand, the enzymes employed are very expensive and can jeopardize the manufacturing of affordable protein products derived from animal raw materials. Additionally, the enzymatic hydrolysis requires using large amounts of water (in a weight ratio between animal raw material / steam ranges around 1:1), which represents a significant waste of water and increases the energy costs and complexity of the drying step. On the other hand, depending on the enzymes employed, different protein products are obtained.

[0008] In relation to acid / basic hydrolysis, it is related with safety and environmental risks and increases the complexity of the manufacturing process.

[0009] Additionally, the processes described in the prior art often lead to hydrolysed proteins with high degree of hydrolysis, resulting in the loss of structural properties necessary for encapsulating active compounds. Achieving high digestibility without compromising functionality remains a challenge.

[0010] These facts are disclosed to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0011] The present disclosure seeks to address the challenges identified in the prior art by providing an enzymatic-free process for producing improved hydrolysed protein (hydrolysed protein products) from animal raw materials, which offers several advantages, including but not limited to cost-effectiveness, sustainability, safety and robustness of the manufacturing process.

[0012] In the present disclosure, an enzymatic-free process is understood as a process that does not require adding enzymes in any step of the process to obtain the final products.

[0013] Animal raw material refers to any natural substance or resource derived from animals that can be used for various purposes. These materials can come from different parts of an animal, including its body, organs, or by-products. Preferably, the animal raw material used in the process of the present disclosure is animal byproducts.

[0014] Animal by-products (ABPs) are materials obtained from animals which are not intended for human consumption, in particular, slaughterhouse waste (skin, bones, horn and hooves, blood, fat and offal).

[0015] The process of the present disclosure allows to avoid the use of any external agents (whether chemical or enzymatic) to carry out the hydrolysis step, except water in the form of steam. Thus, when compared with enzymatic hydrolysis, it is eliminated the addition of an external and high-cost agent (e.g., enzymes). When compared to acid / base hydrolysis, the present manufacturing process eliminates the introduction of these chemical reagents that degrade the protein and introduce salts into the final hydrolysed product, requiring additional purification steps and reducing the quality of the final product.

[0016] An aspect of the present disclosure relates to an enzymatic-free process for producing hydrolysed protein from animal raw material, preferably wherein the digestibility of the hydrolysed protein is superior to 90%, comprising the following steps: providing grinded and / or minced animal raw material; extracting proteins from the grinded / minced animal raw material using steam for obtaining an aqueous phase, a solid phase and a fat phase, wherein said aqueous phase comprises hydrolysed proteins; collecting the aqueous phase in order to obtain the hydrolysed protein; wherein the protein extraction (step of extracting proteins from the grinded / minced animal raw material) is performed at a temperature ranging from 150 - 190°C and at a pressure ranging from 7 - 9 bar, during at least 30 minutes;wherein the weight ratio between animal raw material / steam ranges from 1:0.1 to 1:0.5;

[0017] The process of the present disclosure provides several advantages:- Obtention of a legally accepted hydrolysed protein with the ability to microencapsulate hydrophilic and hydrophobic compounds;- Free of use of any external reagent to carry out the hydrolysis reaction and therefore providing a totally natural and cost-effective process;- It is applicable to the hydrolysis of raw material / by-products of various terrestrial animal species (pig, cattle, sheep, goats, poultry, among others...);- It does not require the addition of large amounts of water into the process, so it becomes much more economical (high cost of evaporation of water in the final product) and sustainable.

[0018] The specific parameters of the process of the present disclosure allows to obtain hydrolysed protein with improved balanced characteristics.

[0019] Surprisingly, the resulting hydrolysed protein comprises an optimal balance between digestibility and degree of hydrolysis. With a degree of hydrolysis below 50% (preferably below 45%), the protein is uniquely suited for encapsulating both hydrophobic and hydrophilic substances, a feature not achievable with highly hydrolysed proteins. Simultaneously, the protein demonstrates exceptional digestibility, exceeding 90%, making it ideal for animal feed formulations where high nutrient absorption is vital.

[0020] Moreover, the hydrolysed protein obtained through the process of the present disclosure also exhibits enhanced antioxidant activity as measured by ORAC (above 100 mg / Trolox / g), high solubility in water and a molecular weight below 5 kDa (preferably below 3KDa).

[0021] In a preferred embodiment, the temperature of the protein extraction ranges from 170 - 190°C.

[0022] In a preferred embodiment, the pressure of the protein extraction is 8 bar.

[0023] In a preferred embodiment, the degree of hydrolysis of the hydrolysed protein is below 50%.

[0024] In a preferred embodiment, the weight ratio between animal raw material / steam ranges from 1:0.2 to 1:0.3.

[0025] In a preferred embodiment, the protein extraction is performed in a closed reactor, preferably a cooking vessel.

[0026] In a preferred embodiment, the protein extraction is performed for at least 45 minutes; preferably ranging from 45-90 minutes; even more preferably 60 minutes.

[0027] In a preferred embodiment, the grinded / minced animal raw material comprises particles with granulometry below 12 mm; preferably below 10mm; more preferably below 8 mm.

[0028] The measurement of the granulometry / particle size of the grinded animal raw material may be carried out in various ways, in this disclosure the measurement granulometry / particle size was carried out based on the standard the granulometry analysis by mechanical sieving. In particular, concerning the sizes of the particles obtained by the sieves with opening mesh size of 12 mm, 10 mm and / or 8 mm.

[0029] In a preferred embodiment, the step of collection the aqueous phase is performed through decantation; preferably through a 3-phase decanter.

[0030] In a preferred embodiment, the process herein described further comprises a step of purification of the aqueous phase comprises hydrolysed proteins, preferably by centrifugation, obtaining a purified aqueous phase comprising hydrolysed proteins.

[0031] In a preferred embodiment, the centrifugation step is performed at 5000- 10000 g during 10-30 minutes. In another embodiment, the centrifugation is continuous flow centrifugation.

[0032] In a preferred embodiment, the process further comprises a step of drying the purified aqueous phase comprising hydrolysed proteins. Preferably, the step of drying is spray drying, freeze-drying, or air drying; more preferably spray drying.

[0033] In a preferred embodiment, the animal raw material is animal by-products, preferably slaughterhouse waste. In a preferred embodiment, the animal is selected from the group consisting of beef, pork, chicken, or mixtures thereof; preferably bonecontaining animal raw material.

[0034] Another aspect of the present disclosure relates to a hydrolysed protein composition, wherein the hydrolysed protein is able to encapsulate hydrophobic and hydrophilic substances.

[0035] In a preferred embodiment, more than 50 % of the hydrolysed protein comprise a molecular weight below 5 KDa, preferably more than 50 % of the hydrolysed protein comprise a molecular weight below 4 KDa, more preferably more than 50 % of the hydrolysed protein comprise a molecular weight below 3 KDa, even more preferably more than 50 % of the hydrolysed protein comprise a molecular weight ranging from 0.1-2.9 KDa.

[0036] In a preferred embodiment, the digestibility of the hydrolysed protein is superior to 90%; preferably the digestibility of the hydrolysed protein is superior to 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0037] In a preferred embodiment, the digestibility of the hydrolysed protein ranges from 95-99.9%.

[0038] In a preferred embodiment, the degree of hydrolysis of the hydrolysed protein is below 50%; preferably below 45%; more preferably below 40%.

[0039] In a preferred embodiment, the degree of hydrolysis of the hydrolysed protein ranges from 40-49%.

[0040] In a preferred embodiment, the biological activity (ORAC) of the hydrolysed protein is above 100 mg Trolox / g; preferably above 115 mg Trolox / g; more preferably ranging from above 115-200 mg Trolox / g.

[0041] Another aspect of the present disclosure relates to a powdered protein composition comprising the hydrolysed protein composition herein described.

[0042] The hydrolysed protein obtained through the process of the present disclosure comprises the following properties:- High protein content;- Hydrolysed protein with very high in vitro and in vivo digestibility;- Hydrolysed protein with sensorial advantages related with palatability or appetite;- Ability to encapsulate lipophilic and lipophobic compounds in a protein microcapsule;- Hydrolysed protein containing its own bioactive properties (e.g., antioxidant activity);- Hydrolysed protein highly soluble in water- Hydrolysed protein free of additives whatsoever.

[0043] Another aspect of the present disclosure relates to an animal feed comprising the hydrolysed protein composition herein described or the powdered protein composition herein described.

[0044] Another aspect of the present disclosure relates to an ingestible product comprising the hydrolysed protein composition herein described or the powdered protein composition herein described, preferably the ingestible product is a food product, a nutraceutical product, a pharmaceutical product or a dietary supplement.BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0046] Figure 1: Fluxogram of the enzymatic-free process of the example lof the present disclosure, wherein:1: represents animal raw material2: represents the grinding (of the raw material)3: represents the grinded raw material4: represents the closed reactor5: represents the enzymatic free hydrolysis6: represents the hydrolysed mixture7: represents the horizontal separator8: represents the physical separation9: represents the aqueous phase containing soluble and hydrolysed protein10: represents the solid phase containing bones and other non-hydrolysed solids11: represents the lipid phase containing animal fat12: represents the centrifugation13: represents the purified aqueous phase containing soluble and hydrolysed protein14: represents the evaporation process15: represents the aqueous phase concentrated in protein16: represents the spray drying process17: represents the dried powdered hydrolysed protein

[0047] Figure 2: Simplified fluxogram of the enzymatic-free process of the example lof the present disclosure.DETAILED DESCRIPTION

[0048] The present disclosure relates to a novel enzymatic-free process for the production of hydrolysed protein derived from animal raw materials. Moreover, the present disclosure relates to the hydrolysed protein obtained with such process and to ingestible products comprising said products.

[0049] The enzymatic-free hydrolysis step of the present disclosure is performed in a closed reactor (cooking vessel) with steam inlet; preferably with a plurality of steam inlet distributed along the reactor. Such reactor is resistant to a pressure of at least 11 bar and temperature of at least 210 °C. The reactor comprises mixing means (e.g., a mixing propeller) and a plurality of steam entries, for which the steam is injected into the reactor.Example 1

[0050] In example 1, 100 kg of pork raw material were processed with the enzymatic- free process of the present disclosure, comprising the following steps:- providing animal raw material (1)- fine grinding (2) of the raw material (e.g., pork raw material), with a crusher and a mincer in order to obtain a finely grinded raw material (3) with particles of dimension below 8 mm;- enzymatic-free hydrolysis (5) in a closed reactor (cooking vessel) (4) of 0.4 m3to which the finely grinded raw material (3) is pumped, where 0.02 m3(20 L) of direct water vapour (steam) (corresponding to a weight ratio between animal raw material / water vapour of 1:0.2) is introduced up to a pressure of 8 bar and up to a temperature of 180°C. These conditions are maintained for 60 minutes under constant mixing / agitation of the raw material, preferably 15 rpm, and a hydrolysed mixture (6) is obtained;- Physical separation (8) of the hydrolysed mixture (6) through a horizontal separator (7), preferably using a 3 phase decanter (e.g., Tricanter® from Flottweg). With this equipment, the reaction mixture is separated into three phases: aqueous phase containing soluble and hydrolysed protein (9); solid phase containing bones and other non-hydrolysed solids (10); and lipid phase containing animal fat (11).- focusing on the aqueous phase containing soluble and hydrolysed protein (9), it is purified through a centrifugation (12) that reduces mineral contaminants and fats, obtaining a purified aqueous phase containing soluble and hydrolysed protein (13). This purified aqueous phase containing soluble and hydrolysed protein (13) will then be subjected to an evaporation process (14) to reduce the amount of water and increase the amount of soluble protein up to at least 50% dry matter, obtaining an aqueous phase concentrated in protein (15).- The aqueous phase concentrated in protein (15) will then be subjected to a spray drying process (16), leading to a dried powdered hydrolysed protein (17).

[0051] Table 1 compares the characteristics of the product obtained through a process comprising enzymatic hydrolysis (comparative example 1, as described in: Borges, S., Piccirillo, C., Scalera, F. et al. Valorization of porcine by-products: a combined process for protein hydrolysates and hydroxyapatite production. Bioresour. Bioprocess. 9, 30 (2022). https: / / doi.org / 10.1186 / s40643-022-00522-6) and the product obtained through the enzymatic-free process herein disclosed in example 1.Table 1. Characteristics of the product obtained through a process comprising enzymatic hydrolysis (comparative example 1) and the product obtained through the enzymatic-free process of example 1.N / A: information not available

[0052] Additionally, it was also observed that more than 50 % of the hydrolysed protein of example 1 of the present disclosure comprise a molecular weight below 5 KDa (in particular, more than 50 % of the hydrolysed protein of example 1 comprise a molecular weight below below 3KDa.Example 2 - Encapsulation of compounds

[0053] The method consists of obtaining a liquid solution of the compound to be encapsulated, in a known quantity, and mixing it with the aqueous phase concentrated in protein (15). After checking the miscibility of the compound to be encapsulated and the aqueous phase concentrated in protein (15), this mixture is spray dried. Then, the spray dried mixture is washed with a non-aqueous agent that solubilizes the compound that has not been encapsulated, and a washed mixture is obtained. Then, the non-encapsulated compound, solubilized in the washing liquid, is quantified. At the same time, an FTIR or other analysis must be carried out on the washed mixture to obtain a spectrum identifying and quantifying the encapsulated compound.

[0054] When considering hydrolysed proteins obtained through other processes described in the prior art, such proteins are not able to encapsulate compounds, due to the small size of the peptides obtained, in particularwhen an enzymatic process is applied. The high degree of digestibility and small size of the peptides obtained when using the processes described in the prior art make it impossible to such peptides contain polar and non-polar portions in the same chain, therefore they are not capable of mixing apparently immiscible mixtures.Example 3- Molecular weight distributionThe molecular weight distribution parameter (assessed through high-performance liquid chromatography (HPLC)) showed that the enzymatic hydrolysis hydrolyses (or breaks) the proteins in a more exhaustive way, leading to obtaining protein fragments or peptides with molecular weights on average lower than those obtained through the enzymatic free process of the present disclosure.

[0055] It was surprisingly found that the process of the present disclosure allows to obtain a product with a percentage of protein higher than the one obtained with processes comprising enzymatic hydrolysis, herein depicted in comparative example 1. Additionally, the presented process for protein hydrolyzation without using enzymes generates a protein able to encapsulate compounds, an ability which is not present in hydrolysed proteins obtained through other processes described in the prior art, in particular enzymatically hydrolysed proteins.

[0056] These results indicate that the two hydrolysis processes (enzymatic free process of the present disclosure and process comprising enzymatic hydrolysis) do not work in the same way. Hence, it is observed that an enzymatic hydrolysis leads to obtaining a more hydrolysed protein, or with a higher degree of hydrolysis, or to an average of molecular weights of the peptides lower than a protein hydrolysed through the enzymatic-free process of the present disclosure.

[0057] Using the enzymatic-free process of the present disclosure it is obtained a product with distinct characteristic: the peptides obtained by the process of the present disclosure, besides their high digestibility above 90%, they also present a characteristic size that allows them to encapsulate hydrophilic and hydrophobiccompounds when dehydrated. The ability to encapsulate compounds is very important particularly when there is a need to protect and deliver sensitive compounds, such as vitamins, when feeding an animal. Furthermore, this ability may also be used to protect by encapsulation and deliver active pharmaceutical ingredients to an organism, and the carrier (hydrolysed protein) may be called a highly functional excipient.

[0058] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0059] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0060] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable.

[0061] The following dependent claims further set out particular embodiments of the disclosure.

Claims

C L A I M S1. Enzymatic-free process for producing hydrolysed protein from animal raw material, wherein the digestibility of the hydrolysed protein is superior to 90%, comprising the following steps: providing grinded and / or minced animal raw material; extracting proteins from the grinded / minced animal raw material using steam for obtaining an aqueous phase, a solid phase and a fat phase, wherein said aqueous phase comprises hydrolysed proteins; collecting the aqueous phase in order to obtain the hydrolysed protein; wherein the protein extraction is performed at a temperature ranging from 150 - 190°C and at a pressure ranging from 7 - 9 bar, during at least 30 minutes; and wherein the weight ratio between the animal raw material / steam ranges from 1:0.1 to 1:0.5.

2. Hydrolysed protein composition according to the previous claim, wherein the degree of hydrolysis of the hydrolysed protein is below 50%.

3. The process according to any of the previous claims wherein the temperature of the protein extraction ranges from 170 - 190°C; preferably is 180°C.

4. The process according to any of the previous claims wherein the pressure of the protein extraction is 8 bar.

5. The process according to any of the previous claims wherein the weight ratio between animal raw material / steam ranges from 1:0.2 to 1:0.3.

6. The process according to any of the previous claims wherein the protein extraction is performed for at least 45 minutes; preferably ranging from 45-90 minutes; even more preferably 60 minutes.

7. The process according to any of the previous claims wherein the grinded / minced animal raw material comprises particles with granulometry below 12 mm; preferably below 10 mm; more preferably below 8 mm.

8. The process according to any of the previous claims wherein the protein extraction is performed in a closed reactor; preferably a cooking vessel.

9. The process according to any of the previous claims wherein the step of collection the aqueous phase is performed through decantation; preferably through a 3- phase decanter.

10. The process according to any of the previous claims further comprising a step of purification of the aqueous phase comprises hydrolysed proteins, obtaining a purified aqueous phase comprising hydrolysed proteins.

11. The process according to the previous claim wherein the step of purification comprises a centrifugation step; preferably at 5000-10000 g during 10-30 minutes.

12. The process according to the previous claim 10 wherein the step of purification comprises a continuous flow centrifugation.

13. The process according to any of the previous claims 10-12 further comprising a step of drying the purified aqueous phase comprising hydrolysed proteins; preferably the step of drying is spray drying, freeze-drying, or air drying; more preferably spray drying.

14. The process according to any of the previous claims wherein the animal raw material is animal by-products; preferably slaughterhouse waste.

15. The process according to the previous claims wherein the animal is selected from the group consisting of beef, pork, chicken, or mixtures thereof; preferably bonecontaining animal raw material.

16. Hydrolysed protein obtained through the enzymatic-free process according to any of the previous claims.

17. Hydrolysed protein wherein the hydrolysed protein is able to encapsulate hydrophobic and hydrophilic substances.

18. Hydrolysed protein according to any of the previous claims 16-17 wherein more than 50 % of the hydrolysed protein comprise a molecular weight below 5 KDa, preferably more than 50 % of the hydrolysed protein comprise a molecular weight below 4 KDa; more preferably more than 50 % of the hydrolysed protein comprise a molecular weight below 3KDa; even more preferably more than 50 % of the hydrolysed protein comprise a molecular weight ranging from 0.1 to 2.9 KDa.

19. Hydrolysed protein according to any of the previous claims 16-18, wherein the digestibility of the hydrolysed protein is superior to 90%; preferably the digestibility of the hydrolysed protein is superior to 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

20. Hydrolysed protein according to any of the previous claims 16-19, wherein the degree of hydrolysis of the hydrolysed protein is below 50%, preferably below 45%; more preferably below 40%.

21. Powdered protein composition comprising the hydrolysed protein according to any of the previous claims 16-20.

22. Animal feed comprising the hydrolysed protein according to any of the previous claim 16-20 or the powdered protein composition according to the previous claim 21.

23. Ingestible product comprising the hydrolysed protein according to any of the previous claims 16-20 or the powdered protein composition according to the previous claim 21, preferably the ingestible product is a food product, a nutraceutical product, a pharmaceutical product or a dietary supplement.

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