Hydrolysis of Rest Raw Material From Chicken: Effect of Processing Conditions on Yields and Product Properties, With Extended Focus on the Sediment Fraction

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NTNU Norwegian University of Science and Technology Faculty of Natural Sciences Department of Biotechnology and Food Science

Daniel Fjelldal ForshaugHydrolysis of Rest Raw Material From Chicken

Daniel Fjelldal Forshaug

Hydrolysis of Rest Raw Material From Chicken

Effect of Processing Conditions on Yields and Product Properties, With Extended Focus on the Sediment Fraction

Master’s thesis in Chemical Engineering and Biotechnology Supervisor: Turid Rustad

Co-supervisor: Kathrine Five June 2021

Master ’s thesis

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Daniel Fjelldal Forshaug

Hydrolysis of Rest Raw Material From Chicken

Effect of Processing Conditions on Yields and Product Properties, With Extended Focus on the Sediment Fraction

Master’s thesis in Chemical Engineering and Biotechnology Supervisor: Turid Rustad

Co-supervisor: Kathrine Five June 2021

Norwegian University of Science and Technology Faculty of Natural Sciences

Department of Biotechnology and Food Science

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Preface

This master’s thesis was written during the spring of 2021 for the department of Biotech- nology and Food Science. It marks the end of 5 years of studying at Chemical Engineering and Biotechnology. The master’s thesis is a continuation of a specialisation project written during the fall semester of 2020.

I would like to express gratitude towards my supervisor, Turid Rustad, for her guidance and support throughout the last year. She has also shown an inhuman capacity for proofreading several master’s theses all at once. I would like to congratulate Kathrine Five on her newborn baby and thank her for giving me the opportunity to write this thesis. Lab engineer Siri Stavrum deserves praise for helping with lab procedures, as well as processing large quantities of HPLC samples for us. Our master’s theses would have been a lot shorter without her.

I want to thank my parents for supporting me and having faith in me during all these years. The last year would not have been the same without my girlfriend Ingvild, who, with her infectious laugh, has made though times more tolerable. Lastly, I would like to express gratitude to all my fellow students for making these last five years the most memorable years of my life. We did not get the ending we deserved, but I am sure we will make it up in the future.

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Abstract

Global production of poultry meat is expected to reach 102.9 million tons by the end of 2021. This will generate a large amount of rest raw material, which has the potential of being processed into value-added products. Through enzymatic hydrolysis, products such as chicken protein hydrolysate (CPH), lipids and an insoluble fraction called sediment can be extracted from the rest raw material. The objective of this master’s thesis was to hydrolyse rest raw material from chicken using various processing conditions and analyse the product yields and properties, with a special focus on the sediment. The processing conditions studied were hydrolysis time, raw material type, the added water content in the hydrolysis mixture and pretreatment methods. The pretreatment methods included thermal inactivation of endogenous enzymes and thermal separation of lipids from the raw material (two-stage processing).

Endogenous enzyme activity had considerable effects on dry matter yields and protein recoveries. Hydrolysing untreated viscera resulted in high dry CPH yields as well as low dry sediment yields. Hydrolysing without endogenous enzymes had the opposite results, which was reinforced with only 10% added water. Endogenous enzyme activity contributed significantly to increasing the protein recovery in the CPH. High CPH protein recoveries were obtained faster when hydrolysing with 50% added water instead of 10%. However, after 120 minutes of hydrolysis with both endogenous enzymes and Endocut02L, similar protein recoveries were reached with 10% and 50% added water. Enzyme activity from Endocut02L and endogenous enzymes resulted in a highly hydrolysed CPH and sediment.

After 120 minutes, the degree of hydrolysis (DH) was 48.6% and 35.2% for the hydrolysate and sediment, respectively. The DH of the sediment from pretreated viscera had little to no development.

Hydrolysis of untreated viscera also resulted in a high free amino acid content (FAA) in the hydrolysate. Increasing the amount of added water to 50% resulted in a significantly higher FAA content in the sediment. The essential amino acids lysine and leucine dominated the FAA content of both sediment and CPH. An attempt was made to analyse the molecular weight distributions of the sediment protein with SDS-PAGE. However, no visible bands were formed, possibly due to either the proteins having too low molecular weights (M_W <

14400 Da) or that the sediment proteins were not soluble.

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Sammendrag

Den globale produksjonen av kyllingkjøtt forventes ˚a n˚a 102,9 millioner tonn innen ut- gangen av 2021. Dette vil generere en stor mengde restr˚astoff, som kan bli bearbeidet til produkter av høyere verdi. Gjennom enzymatisk hydrolyse kan produkter som kyllingpro- teinhydrolysat (KPH), lipider og sediment ekstraheres fra restr˚astoffet. M˚alet med denne masteroppgaven var ˚a hydrolysere restr˚astoff fra kyllingen ved hjelp av ulike prosess- betingelser. Deretter skulle produktutbyttet og produktegenskapene analyseres, med spesielt fokus p˚a sedimentet. Prosessbetingelsene som ble undersøkt var hydrolysetid, r˚amaterialetype, vannmengde tilsatt i hydrolyseblandingen og forbehandlingsmetoder. For- behandlingsmetodene inkluderte termisk inaktivering av endogene enzymer og termisk separasjon av lipider fra r˚amaterialet (to-trinns prossesering).

Endogen enzymaktivitet hadde betydelig innvirkning p˚a tørrstoffutbyttet og proteinutbyttet. Hydrolyse av ubehandlede innvoller resulterte i høye tørrstoffutbytter av KPH og lave tørrstoffutbytter av sediment. Hydrolyse uten endogene enzymer hadde motsatt resultat, med høye tørrstoffutbytter av sediment og lave tørrstoffutbytter av KPH. Dette ble forsterket med bare 10% tilsatt vann. Endogen enzymaktivitet bidro betydelig til ˚a øke proteinutbytte fra KPH. Proteinutbyttet i KPH økte raskere ved ˚a hydrolysere med 50%

tilsatt vann i stedet for 10%. Etter 120 minutters hydrolyse med b˚ade endogene enzymer og Endocut02L oppn˚adde hydrolysene med 10% og 50% tilsatt vann, lignende proteinut- bytter. Endogen enzymaktivitet resulterte i de mest hydrolyserte KPH- og sediment- fraksjonene. Etter 120 minutters hydrolyse var hydrolysegraden henholdsvis 48.6% og 35.2% for hydrolysatet og sedimentet. Hydrolysegraden av sedimentet, fra hydrolyserte benfraksjoner og varmebehandlede innvoller, hadde liten eller ingen utvikling over tid.

Hydrolyse av ubehandlede innvoller resulterte ogs˚a i et høyt innhold av frie aminosyrer i hydrolysatet. ˚A øke mengden tilsatt vann til 50% resulterte i et betydelig høyere innhold av frie aminosyrer i sedimentet. De essensielle aminosyrene lysin og leucin dominerte det frie aminosyreinnholdet i b˚ade sediment og CPH. Det ble gjort et forsøk p˚a ˚a analysere molekylvektfordelingen av sediment-proteinet med SDS-PAGE. Ingen synlige b˚and ble dannet, muligens p˚a grunn av at proteinene hadde for lave molekylvekter (MW <14400 Da) eller at proteinene i sedimentet ikke var løselige.

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List of Figures

1 A short description of the by-product categories. The appropriate disposing

method for each category is also shown[37] . . . 2

2 The mode of action for both endopeptidases and exopeptidases.[6] . . . 4

3 The main differences between enzymatic hydrolysis, chemical hydrolysis and the rendering process. . . 4

4 The four phases after centrifugation of the hydrolysis product. . . 6

5 Some of the most important processing conditions for enzymatic hydrolysis. The processing conditions are: Pretreatment of raw materials, raw material type, enzymes, temperature, hydrolysis time and added amount of water to the hydrolysis mixture. . . 9

6 Experimental setup, which included a 2L reactor with heating jacket. . . 16

7 Flow sheet of the process . . . 17

8 The separation of products after hydrolysis. . . 19

9 Overview of the analyses done for the master’s thesis and specialisation project. The analyses were done for both CPH and sediment from the hydrolyses with 10% added water (LW). . . 20

10 Overview of the analyses done for the master’s thesis. The analyses were done only for the sediment from the hydrolyses focusing on different pretreatments (done by Gabriel Johan Roland). . . 21

11 Dry phase yield of the CPH from LW hydrolyses . . . 26

12 Wet phase yields of lipid, emulsion, CPH and sediment, relative to total wet weight. The wet phase yield is shown for Bones-EC-LW[18]. . . 27

13 Transition phase . . . 28

14 Dry phase yields for the CH produced by Roland . . . 30

15 Dry phase yields for the CPH produced by F˚alun . . . 31

16 Dry phase yields of sediments produced in LW-hydrolyses . . . 32

17 Dry sediment yield produced by Roland . . . 34

18 Dry sediment yields produced by F˚alun . . . 35

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19 Dry matter balance of the hydrolysis of untreated viscera, with added En-

docut02L and 10% water, plotted for each sample (t = 00, 0, 60, 120). . . . 36

20 Dry matter balance of the hydrolysis using thermally pretreated viscera, with added Endocut02L and 10% water (VIS-INACT-EC-LW) plotted for each sample (t = 00, 0, 60, 120) . . . 37

21 Dry matter balance for Bones-EC-LW . . . 38

22 Protein concentration for CPH and sediment from LW-hydrolyses . . . 40

23 Protein concentration in the sediment of the hydrolyses done by Roland . . 41

24 Protein recovery for the CPH from the LW hydrolyses . . . 42

25 Protein recovery for the sediment from the LW hydrolyses . . . 44

26 Protein recovery for the sediment from the hydrolyses done by Gabriel Jo- han Roland . . . 45

27 The mass balance for proteins from VIS-EC-LW . . . 46

28 The mass balance for proteins from VIS-INACT-EC-LW . . . 47

29 The mass balance for proteins from Bones-EC-LW . . . 48

30 Degree of hydrolysis for both CPH and sediment from the LW-hydrolyses . 50 31 Degree of hydrolysis for the sediment fractions produced by Roland . . . 52

32 the total free amino acid content . . . 54

33 FAA profile for the essential amino acids in the CPH, extracted from the LW hydrolyses, at t = 60. . . 56

34 FAA profile for the essential amino acids in the sediment, extracted from the LW hydrolyses, at t = 60. . . 57

35 The free amino acid content is plotted for every amino acid present, in both CPH and sediment from VIS-EC-LW. . . 58

36 FAA profile for the essential amino acids in the sediment, extracted from the hydrolyses done by Gabriel Johan Roland, at t = 60. . . 59 37 The first attempt of molecular weight distribution analysis, using the sedi-

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39 The fifth attempt of molecular weight distribution analysis, using the sedi-

ment from VIS-INACT-EC-LW . . . 62

40 The sixth attempt of molecular weight distribution analysis, using the sediment from VIS-INACT-EC-LW . . . 63

G.1 The total amino acid content for both CPH and sediment from the hydrolyses with 10% added water, at t = 60. . . 86

G.2 HPLC-spectre analysing the free amino acid content . . . 87

G.3 HPLC-spectre analysing the total amino acid content . . . 87

H.1 FAA in the CPH of VIS-EC-LW . . . 89

H.6 Bones-EC-LW . . . 89

H.2 FAA in the the sediment of VIS-EC-LW . . . 90

H.3 FAA in the CPH of VIS-INACT-EC-LW . . . 90

H.4 FAA in the sediment of VIS-INACT-EC-LW . . . 91

H.5 FAA in the CPH of Bones-EC-LW . . . 91

I.1 The standard curve created for measuring the protein concentration in teh sediment (Biorad method). . . 93

List of Tables

1 The weight percentages of each bone fraction relative to whole chicken . . . 13

2 The processing conditions for the hydrolyses with 10% added water in the hydrolysis mixture . . . 14

3 The processing conditions for the hydrolyses focusing on pretreatments (done by Roland) . . . 15

4 Mass fractions of dry matter, ash, dry matter excluding ash, lipid, and protein content in the raw material . . . 25

5 Lipid yield for LW-hydrolyses . . . 39

6 Dry emulsion yields and an estimate of the protein recovery in the emulsion, for VIS-EC-LW and VIS-INACT-EC-LW. . . 49

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A.1 The percentage of each phase relative to the total weight of each sample,

for VIS-INACT-EC-LW . . . 73

A.2 The percentage of each phase relative to the total weight of each sample, for VIS-EC-LW . . . 73

A.3 The percentage of each phase relative to the total weight of each sample, for Bones-EC-LW . . . 74

B.1 Dry matter content in hydrolyses with 10% added water . . . 75

B.2 Ash content in hydrolyses with 10% added water . . . 76

C.1 Dry phase yields with 10% added water . . . 77

C.2 Dry phase yields done by Gabriel Johan Roland . . . 78

C.3 Dry phase yields of hydrolyses done by Ingvild F˚alun . . . 79

D.1 Protein concentrations . . . 80

D.2 Protein concentrations for hydrolyses done by Roland . . . 81

E.1 Protein recoveries . . . 82

E.2 Protein recoveries for the hyrolyses done by Roland . . . 83

F.1 The degree of hydrolysis for the sediment and CPH from the hydrolyses with 10% added water. Hydrolyses: VIS-EC-LW, VIS-INACT-EC-LW and Bones-EC-LW. . . 84

F.2 The degree of hydrolysis for the hydrolyses done by Roland[45]. Hydrolyses: VIS-EC, VIS-INACT-EC and VIS-T-EC. . . 85

H.1 Total content of free amino acids with 10% added water . . . 88

H.2 FAA content in sediment produced by Gabriel Johan Roland . . . 92

I.1 SDS-PAGE standards . . . 93

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Abbreviations

Bones Bone fractions CPH Chicken protein hydrolysate

EC Endocut02L

EM Emulsion

FAA Free amino acid

INACT Thermal inactivation

LW Low water content

LWC Low water content RRM Rest raw material

SED Sediment

TAA Total amino acid

VIS Viscera

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1 Introduction

The world population is expected to reach 9.3 billion inhabitants by 2050, which will significantly increase food demand. In order to sustain this future world population, it is estimated that global food production has to be increased by 70%[28]. One way to accommodate this is to utilise the food resources more efficiently. Therefore, one should try to reduce food loss and waste. Instead, one should try to keep as much of this in the food chain as possible[28]. 40-60% of the weight of animals and fish are considered residuals and inedible[2]. The chicken meat industry is expected to reach a record high production of 102.9 million tons of ready-to-cook products by the end of 2021[17]. Ready- to-cook means that parts like feathers, viscera, head and feet have been removed and considered residuals[17]. The chicken production could therefore generate between 68 - 154 million tons of residuals.

These types of residuals have received increased focus, partly due to bioeconomic principles[12].

With the potential for increased value creation, some of these residuals have been redefined to rest raw materials (RRM). It is estimated that the Norwegian chicken co-stream sec- tor may generate 80 million NOK per year through an innovating co-product utilisation called enzymatic hydrolysis[13]. Through this procedure, more value-added products can be created, and at the same time, contribute to increasing feed/food production.

1.1 Rest raw materials

SINTEF defines rest raw material as ”material that is not considered the primary product when utilising raw material”[38]. This material has previously been considered waste, but new applications have been found due to the focus on sustainability. Norway and Iceland are already at the forefront of utilisation of the rest raw material from fish, but other sources of rest raw material are worth exploring[56]. Production of poultry meat globally is expected to increase by 2% by the end of 2021, with continued growth in the future[17], and will, in turn, result in more rest raw material.

In Norway, animal by-products or rest raw materials are grouped based on the potential health risks they are associated with. They are arranged into three categories: Category 1, category 2 and category 3. Raw material belonging to category 1 and 2 is considered high risk and must be excluded from the food chain. One of the main differences between the two categories is the disposing methods allowed for each category. Raw material from category 1 must be combusted or disposed of in a landfill. Raw material from category

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2[37]. Figure 1 shows the main difference between each by-product category.

Figure 1: A short description of the by-product categories. The appropriate disposing method for each category is also shown[37]

Rest raw material from chicken mainly consists of heads, bones, blood, skin, viscera, feet and feathers, and are mostly excluded from the food chain[60]. Still, the rest raw materials generated from the poultry industry is generally considered low risk. Therefore, it can be treated as a category 3 by-product. Businesses can still use methods like landfilling, com- bustion and dumping to dispose of category 3 by-products[34]. It is, therefore, necessary to find more sustainable applications that can also generate profit. Possible applications could be producing high-value products for animal feed or even human consumption[13].

1.2 Processing of rest raw material

A customised process is required for specific applications. One of the most common processing methods is the rendering process. The rendering process utilises heat and pressure treatment to separate lipids and proteins. The method operates at around 133^◦C, with a pressure of 3 bars and a cooking time of ca. 20 minutes[48]. Other processing methods are composting and conversion to biofuels[34]. Hydrolysis is a process where chemical bonds are broken, and water molecules are taken up[48][35]. Examples of molecules that can be broken down are carbohydrates, lipids and proteins. These molecules are usually trapped within structures of biological tissues. They can therefore also be found in rest raw materials[34]. Through hydrolysis, these molecules are more easily separable.

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Hydrolysis needs a driving force to break down the tissue. Acid or base could be an effective driving force for hydrolysis, however, with some drawbacks. The acid/base works as a catalyst for the hydrolysis[33]. The reaction usually takes place under severe conditions, and the process can be difficult to control. The nutritional value of alkaline hydrolysis products is lower than acidic hydrolysis products due to the formation of toxic compounds.

Still, the acidic hydrolysis process also has a negative influence on the nutritional value of the products. The acid catalysed decomposition of proteins could cause the destruction of essential amino acids like tryptophan and cysteine, as well as the conversion of glutamine and asparagine into glutamic and aspartic acid[43][2]. An alternative solution to this problem could be to use a more gentle catalytic driving force, namely enzymes.[1]

1.3 Proteolytic enzymes

Proteolytic enzymes or proteases are enzymes that catalyse the hydrolytic cleavage of peptide bonds[36]. The cleavage of peptide bonds will reduce proteins into smaller peptides and free amino acids. This is advantageous as the body has to digest dietary proteins into amino acids and/or small peptides in order to absorb them[62]. Smaller peptides and free amino acids, therefore, have a higher nutritional value compared to whole proteins [5]. Some proteases only cleave peptide bonds adjacent to a specific amino acid, thereby predictably fragmenting the polypeptide. Others are less specific and have a broader spectrum of cleaving sites[36]. There are four major known classes of proteases: serine, thiol, carboxyl and metalloproteases. They are classified based on their principal functional group in their active site, for example, the thiol group[33].

Furthermore, these enzymes are classified as either exopeptidase or endopeptidase. En- dopeptidases cleave peptide bonds within the peptide chain. Exopeptidases systematically cleave peptide bonds at either the N terminus or the C terminus of the peptide chain. Both modes of action are illustrated by Figure 2. Many different proteases can be applied for enzymatic hydrolysis. Among these are chymotrypsin, trypsin, pepsin, bromelain, papain and Protamex, to mention a few[43]. These will be described further below.

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Figure 2: The mode of action for both endopeptidases and exopeptidases.[6]

1.4 Enzymatic hydrolysis

The main driving force in enzymatic hydrolysis is enzyme activity. With the enzyme’s ability to catalyse a specific reaction at a specific part of a molecule, the process is easier to control than chemical hydrolysis. The process is also significantly milder than chemical hydrolysis. It results in a relatively high product yield, without compromising the nutritional quality[43][9]. For enzymatic hydrolysis, enzymes are needed. Enzymes have very different properties. Choosing enzymes is therefore very important as it should be tailored to the intended product[43]. In the case of enzymatic hydrolysis of rest raw material, proteases are the main group of enzymes that is utilised[2].

Figure 3: The main differences between enzymatic hydrolysis, chemical hydrolysis and the rendering process.

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1.4.1 Exogenous and endogenous proteases

Exogenous enzymes are enzymes extracted from an external source and applied to the hydrolysis mixture. They are commercial enzymes acquired for industrial use. Examples of exogenous enzymes are bromelain, papain and Protamex. They can be extracted from the animal, plant and microbial sources and applied to industrial processes. The benefit of exogenous enzymes is that the product qualities are usually very consistent. However, applying these enzymes can be costly with more efficient alternatives available[31]. En- dogenous enzymes are one such alternative. They are already present in the rest raw material and do not involve the extra expenses required for exogenous enzymes. The main source of endogenous enzymes found in chicken rest raw material is the viscera. Examples of endogenous enzymes are chymotrypsin, trypsin and pepsin.[9][22][43] Endogenous enzymes are known to hydrolyse to a higher degree than commercial enzymes[18][45][52][31].

Extensive hydrolysis could result in a higher product yield, but it could also negatively impact product quality. This will be discussed further in Section 1.5.3.

1.5 Hydrolysis products

By breaking down the rest raw material, mainly four products are liberated from the tissue[33]. These products are lipids, emulsion, soluble proteins and a solid phase called sludge or sediment. The fractions could be processed and used in microbiology, medicine, pharmaceuticals, human nutrition and cosmetics, and feed[4]. They can be obtained and separated from each other through centrifugation and decantation. Centrifugation is a useful method of separating fractions, based on their differing densities, by spinning them in solution around an axis at high speed[54]. The placement of each phase can be seen in Figure 4.

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Figure 4: The four phases after centrifugation of the hydrolysis product.

1.5.1 Lipids

After centrifugation, the lipid phase will be found as the top layer. The lipid phase has the lowest density of the four phases and will be found above the rest. Lipids are important nutrients required in human nutrition[33]. Fatty acids are the building blocks of lipids.

Essential fatty acids like the polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA) are in high demand due to their health benefits[44]. With an increased focus on health and an increasing population, researchers are looking for new sources for PUFAs[34]. One such source is rest raw material from chicken. Lipids extracted from chicken rest raw material is shown to contain 29.7% PUFAs, and 47.22%

monounsaturated fatty acids (MUFA)[30]. These fatty acids have the potential to either be utilised for feed or human consumption[44].

1.5.2 Emulsion

The emulsion phase is denser than the lipid phase and is therefore beneath the lipid phase in the centrifuged product. An emulsion is a system of two immiscible liquids, where one phase is dispersed in the other, the continuous phase. The main types of emulsions are water-in-oil or oil-in-water emulsion. Emulsifiers are compounds that help stabilise the emulsion. Emulsifiers decrease the surface tension between two phases by having the

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ability to attract both phases[8]. Some proteins are suitable emulsifiers. The general understanding is that lowering the protein size lowers the emulsion stability[19]. However, the smallest peptides cannot unfold and reorient at the interface and are, therefore, less efficient at reducing the interfacial tension[57]. Larger peptides are also able to act as emulsifiers, but partly hydrolysed peptides are often preferable[19]. The main reason is that the protein solubility increases by decreasing the protein size. The peptides need to solubilise and be able to rapidly migrate to and be adsorbed by the interface[33].

1.5.3 Chicken protein hydrolysate

A water-soluble phase consisting of soluble proteins is found beneath the emulsion phase in the centrifuged hydrolysis mixture. The soluble protein phase from chicken is called chicken protein hydrolysate (CPH) and is generally considered the most valuable product from hydrolysing rest raw material. Hydrolysis of proteins reduces their size, which is usually accompanied by an increase in peptide solubility[42]. The extent of hydrolysis is often described by the Degree of Hydrolysis (DH). It is the relationship between the amount of cleaved peptide bonds and the total amount of peptide bonds. The degree of hydrolysis is measured in different ways. One way is to utilise formol titration. The resulting free protons from the hydrolysis causes a decrease in pH. The amount of base required to increase the pH has a direct relationship to the number of peptide bonds hydrolysed[46]. The degree of hydrolysis can be calculated using Equation 1, where D is the percentage of free amino groups and E is the percentage of nitrogen[1].

DH = D·100%

E (1)

Partially hydrolysed proteins have many beneficial functional properties, like solubility, fat absorption, foaming capacity and emulsifying properties[32]. Peptides and amino acids are also important flavour precursors, which makes hydrolysate a cheap and abundant source for the flavour industry[59]. However, as mentioned in Section 1.4.1, extensive hydrolysis could negatively impact product quality. It could generate a bitter taste in the product.

The bitter taste is often associated with smaller peptides of less than 1000 Da, with hydrophobic and/or aromatic amino acids[47]. Another study found that fish hydrolysate with a DH of 4%-40% had a high risk of tasting bitter. DH above 40% or less than 4%

was less bitter. Therefore, one could aim for complete hydrolysis, resulting in as many free amino acids as possible[11].

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proteins and minerals[34]. The sediment contains relatively large amounts of protein.

These proteins have high molecular weights and are generally less soluble than hydrolysate proteins. Due to its insoluble properties, hydrophobic proteins concentrate within the phase. These proteins can contribute to a bitter-tasting product[11]. This is one of the main issues with the sediment phase.[34] Nevertheless, the sediment is a protein-rich fraction with relatively high content of desirable amino acids.

The sediment generally constitutes the majority of the product generated after hydrolysing rest raw material from chicken[18][58][29]. Still, the sediment is generally considered unwanted and is in some cases discarded[34]. In earlier studies, little attention has been given to the sediment phase[34]. As a significant hydrolysis product, the sediment should be analysed and characterised. Furthermore, possible applications such as pet food and feed should be considered.

1.6 Processing conditions

Optimal processing conditions have been the main focus in several studies. The overall goal is to produce both lipids and hydrolysate in large quantities and at the same time maintaining high-quality products. This has proven to be difficult, as increasing product quantity can sometimes decrease product quality. In addition, certain processing conditions are only favourable for one desired product whilst disadvantageous for others[15].

This section will explore what the effects of different processing conditions have on the products. Previous studies have focused on different pretreatment methods, different raw material compositions and different amounts of water in the hydrolysis mixture. This is summarised in Figure 5.

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Figure 5: Some of the most important processing conditions for enzymatic hydrolysis. The processing conditions are: Pretreatment of raw materials, raw material type, enzymes, temperature, hydrolysis time and added amount of water to the hydrolysis mixture.

1.6.1 Inactivation

As mentioned in Section 1.4.1, it has been reported that endogenous enzymes are effective at producing high yields of protein hydrolysate but could also negatively influence lipid and CPH quality. Endogenous enzyme activity from lipases can break down the lipids to smaller fatty acids and free fatty acids. This can make them more susceptible to oxidation.

Extensive hydrolysis of proteins into small peptides and free amino acids could also limit their functional properties. By limiting the hydrolysis, this can be prevented. A solution to this could be to inactivate the endogenous enzymes using heat and subsequently adding only commercial enzymes[50]. In Martin-Kristofer Helgeland’s specialisation project, he investigated the endogenous enzyme activity while increasing the temperature. The results showed that after reaching a temperature of 80^◦C, no enzyme activity was detected[26].

However, using temperatures that high can cause protein denaturation. Studies on fish have shown that denatured protein is resistant to enzymatic breakdown.[51] Studies using

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should, therefore, be optimised towards producing the desired product.

1.6.2 Thermal separation

Thermal separation is a pretreatment method to separate as much oil/lipids as possible from the raw material before hydrolysis. Thus, it is called two-stage processing, with thermal separation as step one and hydrolysis as step two. Separating the lipids from the raw material can improve the lipid yield and quality. The lipids are less exposed to both lipases and heat over a long period. Removing lipids from the raw material before the hydrolysis will also reduce the amount of raw material directed for hydrolysis. This can reduce the amount of enzymes and water that needs to be added.

High lipid yields have been obtained by utilising two-stage processing. More than 85%

of the oil present in rest raw material from fish was extracted using 40^◦C[53]. Rest raw material from chicken has more saturated fatty acids than rest raw material from fish.

Due to the higher content of saturated lipids, the temperature to melt the chicken fat is significantly higher than fish oil. Because of this, 65^◦C has been used to separated oil from chicken rest raw material, resulting in a lipid recovery of 37%[45]. With high temperatures such as 65^◦C, there is a risk of protein denaturation and formation of protein- lipid complexes.

1.6.3 Water content

To extract water-soluble proteins, the presence of water is necessary. Water acts as a reaction medium and is especially important for enzymatic reactions. Water also helps to homogenise the mixture of raw material and enzymes. This is important in order for enzyme and substrate to make contact[9]. The general practice for enzymatic hydrolysis is to use 50% water, and 50% raw material[53][58]. Studies suggest that by reducing the amount of water in the hydrolysis mixture, the yield of protein hydrolysate decreases. This is likely caused by a feedback inhibition generated by high product concentrations. Due to the inhibition of enzymes, the yield of sediment increased in comparison to the sediment yield while using 50% water[50][51]. However, adding less water to the hydrolysis has improved the lipid yield in studies using the rest raw material from fish[51][15]. Lowering the water content in the hydrolysis mixture also decreases the unwanted emulsion yield.

It has been observed that high concentrations of proteins can prevent the emulsion from forming. By adding little to no water to the hydrolysis, one can achieve high concentrations of proteins, thus reducing the emulsion yield[51][53].

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1.7 Raw material

The type of raw material might be the most important factor influencing product properties and yields. In an industrial process, the composition of different raw material types may vary. Fractions like bone tissue, skin tissue and viscera, have different compound compositions. The proportion of these fractions can heavily influence the final product[51].

Even with control of the raw material composition, there can still be significant differences within the raw material. This can be due to variability between raw material batches.

Nevertheless, several studies have tried to outline some trends regarding raw material composition. As mentioned in Section 1.4.1, the viscera contains endogenous enzymes, which could increase protein hydrolysate yield. However, studies have shown that it could also increase the amount of lipids in the protein hydrolysate, which negatively influences the hydrolysate quality.[33][50] A study that hydrolysed fish viscera showed that the composition of raw materials influenced the lipid and hydrolysate yield. If the raw material contained more than 17 g protein per 100 g raw material, the lipid phase could be prevented from appearing.[51] Studies on fish also revealed that high lipid content in the raw material could increase the amount of lipids in the hydrolysate.[52]

The bone fractions in chicken are generally very rich in protein. The main protein in bone is collagen, which along with hydroxyapatite, are the dominant compounds in bone tissue[40][39]. Collagen is the most abundant protein found in the animal body. Partial hydrolysis of collagen could result in gelatin, which is primarily extracted from bovine hide and pig skin[21]. The bone fractions of, for example, chicken can therefore supply collagen. Aside from protein, the hydrolysis products contain a more significant amount of ash when using bone fractions rather than viscera[18][16][22]. The ash consists of inorganic compounds like phosphorous and calcium, with potential as nutritional supplement[40].

1.8 Objective

The objective of this master’s thesis was to study the yield and quality of the enzymatic hydrolysis products produced by using different processing conditions. The sediment has had little focus in previous studies. A higher number of sediment fractions was, therefore, analysed. The different processing conditions were:

• Water content in hydrolysis mixture.

– 50% water and 50% raw material – 10% water and 90% raw material

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• Pretreatment

– Thermal inactivation

– Thermal separation of lipids (two-stage processing) – No pretreatment

By varying these conditions, the produced sediment and hydrolysate was analysed to find the following information:

• Yields

– Dry matter yield – Protein recovery

• Properties and qualities – Total amino acid content – Free amino acid content – Molecular weight distribution

The yields and properties of hydrolysate and sediment were compared to propose ideal processing conditions.

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2 Materials and Methods

2.1 Preparation of rest raw material

The chicken raw material was obtained fromNorsk Kylling in Støren, 31. August 2020.

The rest raw material was separated from the chicken and gathered in plastic bags. Im- mediately after gathering the rest raw material, it was transported (1 hour) to NTNU Kavlskinnet. The rest raw material was cooled with ice during transport and was put in cold storage (4^◦C) on arrival. The individual parts of each fraction were weighed and then ground in a meat grinder (Savioli meat grinder 32 classic, 6mm steel plate). The ground rest raw material was distributed in bags weighing 1 kg. The bags were then frozen in a freezer (−25^◦C). At 21:00 the same day, the bags were transferred to a −80^◦C freezer.

Rest raw material from chicken was separated into two types, viscera and bone fractions.

The viscera consists of chicken’s inner organs. The bone fractions is a term used for the chicken bones and the outer tissue that was not used for human food. The bone fraction composition was characterised and provided by Martin-Kristofer Helgeland-Rossavik[26].

Table 1 shows what the bone fractions consist of, as well as their weight percentage. The weight percentage represents the weight of each fraction relative to the total weight of the bone fractions. The same proportions can be found in the bone fractions of a whole chicken[26].

Table 1: The name of each bone fraction and their respective weight percentages. The percentages are based on the proportions found in the bone fractions of a whole chicken.

Bone fraction Percentage of total weight [%]

Heads 6.3

Thigh bone 14.5

Feet 8.9

Upper back 11.8

Lower back 19.3

Neck with skin 9.2

Carcass 11.9

Wing tip 1.8

Wish bone 2.0

Chest skin 6.3

Thigh skin 8.0

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2.2 Hydrolysis

As mentioned in Section 1.8, hydrolyses with different processing conditions were carried out. These conditions were mainly pretreatments, raw material types, and different water contents in the hydrolysis mixture. They were all carried out during the fall of 2020 for the specialisation project[18]. Seven different hydrolyses were carried out and split into two sets. The two sets of hydrolyses will be explained further in Sections 2.2.1 and 2.2.2. All hydrolyses were carried out in two parallels (A and B). In total, there were 14 hydrolyses.

All hydrolyses used 55^◦C and homogenised the mixture with an impeller. The impeller had a rotation speed of 120 RPM.

2.2.1 Hydrolyses with 10% water

Three hydrolyses were done with 10% water and 90% wet RRM[18]. Two hydrolyses used viscera as raw material. One of the two hydrolyses with viscera was pretreated with heat to inactivate the endogenous enzymes and was added Endocut02L (VIS-INACT-EC-LW).

The other was not pretreated and hydrolysed with added Endocut02L (VIS-EC-LW). The final hydrolysis used bone fractions as raw material and was added Endocut02L (Bones- EC-LW). The three hydrolyses with 10% water content were labelled low water content hydrolyses (LW). The setup for the LW hydrolyses is presented in Table 2. Sample weight is explained in more detail in Section 2.2.3.

Table 2: The processing conditions for the hydrolyses with 10% added water in the hydrolysis mixture. Untreated viscera (VIS-EC-LW), thermally pretreated viscera (VIS-INACT- EC-LW) and bone fraction (Bones-EC-LW) was hydrolysed with added Endocut02L[18].

VIS-EC-LW VIS-INACT-EC-LW Bones-EC-LW

RRM type Viscera Viscera Viscera

RRM/Water[%/%] 90/10 90/10 90/10

Weight RRM/Water 1260g/140g 1250g/139g 1250g/139g

Sample weight 467g 463g 463g

Pretreatment None Inactivation None

Added enzyme Endocut02L Endocut02L Endocut02L

2.2.2 Pretreatments

Gabriel Johan Roland performed four hydrolyses for his thesis and specialisation project[45].

The main focus was on the effect of pretreatments. The four hydrolyses used 50% water and 50% raw material in the hydrolysis mixture. Only viscera was used as raw material.

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One hydrolyses used untreated viscera with added Endocut02L (VIS-EC). For two hydrolyses, the endogenous enzymes in the raw material were thermally inactivated. One of the two hydrolyses added Endocut (VIS-INACT-EC), and the other did not add any enzymes (VIS-INACT). The final hydrolysis used thermal separation to remove lipids from the raw material and subsequently added Endocut02L (VIS-T-EC). In this thesis, the thermal separation of lipids will be called two-stage processing. The processing conditions are explained further in Table 3.

Table 3: The processing conditions for the hydrolyses focusing on pretreatments (done by Roland)[45]. Untreated viscera (VIS-EC), thermally pretreated viscera, with and without Endocut02L (VIS-INACT-EC and VIS-INACT), and two-stage processed viscera (VIS-T- EC) was hydrolysed with 50% added water and 50% wet RRM[45].

VIS-EC VIS-INACT VIS-INACT-EC VIS-T-EC

RRM type Viscera Viscera Viscera Viscera

RRM/Water[%/%] 50/50 50/50 50/50 50/50

Weight RRM/Water 1000g/1000g 1000g/1000g 1000g/1000g 1000g/1000g

Sample weight 667g 667g 667g 667g

Pretreatment None Inactivation Inactivation Thermal separation Added enzyme Endocut02L Endocut02L Endocut02L Endocut02L

2.2.3 Experimental setup

The experimental setup for the hydrolyses is shown by Figure 6, which includes two reactors for each parallel (A and B) and other equipment used during the hydrolysis.

The reactors were equipped with a heating jacket to stabilise temperature, an impeller for homogenisation and had a capacity of 2 litres. The effect of time was analysed by taking three samples out of the reactor. The first reactor sample was taken when the raw material was heated to 55^◦C. This was defined as the starting point of the reaction and labeled t = 0. After the 0-sample was taken, Endocut02L was added to the reactor. After 60 minutes, another sample was taken (t = 60), and after 120 minutes, the last sample was taken (t = 120). The weight of each sample was approximately a third of the content of the reactor. Thereby emptying the reactor during the final sampling.

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Figure 6: Experimental setup, which included a 2L reactor with heating jacket.

However, some hydrolysis could occur in the time between adding the raw material to the reactor and the reactor reaching 55^◦C. A sample was created to imitate the hydrolysis mixture before it was added to the reactor and heated. Due to a limited reactor volume, the sample had to be made outside the reactor. The sample was created by mixing raw material and water so that they corresponded with the RRM/water ratio of the hydrolysis.

The weight of the sample also corresponded to the same weight of the other samples (see Tables 3 and 2). The sample was labeled t = 00.

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2.2.4 Experimental procedure of hydrolysis

The hydrolysis process is presented in Figure 7. The flow sheet is described in more detail in the list below.

Figure 7: Flow sheet of the hydrolysis process. The flow sheet was also used in the specialisation project[18].

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1. 3 kg of RRM was taken from a freezer at −80^◦C, and thawed overnight at 4^◦C.

The RRM was then thawed at room temperature (25^◦C) for 1-2 hours before the hydrolysis.

2. Three samples of pure raw material were taken for further analysis. Approximately 15 grams for protein analysis and 15 grams for dry matter and ash content analysis were taken, in addition to approximately 40 grams for lipid content analysis.

3. The sample labelled the 00-sample was created by mixing RRM with water, accord- ing to the same water and RRM ratio as planned for the hydrolysis. The weight of the 00-sample had the same weight as the consecutive samples. The 00-samples was transferred to preweighed centrifugation cups (300ml) and inactivated (at 90^◦C, for 10 minutes).

4. The reaction slurry for the hydrolysis was created with the RRM and water fractions required for the specific reaction and transferred to the reactor. The slurry was heated to 55^◦C, with continuous homogenisation.

5. When the slurry reached 55^◦C, a sample was taken and transferred to preweighed 300 mL centrifugation cups and inactivated (at 90^◦C, for 10 minutes). The sample was labeled as 0-sample since it is considered the start of the reaction, t = 0. After the sampling, Endocut02L was added to the reactor with a total concentration of 0.1g per 100g rest raw material. The concentration was calculated based on the rest raw material weight after taking out the 0-sample.

6. After 60 minutes, the 60-sample was taken and transferred to 300 mL centrifugation cups and inactivated (at 90^◦C, for 10 minutes).

7. After 120 minutes, the 120-sample was taken and transferred to 300 mL centrifugation cups and inactivated (at 90^◦C, for 10 minutes)

8. All the samples were transferred to 50 mL centrifugation tubes and centrifuged for 15 minutes, at 5000 xg and 40^◦C.

9. After centrifugation, the tubes with their content were frozen in a−80^◦C freezer.

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2.2.5 Separation of phases

When the tubes and their contents were frozen, the phases were ready to be separated.

This was done by hand, by emptying the frozen content and splitting the phases with a scalpel and distributing the fat, emulsion, hydrolysate, and sediment to separate preweighed containers. The containers were then weighed with their content to calculate the yield of the different phases. The lipid phase was stored in 50 mL centrifuge tubes in a freezer at −80^◦C. Small samples of approximately 10 mL of the remaining phases were taken to measure dry matter and ash content from the remaining phases. The remaining phases were then freeze-dried for further analysis. The separation process is described in Figure 8[18].

Figure 8: Flow sheet showing the centrifugation and subsequent separation of products into fat, emulsion, a water phase containing CPH, and sediment. After separation the emulsion, CPH and sediment was freeze dried into a powder[18].

2.3 Analyses

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in the hydrolysis mixture. The analyses represented in Figure 10 were only done for the sediment from the hydrolyses focusing on pretreatment methods (done by Gabriel Johan Roland)[45]. Due to time constraints and limited analysis capacity, not all samples (00-, 0-, 60- and 120-sample) were analysed during neither the specialisation project nor the master’s thesis. Because of this, the analyses done for each sample is specified in the figure. However, there are exceptions to the overview, which will be explained during the results.

Figure 9: Overview of the analyses done for the master’s thesis and specialisation project.

The analyses were done for both CPH and sediment from the hydrolyses with 10% added water (LW). The blue area represents what was done for the master’s thesis, while the orange area represents what was done for the specialisation project.

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Figure 10: Overview of the analyses done for the master’s thesis. The analyses were done only for the sediment from the hydrolyses focusing on different pretreatments (done by Gabriel Johan Roland). The blue area represents what was done for the master’s thesis, while the orange area represents what was done for the specialisation project. The dry matter and ash content was measured by Gabriel Johan Roland[45].

2.3.1 Dry matter and ash analysis

Dry matter analysis was carried out by drying three samples (2-4 grams) of each phase, excluding lipid, in preweighed crucibles. The crucibles were dried in an oven at 105^◦C for 22-24 hours. When the drying was complete, the crucibles were cooled down in a desiccator before being reweighed. The dry matter content was determined by subtracting the crucible weight from the final weight. To measure the ash content, the same samples with crucibles were heated at 550^◦C for a minimum of 10 hours. The crucibles were then cooled down in a desiccator before being weighed. The dry matter content was decided by subtracting the crucible weight from the final weight[27].

2.3.2 Kjeldahl method

The protein content of the raw material was determined by the Kjeldahl method. It was determined during the specialisation project. The method analyses the quantitative content of nitrogen in organic material, including inorganic materials such as ammonia and ammonium. The method was applied by digesting 1.4 - 1.7 grams of raw material

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2.3.3 C/N-analysis

The protein concentration in the dry CPH and dry sediment samples were analysed by first finding the nitrogen concentration. Three parallels were made for each sample. 0.1−1 mg of each sample was weighed in tin cups, then sent toNTNU Trondheim Biologiske Stasjon.

The protein concentration was determined by multiplying the nitrogen concentration by 6.25.

2.3.4 Degree of hydrolysis

The degree of hydrolysis (DH) was determined by using the formol titration method, in accordance with Taylor[55]. The degree of hydrolysis was analysed by weighing out 0.5 g of CPH or sediment and adding water to a total mass of 50 g. The solution was added NaOH (0.1 M) until a neutral pH of 7 was achieved. 10 mL of formaldehyde (pH 8.5) was then added, and the mixture was left for 5 minutes under constant stirring. The solution was then titrated NaOH (0.1 M) until it reached a pH of 8.5. Three parallels were done for each sample. The amount of free amino groups was first calculated, then the DH by dividing the amount of free amino groups by the total amount of nitrogen concentration in the sample (see appendix F)[46].

2.3.5 Free amino acid content

The free amino acid content was determined by separating proteins and free amino acids, in accordance with Osnes and Mohr, and analysed through reversed high-performance liquid chromatography (HPLC)[41]. A protein extract was made by dissolving 0.1 g sample in 10 mL water. The extract was filtrated through a filter with a pore size of 70 mm. By adding sulfosalicylic acid, proteins precipitated, and the free amino acids were isolated in the supernatant. The supernatant was then diluted 1:100 and filtrated through a 0.2µm filter. Finally, 0.205 mL of the filtered and diluted supernatant was transferred to HPLC vials and analysed using HPLC. The HPLC analysis was performed by Siri Stavrum using aDIONEX ULTIMATE 3000 UHPLC (Thermo Fisher Scientific Inc. USA) with RF 2000 FLUORESCENCE DETECTOR(Dionex Corporation, USA). o-phthalaldehyde (OPA) was used as a derivatisation agent. The column used was WATER SNOVA- PAK C184um 9.9·150mm column. Methanol and sodium acetate was used as eluents.

Alpha-butyric acid was used as an internal standard. For each sample, three parallels were analysed.

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2.3.6 Total amino acid content

This method was performed by hydrolysing samples of sediment and CPH in 6 M HCl. 1 mL of HCl was added to 0.05 g of CPH, and 2 mL HCl was added to 0.1 g of sediment. The tubes were hydrolysed in a heating chamber (100^◦C) for 22 hours. The acidic solutions were neutralised (pH 7) using NaOH. Then the hydrolysed samples were filtrated through a Whatman glass microfilter (pore size of 25 mm), using suction. The samples were diluted 1:500 using double distilled water and filtrated through a 0.2µm. They were then analysed using HPLC, the same way as described in Section 2.3.6.

2.3.7 Molecular weight distribution

The molecular weight distribution of the peptides was determined using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). A sediment solution was made and added (30 µL) to an eppendorf tube. A prepared mixture of SDS, dithiolthreitol (DTT) and glycerol was added (10µL) to the same eppendorf tube. The tubes were heated (10 minutes, 70^◦C) to disrupt the secondary and tertiary structure of the proteins.

10µL of each solution was transferred to a well in the gel. The gel was applied 185 volts for approximately one hour. The gel was subsequently stained by eStain L1 Protein Staining Device, using Coomassie Blue.

2.3.8 Bligh and Dyer

The lipid content in the raw material was analysed using the Bligh and Dyer method.

The raw material was homogenised in methanol and chloroform and further diluted with water and chloroform to separate the homogenate into two phases. A sample from the chloroform layer was extracted into preweighed tubes, and the chloroform was evaporated.

The lipid content was then calculated.[3]

2.3.9 Statistical analysis

For the statistical analysis, standard deviations and statistical significance were calculated using the STDAV.S command in Microsoft Excel 2021. The significance level was chosen to be 5%, and depending on methods, two, three or one parallels were utilised.

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3 Results and discussion

3.1 Raw material analysis

The raw material was analysed to determine the protein, lipid, dry matter and ash content.

These analyses were done for the specialisation project, but will also be presented in this thesis to give an understanding of the raw material. The dry matter, ash, dry matter excluding ash, lipid, and protein content can be seen in Table 4. The table shows the raw materials for the hydrolyses with 10% added water and the hydrolyses done by Gabriel Johan Roland. The raw materials used in the hydrolyses had considerable differences.

Viscera generally had dry matter contents around 34 g/100g wet RRM and ash contents around 0.9 g/100g wet RRM. The untreated viscera hydrolysed with 10% added water (VIS-EC-LW) had a dry matter content of 28.9 g/100g wet RRM (±0.1), which was lower compared to raw material used in other hydrolyses.

The bone fractions (Bones-EC-LW) had the highest dry matter content with 40.5 g/100g wet RRM (±1.2), as well as the highest ash content of 4.6 g/100g wet RRM (±0.7).

The hydrolysis using thermally pretreated viscera with 50% added water and no added commercial enzyme (VIS-INACT) also had a relatively high dry matter content (38.9 g/100g wet RRM). The bone fractions were also the raw material with the highest protein and lipid content (27.0 ±1.9 and 15.5 ±0.2 g/100g wet RRM, respectively). The lipid content in the remaining raw materials varied between 12.8-14.0 g/100g wet RRM. The protein content in the remaining raw materials varied between 12.0-14.0 g/100g wet RRM.

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Table 4: Dry matter, ash, dry matter excluding ash, protein and lipid content in the raw material (%: g/100g wet RRM). The raw materials analysed were used in the hydrolyses with 10% added water (LW), as well as the hydrolyses focusing on the effect of pretreatment methods (done by Gabriel Johan Roland). LW-hydrolyses: Bone fractions (Bones-EC-LW), untreated viscera (VIS-EC-LW) and viscera with thermally inactivated endogenous enzymes (VIS-INACT-EC-LW). Hydrolyses done by Roland[45]: untreated viscera, thermally pretreated viscera, both with and without added Endocut02L (VIS- INACT-EC and VIS-INACT) and two-stage processed viscera (VIS-T-EC).

Hydrolysis Dry matter (DM) [%] Ash [%] DM - ash [%] Lipid [%] Protein [%]

VIS-INACT-EC-LW 34.3 (±1.0) 0.95 (±0.04) 33.4 (±1.0) 19.73 (±0.82) 14.0 (±0.3) VIS-EC-LW 28.9 (±1.6) 0.77 (±0.01) 28.1 (±0.77) 15.65 (±0.41) 12.8 (±0.6) Bones-EC-LW 40.5 (±1.2) 4.60 (±0.70) 35.9 (±1.4) 27.0 (±1.9) 15.5 (±0.2) VIS-EC 33.3 (±0.39) 0.9 (±0.01) 32.4 (±0.39) 18.4 (±3.5) 12.0 (±0.4 VIS-INACT-EC 34.7 (±1.5) 0.9 (±0.008) 33.8 (±0.1.5) 20 (±2.2) 12.8 (±0.2) VIS-INACT 38.9 (±0.67) 0.9 (±0.01) 38 (±0.67) 18.1 (±0.7) 13.5 (±0.6) VIS-T-EC 35.3 (±1.4) 0.8 (±0.06) 34.5 (±1.4) 19.4 (±0.2) 12.2 (±0.2)

3.2 Dry phase yield

3.2.1 Chicken protein hydrolysate

The results from the specialisation project include the dry phase yields of CPH and sediment for 0- and 60-samples. The dry phase yields of emulsion, CPH and sediment for 00- and 120-samples were analysed for the master’s thesis. The results from the specialisation project and master’s thesis will be compared to answer some questions raised in the specialisation project. The sediment and CPH dry phase yields will also be compared.

Figure 11 shows the development of the dry CPH yields from the hydrolyses with 10%

added water (Low water content:LW). The dry phase yields calculated during the specialisation project is marked with a circle, while the dry phase yields calculated during the master’s thesis is marked with a triangle. The hydrolysis with untreated viscera, with added Endocut02L and 10% water (VIS-EC-LW), had the highest dry CPH yields.

The thermally inactivated viscera with 10% added water (VIS-INACT-EC-LW) showed a similar trend as the hydrolysed bone fractions with 10% added water (Bones-EC-LW). VIS- INACT-EC-LW and Bones-EC-LW started at 3.09 and 2.51 g/100g RRM, respectively.

The dry CPH phase yield of VIS-INACT-EC-LW did not change significantly (P <0.05)

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increased dry emulsion yield. This will be explored further in Section 3.2.3.

Figure 11: Dry phase yield, given in g/100g wet RRM. The dry phase yields of hydrolysed viscera, both untreated (VIS-EC-LW) and thermally treated (VIS-INACT-EC-LW), and hydrolysed bone fractions (Bones-EC-LW) is shown in the graph for 00-, 0-, 60- and 120 sample. The 00- and 120-sample were analysed during the master’s thesis and is marked with triangles, while 0- and 60-sample is marked with circles.

The dry CPH yield of Bones-EC-LW decreased significantly (P <0.05) from 2.51 to 1.31 g/100g RRM between t = 00 and t = 0, respectively. After 60 minutes of hydrolysis with Endocut02L, it reached 5.65 g/100g RRM at t = 60. After another hour (t = 120) it decreased, however not significantly (P < 0.05). In the specialisation project, it was hypothesised that the dry CPH phase yield of Bones-EC-LW and VIS-INACT-EC-LW would continue to increase after 60 minutes. Figure 11 shows that this did not happened.

The reasoning behind the hypothesis was that the raw material from both Bones-EC-LW and VIS-INACT-EC-LW contained more proteins than the raw material of VIS-EC-LW.

Given enough hydrolysis time, they would produce higher dry CPH yields than VIS- EC-LW. Neither VIS-INACT-EC-LW nor Bones-EC-LW had enzyme activity as high as VIS-EC-LW. VIS-INACT-EC-LW had its enzymes thermally inactivated, and the bone fractions lack any significant enzyme activity. It might be that Endocut02L is not efficient at hydrolysing the raw material and that endogenous enzymes are required.

As mentioned in the specialisation project, there were some problems achieving a homo- geneous hydrolysis mixture when the bone fractions were added (t = 0) to the reactor.

The decrease in dry CPH phase yield from t = 00 to t = 0 could be caused by a lack of homogenisation in the reactor. The impeller was unable to rotate when fully lowered into the suspension in the reactor. The temperature of the reactor content never reached 55^◦C,

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and the 0-sample had to be removed earlier than planned[18]. After the 0-sample was removed and Endocut02L added, the impeller managed to rotate, still with some issues with homogenising the reactor’s content. However, this improved throughout the hydrolysis, as the raw material was digested into smaller pieces. The wet phase yields for Bones-EC-LW, originally presented in the specialisation project, supports this. By viewing Figure 12, one can observe a decrease in the wet CPH phase yield, from t = 00 to t = 0. The 00-sample and the 0-sample should contain the same amounts of both wet and dry CPH.

Figure 12: Wet phase yields of lipid, emulsion, CPH and sediment, relative to total wet weight. The wet phase yield is shown for Bones-EC-LW[18].

In the specialisation project, it was hypothesised that the hydrolysis with untreated viscera, with added Endocut02L, and 10% added water (VIS-EC-LW) stabilised at 7.3 (±1.9) g/100g wet RRM (T = 60). However, this was not the case. From 60- to 120-sample, the dry CPH phase yield increased significantly (P <0.05) from 7.3 to 10.4 g/100g wet RRM (±1.5). Studies on hydrolysis of rest raw material from fish have observed a stabilisation of dry hydrolysate yield[51][49]. This has also been observed for hydrolysate from chicken rest raw material[22][16]. For chicken this has been observed after 60 minutes and for fish after 30 minutes[58][16][51][49]. It should be noted that there have been varying results with studies also showing no stabilisation of dry CPH development[58]. The yield of CPH for VIS-EC-LW did not stabilise after 60 minutes and increased almost linearly with time.

Industrial processes could hydrolyse the RRM for longer than 60 minutes while using viscera, Endocut02L and 10% added water. However, the cost of running the process for an extended time could likely discourage that.

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problematic for VIS-EC-LW. During the separation of the frozen phases in the 60- and 120-samples, there were difficulties separating the CPH and sediment phases from each other. In addition to the four phases: lipid, emulsion, CPH and sediment, there was an additional phase, which in the specialisation project was called a transition phase. This is illustrated by Figure 13. Visually it looked like the CPH phase ended at the interface between yellow and dark brown. However, the consistency of the dark brown part strongly resembled the characteristic consistency of CPH. By gradually examining the consistency, downwards towards the tip, the consistency became more like that of sediment. At the tip, the consistency was very clearly that of sediment[18].

Figure 13: The transition phase that was observed for 60- and 120-samples. The samples was extracted from the hydrolysis using untreated viscera, and added Endocut02L and 10% water (VIS-EC-LW).

Even though the consistency became gradually more like sediment, the consistency of the transition phase still bore the most resemblance to the CPH. Because of this, it was decided to classify the transition phase as CPH. However, if the hydrolysate phase contained sediment, then it could have significant effects on many parameters. The CPH and sediment would likely possess similar properties. One consequence could be that the dry CPH phase yield was too high due to the presence of sediment. However, the dry CPH yield at t = 120 was similar to other dry CPH yields from untreated viscera, with added

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Endocut02L and 50% water (VIS-EC)[45]. The same can be said for untreated viscera with added Protamex and 50% water, done by Ingvild F˚alun[16]. Compared to other hydrolyses, the CPH from VIS-EC-LW did not have a high ash content (see Appendix B).

It should be mentioned that there were significant (P <0.05) differences between parallel A and B (VIS-EC-LW), at t = 60. The dry CPH yield of A was 9.10 g/100g wet RRM (±0.06), and for B, it was 5.5 g/100g wet RRM (±0.7). Sediment could be present in the CPH of parallel A, resulting in increased dry CPH yield. However, it could be said that the dry CPH yield for parallel B was unusually low. It did not increase from t = 0 to t = 60, but increased significantly (P <0.05) to 9.04 g/100g wet RRM (±0.04), between t = 60 and t = 120. It can not be concluded whether or not the CPH contained any sediment based on dry matter and ash content. Other indicators of sediment in the CPH could be found in the degree of hydrolysis and protein concentration. This will be explored further in Sections 3.5 and 3.3, respectively.

Figures 14 and 15 shows the dry CPH yields obtained by Gabriel Johan Roland and Ingvild F˚alun, respectively. Their data for t = 00 and t = 120 was also determined during the master’s thesis, while the rest were calculated during the specialisation project. The dry CPH yields from the 00- and 120-samples are visually distinguished as triangles from the dots (t = 0 and t = 60). Both Roland and F˚alun added 50% water (high water content:

HWC) to the hydrolysis mixture. Roland hydrolysed only chicken viscera but utilised different pretreatment methods like inactivation with the addition of Endocut02L (VIS- INACT-EC), inactivation without the addition of commercial enzymes (VIS-INACT) and thermal separation of lipids from the viscera (two-stage processing) with the addition of Endocut02L (VIS-T-EC). He also hydrolysed untreated viscera, with the addition of Endocut02L (VIS-EC)[45].

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Figure 14: Dry phase yield, given in g/100g wet RRM. The dry phase yields of VIS-EC, VIS-INACT-EC, VIS-IACT and VIS-T-EC is displayed in the graph for 00-, 0-, 60- and 120 sample. The values were determined by Gabriel Johan Roland. The 00- and 120- sample was analysed during his master’s thesis and is marked with triangles, while 0- and 60-sample was analysed during his specialisation project and marked with circles[45].

Similarly to the LW hydrolyses, pretreated viscera (VIS-INACT-EC and VIS-INACT) resulted in a lower dry CPH yield than untreated viscera (VIS-EC). The hydrolysis two- stage processing (VIS-T-EC) resulted in the second highest dry CPH yields of Roland’s hydrolyses (10.0 g/100g wet RRM). Both VIS-EC and VIS-EC-LW reached approximately the same CPH yields after 120 minutes. At t = 00, the dry CPH yields for VIS-EC and VIS-EC-LW were 6.7 (±1.5) and 2.95 (±0.02) g/100g wet RRM, respectively. As the reaction progressed, the values became more similar. At t = 120, VIS-EC and VIS-EC- LW reached dry CPH yields of 10.6 and 10.4 g/100g wet RRM, respectively.

The same similarity was not observed between VIS-INACT-EC and VIS-INACT-EC-LW.

Their 00-sample had similar dry CPH values of 3.6 g/100g (±0.3) wet RRM for VIS- INACT-EC and 3.1 g/100g wet RRM (±0.4) for VIS-INACT-EC-LW. From there, they developed very differently. VIS-INACT-EC increased steadily and resulted in 7.2 g/100g wet RRM (±1.3), at t = 120. As previously mentioned, VIS-INACT-EC reached its maximum dry CPH phase yield at t = 60 (4.9 g/100g ±0.2). From there, it decreased significantly to 4.0 g/100g wet RRM (±0.5), at t = 120. When lacking endogenous enzymes, the commercial enzymes might be more efficient when the hydrolysis mixture is added 50% water instead of 10% water.

F˚alun investigated different enzymes and their effect on the hydrolysis of viscera and bone fractions. Bone fractions were hydrolysed using Endocut02L (Bones-EC) and Protamex

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(Bones-PR), and the viscera was hydrolysed using only endogenous enzymes (VIS-E) and using Protamex (VIS-PR). Figure 15 shows the dry CPH yield development from t = 00 to t = 120. F˚alun’s results are included to briefly compare with teh other results.

Hydrolysed bone fractions produced significantly more dry CPH yield when adding 50%

water to the hydrolysis mixture instead of 10%. After 60 minutes (t = 60), Bones-EC had a dry CPH yield of 8.4 g/100g wet RRM (±0.3) while Bones-EC-LW had a dry CPH yield of 5.7 g/100g wet RRM (±0.4). Having a high amount of added water (50%) to the hydrolysis was beneficial when hydrolysing without endogenous enzymes.

Figure 15: Dry phase yield, given in g/100g wet RRM. The dry phase yields of VIS-E, VIS-PR, Bones-EC and Bones-PR is plotted for 00-, 0-, 60- and 120 sample. The dry phase yields was determined by Ingvild F˚alun[16]. The 00- and 120-sample was analysed during his master’s thesis and is marked with a triangle, while the 0- and 60-sample was analysed during his specialisation project and marked with a circle.

The hydrolyses using viscera (VIS-PR and VIS-E) had a clear increase in dry CPH yield, between t = 00 and t = 0. This was also observed in the other hydrolyses with untreated viscera (VIS-EC and VIS-EC-LW). This was expected due to the presence of endogenous enzymes in the viscera. The hydrolysis of untreated viscera with no added enzymes (VIS- E) had the lowest maximum dry CPH phase yield of all the hydrolyses using untreated viscera. Its highest dry CPH yield was 7.9 g/100g wet RRM (±0.8), at t = 120. F˚aluns highest dry CPH yield was 10 g/100g wet RRM (±0.8), at t = 60, hydrolysing viscera with Protamex (VIS-PR).

Hydrolysis of Rest Raw Material From Chicken: Effect of Processing Conditions on Yields and Product Properties, With Extended Focus on the Sediment Fraction

Daniel Fjelldal Forshaug

Hydrolysis of Rest Raw Material From Chicken

Effect of Processing Conditions on Yields and Product Properties, With Extended Focus on the Sediment Fraction

Master ’s thesis

Daniel Fjelldal Forshaug

Hydrolysis of Rest Raw Material From Chicken

Effect of Processing Conditions on Yields and Product Properties, With Extended Focus on the Sediment Fraction

Preface

Abstract

Sammendrag

Contents

List of Figures

List of Tables

Abbreviations

1 Introduction

2 Materials and Methods

3 Results and discussion