WO2014207497A1

WO2014207497A1 - A new method to produce marine protein hydrolysate with ultra-low heavy metal contamination and improved physical properties

Info

Publication number: WO2014207497A1
Application number: PCT/IB2013/001390
Authority: WO
Inventors: Bomi FRAMROZE; Roald ROGNE
Original assignee: Hofseth Biocare Asa
Priority date: 2013-06-29
Filing date: 2013-06-29
Publication date: 2014-12-31

Abstract

An improved method to produce soluble marine protein hydrolysate powder with ultra-low heavy metal contamination. This invention relates to a production method that comprises occluding the heavy metal contaminants present in a soluble marine protein hydrolysate solution produced by enzymatic hydrolysis of industrial fish or fish off-cuts, into a lipid particulate structure and separating these particles from the aqueous protein layer. This invention further relates to a method for separating the fatty particulates from the soluble marine protein hydrolysate aqueous layer using a continuously operating automatic disc scrapper or membrane filtering system and evaporating and drying the aqueous fraction into a healthier and safer protein hydrolysate powder for direct and indirect human consumption.

Description

TITLE: A NEW METHOD TO PRODUCE MARINE PROTEIN HYDROLYSATE WITH ULTRA-LOW HEAVY METAL CONTAMINATION AND IMPROVED PHYSICAL PROPERTIES

Field of the Invention This invention relates to a new method to produce soluble marine protein hydrolysate powder with ultra-low heavy metal contamination and improved physical properties. More specifically, this invention relates to a method of production that permits the coagulation of heavy metal particles into a lipid droplet by optimum hydrolysis of the marine protein raw material and separation of the lipid droplets from the aqueous protein-containing solution using a centrifuge or a filter system such as an automatic disc scrapper or membrane filtering system and evaporating and spray drying this aqueous fraction into a healthier and safer protein hydrolysate powder for direct and indirect human consumption.

Background of the Invention The hydrolysis of protein waste byproducts from animal rearing has been described extensively in the prior art. The hydrolysis of proteins has been carried out via the use of acids or the use of basic reagents such as described in Aitken, A. ; Learmonth, M., "The Protein Protocols Handbook, Second Edition : Quantitation of Tryptophan in Proteins ", Humana Press, 2002 and via the use of hydrolyzing protease enzymes as described for example in Baca D. Rebeca et al, "Production of Fish Protein Hydrolysates with Bacterial Proteases; Yield and Nutritional Value" Journal of Food Science, Volume 56, Issue 2, pages 309-314, March 1991. Further the protein waste off-cuts used in the hydrolysis processes have been described to come from both animal and vegetative sources including cattle, swine and aqua culture. Enzymatic processes have shown distinct advantages to other hydrolysis methods allowing for milder conditions and less power consumption which result in a less bitter and easily digestible protein hydrolysate product. These hydrolysate powders have been shown to be useful as animal feed and as a potential source of high quality nutrient for humans. Many types of enzymes have been employed successfully including a mixture of endo and exo enzymes derived from bacterial, fungal and plant sources as described in a recent review article, Shane M. Rutherford, "Methodology for Determining Degree of Hydrolysis of Proteins in Hydrolysates: A Review", Journal of AOAC International, (2010) Volume: 93, Issue: 5, Page(s) : 1515-1522

Aqua-culture and industrial fishing produces a large proportion of the protein consumed directly by humans as well as indirectly used in the form of fish meal fed to cattle, swine, poultry and fish-farms. Fish used for human consumption is increasingly sold as filleted products leaving a large portion of the fish protein as a waste byproducts. This industrial fish and fish off-cuts have been shown to be converted in multiple prior art instances into potentially useful soluble protein hydrolysate powder by enzyme hydrolysis. A process typically described in the art such as in N0332894 and N0332921 involves an enzymatic digestion, followed by a separation of the aqueous solution of amino acids and peptides from the insoluble protein and bones and oil layer using commonly available decanters or filters. However, the protein powder produced from the soluble marine protein hydrolysate typically has a very high level of heavy metal contaminants such as mercury, lead, tin and arsenic which are environmental contaminants found in many marine zones. These heavy metals are extremely detrimental to human health even in parts per million concentrations as shown in Storelli, M.M., "Potential human health risks from metals (Hg, Cd, and Pb) and polychlorinated biphenyls (PCBs) via seafood consumption : Estimation of target hazard quotients (THQs) and toxic equivalents (TEQs)", Food and Chemical Toxicology, Volume 46, Issue 8, August 2008, Pages 2782-2788 and Roser Marti-Cida, Ana Bocioa, Juan M. Llobetb and Jose L. Domingoa, , "Intake of chemical contaminants through fish and seafood consumption by children of Catalonia, Spain : Health risks", Food and Chemical Toxicology, Volume 45, Issue 10, October 2007, Pages 1968-1974 and the successful commercially viable removal of these contaminants would be a significant enhancement of the value of this nutritional product.

Reference is also drawn to Suthasinee Nilsang, Sittiwat Lertsiri, Manop Suphantharika and Apinya Assavanig., "Optimization of enzymatic hydrolysis of fish soluble concentrate by commercial proteases", in Journal of Food Engineering, Volume 70, Issue 4, October 2005, Pages 571-578 that discloses enzyme hydrolysis of industrial fish or fish byproduct.

As apparent from the above state of the art while there are several processes directed to the isolation of marine protein hydrolysate through enzyme hydrolysis, to make it significantly free from heavy metal contaminants and restricting the same to parts per billion concentration in the thus isolated marine protein hydrolysate through an industrially viable and economical process still remains a challenge in the art. Therefore, it is the need of the day to explore for a commercially viable, simple method for hydrolysis of protein waste byproducts that would not involve any sophisticated techniques and materials and yet would substantially free the isolated protein hydrolysate from the heavy metals contaminants to be present only at the levels of less than 1 ppm to thus render it safe and more palatable for human and animal consumption.

Objects of the Invention

It is thus the primary object of the present invention to provide for a method for the production of marine protein hydrolysate with ultra low heavy metal concentration which in being substantially free of any heavy metal contamination would be safer, healthier and tastier for human and animal consumption.

It is another object of the present invention to provide for said method for the production of marine protein hydrolysate with ultra low heavy metal concentration that would be industrially viable and economical in not involving any sophisticated techniques and reagents. It is yet another object of the present invention to provide for said method for the production of marine protein hydrolysate with ultra low heavy metal concentration that would permit the coagulation of heavy metal particles into a lipid droplet and prevent emulsification of the same by way of optimum hydrolysis of the marine protein raw material followed by separation of the same without involving any sophisticated reagent handling techniques and hence would be easy to operate.

Summary of the Invention

Thus according to the basic aspect of the present invention there is provided a process to produce a marine protein hydrolysate powder containing 90% - 99% weight by weight soluble proteins with total heavy metal contamination less than one part per million by enzyme hydrolysis of fish or fish byproducts. The present invention enables a simple and commercially viable method for the removal of heavy metal contaminants from marine protein hydrolysates by involving enzyme hydrolysis of fish or fish byproducts which is based on a surprising advancement whereby more than 99% of the heavy metals in the soluble fish protein hydrolysate remaining occluded in micron sized fatty particulates in the aqueous protein hydrolysate layer which usually gets easily emulsified leading to the formation of microemulsion during processing that could be prevented thus enabling better removal of such high levels of metal contaminants which was heretobefore not possible following the conventional arts. Unexpectedly, it was found by way of the present advancement that by minimizing the formation of microemlusion during processing and simultaneous removal of these fatty (lipid) particulates by centrifuge or ultra-filtration methods specifically after selective concentration of the protein solutions followed by drying the solution into a finished protein powder using simple methods of spray drying, it was possible to provide for desired protein hydrolysate that is significantly free of heavy metals restricted at parts per billion concentrations thus rendering the protein powder much safer, tastier for human and animal consumption.

According to a an aspect of said process the ground solid fish or fish byproducts are mixed with water and enzymes at 50C - 70C for thirty minutes to two hours to produced a hydrolyzed mass.

According to another aspect of said process, the enzymes in the hydrolyzed mass are deactivated in situ by heating to 90C for fifteen minutes. According to yet another aspect of said process, the solids in the hydrolyzed mass are separated from the liquid phases on a sieving system without the use of centrifugal force.

Preferably the liquid phase from the sieve is separated into an oil phase and an aqueous phase using a centrifuge.

According to yet another aspect of said process, the aqueous layer is concentrated from a soluble protein content of 5% - 10% weight by weight to 20% - 60% weight by weight and more preferably between 30% - 40% weight by weight. Preferably, the concentrated soluble protein containing aqueous layer is centrifuged or filtered to give a clear aqueous liquid.

According to an aspect of said process the clear concentrated aqueous solution of marine protein hydrolysate with 20%-60% w/w dry matter is spray dried at 100- 150°C to produce a marine protein hydrolysate powder with total heavy metal concentration less than one part per million.

According to another aspect of the present invention there is provided a composition by way of the said process involving the marine protein hydrolysate powder that is particularly tastier, healthier and safer for direct human consumption.

Detailed Description of the Invention

As discussed hereinbefore the present invention thus provides a simple and commercially viable method to produce marine protein with ultra-low heavy metal contamination. More specifically, this invention involves enzyme hydrolysis of industrial fish or fish byproduct which additionally carefully controls the extent of protein hydrolysis and further provides a very specific and unusual but easy handling of the material post enzyme hydrolysis that allows for the occlusion of heavy metal contaminants present in the aqueous protein solution into separable lipid particles and separating these lipid particles from the aqueous protein layer after concentration of the protein. This invention further relates to a method for separating these lipid particulates from the aqueous soluble protein solution after substantial concentration by using a high-speed centrifuge, automatic disc scrapper or membrane filtering system.

The marine protein hydrolysate produced according to the present invention possess a palatable, highly digestible protein hydrolysate with an ultra-low level of heavy metal contamination which makes it particularly suited for human consumption.

The marine protein hydrolysate produced according to the present invention may optionally be used singly or mixed with other protein, lipid or calcium sources for use in animal and human nutrition. Before describing the present invention in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

The term "and/or" as used herein and in the appended claims, means one or both of the alternatives.

In accordance with the present invention, there is provided a method to produce a palatable hydrolyzed marine protein powder with ultra low heavy metal contamination.

Thus, the method according to the present invention begins with the preparation of industrial fish or. fish byproduct into a size and form most suitable for rapid enzyme hydrolysis via a process of grinding or mashing. Fish byproduct typically consists of the head and backbones of fish after the filleting process. Thus, the first step of the present invention is directed to provide a suitable particle size and composition for carrying out enzymatic hydrolysis using a variety of commercially available enzymes.

Those skilled in the art know that protein hydrolysis can be effected by hundreds of known protease enzymes that can cleave an amide bond in the middle of a protein, such enzymes being commonly known as endo enzymes or cleave a protein from it's terminus one amino acid at a time, such enzymes being commonly known as exo enzymes. Common protease enzymes are commercially available and may be isolated from bacteria, fungi and plants. A preferred mix of endo and exo enzymes used in this invention ranges from a ratio of 2 : 8 to 8: 2 depending on the source and nature of the industrial fish or fish byproduct to be hydrolyzed and the size, concentration and organoleptic properties desired in the marine protein hydrolysate powder produced.

The result of the enzyme hydrolysis of industrial fish or fish off-cuts as carried out in this invention is a reaction mixture comprising of three phases (i) an aqueous phase consisting of soluble amino acids and peptides (ii) a oily phase consisting of a mixture of saturated and unsaturated fatty acids and phospholipids and (iii) an insoluble fraction consisting of small insoluble proteins and bone fragments wherein the aqueous phase also contains heavy metal occluded lipid particulates.

A preferred embodiment of the process of this invention is to carry out the initial separation of the three phases from the enzymatic hydrolysis using a non-centrifugal spinning separation technique. It is well documented in the art that such separations can be carried out by filtration or centrifuge techniques with no preference or benefit in the use of one or the other. However, it was surprisingly found that only non- centrifugal separation of the product of enzymatic hydrolysis, led to a partitioning of the said heavy metal containing lipid particulates into the aqueous phase. Such an apparatus can be employed in batch or continuous manner to arrive at a solid phase and a two-phase liquid layer. The two phase liquid layer can then be separated by centrifugal spinning such as in a centrifuge or two-phase decanter to collect a lipid layer of fish oil and a soluble protein containing aqueous layer.

It is a further embodiment of this invention that such a protein containing aqueous layer containing between 5%- 10% weight by weight soluble protein cannot be used for the separation of the lipid particulates described herein . However, when the aqueous layer when concentrated to 20%-60% weight by weight soluble protein using techniques described in the prior art, becomes surprisingly suitable for separation/filtration of the lipid particulates described herein. Such a concentrated aqueous layer of soluble marine protein hydrolysate when further subjected to separation using a centrifuge or filtration in a continuously operating automatic disc scrapper or membrane filtering system to enable the removal of 2 to 100 micron size lipid particulates in an industrially feasible manner. It was noted by way of studies under the present advancement that the lack of suitable steps directed to the specific removal of lipid particulates where the heavy metal contaminants tend to occlude while producing the marine protein hydrolysate powder resulted in a total heavy metal concentration as measured for mercury, arsenic, lead and tin of approximately 100 ppm whereas following the methods of the present invention described herein and subsequent spray drying of the concentrated aqueous layer containing 20%-60% weight by weight soluble protein into a powder resulted in a marine protein hydrolysis product with less than 5 ppm total heavy metal concentrations making the product much healthier and more suitable for human consumption. A further preferred embodiment of this invention is to purify the oil layer by use of a polishing centrifuge as is commonly known to one skilled in the art and to render the insoluble protein and bone fraction into a dry powder without decomposition of the protein using any one of the drying techniques known to one skilled . in the art, such as a rotary press dryer, vacuum hot air spin flash dryer or tray dryer.

Those skilled in the art of powder formulations will recognize that individual methods for evaporation and drying may be selected in part based on the method of application for the powder being prepared and generally the methods of phase separation and drying that may be used in the process of this invention can be, without intended limitation, any such equipment or method known to one skilled in the art.

Consequently, the three fractions after enzyme hydrolysis, the aqueous soluble protein fraction, the oil fraction and the insoluble protein and bone fractions of this invention can be finally prepared into cosmetic, nutritional and pharmaceutical acceptable forms which are suitable for each such application .

Examples

Example 1 : Preparation of an marine protein hvdrolysate powder with ultra low heavy metal concentration

250 kg of head and back bone of salmon rendered after the filleting process are added into a grinder and processed into 10cm (long axis) irregular pieces. The ground material is transferred to a hydrolyzing reactor containing 250 liters of water and the stirred mixture is heated to 60C. 50 grams of commercially available exo and endo protease enzymes are added into the hydrolyzing reactor in a 1 : 1 ratio and the mixture stirred for 1 hour. The enzymes are rendered inactive by heating the reaction to 90C for 15 minutes and the entire contents of the reactor are separated using a sieve. The liquid phase was passed through a centrifuge (8000 RPM) and separated into a oil layer and an aqueous layer. The aqueous layer containing 7% weight by weight soluble protein was subjected to evaporation to give an aqueous phase containing 35% weight by weight soluble protein. This aqueous phase was passed through a centrifuge (8000 RPM) and a top lipid suspension layer was separated . The aqueous phase was spray dried at 140C to a free flowing off-white powder with 92%+ dry matter. Analysis of the dry powder for heavy metal concentration shows that the levels of mercury (0.25 ppm), lead (0.29 ppm), tin (0.29 ppm) and arsenic (0.23 ppm) are all below 1 part per million (ppm) each.

Example 2 : Preparation of an marine protein hvdrolysate powder with conventional procedures as described in the prior art. 250 kg of head and back bone of salmon rendered after the filleting process are added into a grinder and processed into 10cm (long axis) irregular pieces. The ground material is transferred to a hydrolyzing reactor containing 250 liters of water and the stirred mixture is heated to 60C. 50 grams of commercially available exo and endo protease enzymes are added into the hydrolyzing reactor in a 1 : 1 ratio and the mixture stirred for 1 hour. The enzymes are rendered inactive by heating the reaction to 90C for 15 minutes and the emulsion controlled in the hydrolysis reactor to 2% w/w. The entire contents of the reactor are then fed into a three stage decanter for separation into an aqueous layer of soluble protein, a lipid layer and a solid layer containing bone and insoluble protein mass. The aqueous layer containing 7% weight by weight soluble protein was subjected to evaporation to give an aqueous phase containing 35% weight by weight soluble protein and was spray dried at 140C to a free flowing off-white powder with 90%+ dry matter. Analysis of the dry powder for heavy metal concentration shows the levels of mercury (25 ppm), lead (40 ppm), tin (45 ppm) and arsenic (30 ppm) which are more than 100 times greater than the concentrations of these toxic heavy metals from the process of Example 1.

Example 3: Preparation of an marine protein hydrolysate powder with filtration/centrifuge prior to concentration of the aqueous soluble protein layer

250 kg of head and back bone of salmon rendered after the filleting process are added into a grinder and processed into 10cm (long axis) irregular pieces. The ground material is transferred to a hydrolyzing reactor containing 250 liters of water and the stirred mixture is heated to 60C. 50 grams of commercially available exo and endo protease enzymes are added into the hydrolyzing reactor in a 1 : 1 ratio and the mixture stirred for 1 hour. The enzymes are rendered inactive by heating the reaction to 90C for 15 minutes and the entire contents of the reactor are separated using a sieve. The aqueous layer containing 7% weight by weight soluble protein was again passed through a centrifuge (8000 RPM) and a top lipid suspension layer was separated. The aqueous phase was spray dried at 140C to a free flowing off- white powder with 92%+ dry matter. Analysis of the dry powder for heavy metal concentration shows that the levels of mercury (20ppm), lead (30ppm), tin (40ppm) and arsenic (35ppm) are 100 times greater than the concentrations of these toxic heavy metals from the process of Example 1. Example 4 : Preparation of an marine protein hydrolysate powder with ultra low heavy metal concentration

250 kg of head and back bone of salmon rendered after the filleting process are added into a grinder and processed into 10cm (long axis) irregular pieces. The ground material is transferred to a hydrolyzing reactor containing 250 liters of water and the stirred mixture is heated to 60C. 50 grams of commercially available exo and endo protease enzymes are added into the hydrolyzing reactor in a 1 : 1 ratio and the mixture stirred for 1 hour. The enzymes are rendered inactive by heating the reaction to 90C for 15 minutes and the entire contents of the reactor are separated using a sieve. The liquid phase was passed through a centrifuge (8000 RPM) and separated into a oil layer and aqueous layer. The aqueous layer containing 7% weight by weight soluble protein was subjected to evaporation to give an aqueous phase containing 35% weight by weight soluble protein. This aqueous phase was passed through a automatic disk scrapping filter and the filtrate spray dried at 140C to a free flowing off-white powder with 92%+ dry matter. Analysis of the dry powder for heavy metal concentration shows that the levels of mercury, lead, tin and arsenic are all below 250 parts per billion each.

Example 5 : Preparation of an marine protein hydrolysate powder with ultra low heavy metal concentration

250 kg of head and back bone of salmon rendered after the filleting process are added into a grinder and processed into 10cm (long axis) irregular pieces. The ground material is transferred to a hydrolyzing reactor containing 250 liters of water and the stirred mixture is heated to 60C. 50 grams of commercially available exo and endo protease enzymes are added into the hydrolyzing reactor in a 1 : 1 ratio and the mixture stirred for 1 hour. The enzymes are rendered inactive by heating the reaction to 90C for 15 minutes and the entire contents of the reactor are separated using a sieve. The liquid phase was passed through a centrifuge (8000 RPM) and separated into a oil layer and aqueous layer. The aqueous layer containing 7% weight by weight soluble protein was subjected to evaporation to give an aqueous phase containing 35% weight by weight soluble protein. This aqueous phase was passed through a membrane filter and the filtrate was spray dried at 140C to a free flowing off-white powder with 92%+ dry matter. Analysis of the dry powder for heavy metal concentration shows that the levels of mercury, lead, tin and arsenic are all below 250 parts per billion each.

It is thus possible by way of the present advancement to provide for a simple and commercially viable method to produce a marine protein hydrolysate containing 90% - 99% weight by weight soluble proteins with total heavy metal contamination less than one parts per million that is particularly tastier, healthier and safer for direct human consumption.

Claims

We Claim

1. A process to produce a marine protein hydrolysate powder containing 90% - 99% weight by weight soluble proteins with total heavy metal contamination less than one part per million by enzyme hydrolysis of fish or fish byproducts.

2. A process according to claim 1, wherein the ground solid fish or fish byproducts are mixed with water and enzymes at 50C - 70C for thirty minutes to two hours to produced a hydrolyzed mass.

3. A process according to claim 2, wherein the enzymes in the hydrolyzed mass are deactivated in situ by heating to 90C for fifteen minutes.

4. A process according to claim 3, wherein the solids in the hydrolyzed mass are separated from the liquid phases on a sieving system without the use of centrifugal force.

5. A process according to claim 4, wherein the liquid phase from the sieve is separated into an oil phase and an aqueous phase using a centrifuge.

6. The process according to claim 5, wherein the aqueous layer is concentrated from a soluble protein content of 5% - 10% weight by weight to 20% - 60% weight by weight and more preferably between 30% - 40% weight by weight.

7. The process according to claim 6, wherein the concentrated soluble protein containing aqueous layer is centrifuged or filtered to give a clear aqueous liquid.

8. The process according to claim 7, wherein the clear concentrated aqueous solution of marine protein hydrolysate with 20%-60% w/w dry matter is spray dried at 100- 150°C to produce a marine protein hydrolysate powder with total heavy metal concentration less than one part per million.

9. The composition according to claim 1, wherein the marine protein hydrolysate powder produced is particularly tastier, healthier and safer for direct human consumption.