NTNU Norwegian University of Science and Technology Faculty of Natural Sciences Department of Biotechnology and Food Science
Gabriel Johan Roland
Enzymatic hydrolysis of chicken (Gallus gallus domesticus) rest raw material
The effect of pre-treatments on functional properties, protein quality and yield
Master’s thesis in Industrial chemistry and biotechnology Supervisor: Turid Rustad
Co-supervisor: Kathrine Five June 2021
Master ’s thesis
Gabriel Johan Roland
Enzymatic hydrolysis of chicken (Gallus gallus domesticus) rest raw material
The effect of pre-treatments on functional properties, protein quality and yield
Master’s thesis in Industrial chemistry 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
Preface
This master’s thesis is written for the department of Biotechnology and food science at the Norwegian University of Science and Technology (NTNU). This study was a continuation of the specialisation project conducted fall of 2020. The project was completed as part of a PhD project of Kathrine Five and in collaboration with Nutrimar. The master thesis was part of the study program Industrial chemistry and biotechnology and was written in the spring of 2021.
I would like to thank my supervisor Turid Rustad for all insight, guidance and motivation dur- ing this project. I would also like to thank my co-supervisor Kathrine Five, for her guidance and positive feedback during this master project and specialisation project. I am thankful that I was allowed to be a part of Kathrine Fives PhD and the hydrolysis research group.
I am also very grateful for the help from lab engineer at the department of biotechnology Siri Stavrum. Without her help with the laboratory work, this project would not have been possible.
I would also like to thank my fellow students and friends for all the memories and interesting discussions in the last year. The conversations and lunch breaks was a bright spot in a very different year. Lastly, I would like to thank my family for all the support these last five years, as they have always helped me when I needed it.
Abstract
The chicken production in Norway has increased by 138% since 2000 from 38 000 tons to 90 000 tons a year. This has caused a similar increase in rest raw material produced.
The rest raw material from chicken contains valuable proteins and lipids that could be used for high-quality feed for the aquaculture industry or as ingredients in human food products. By enzymatic treatment of the rest raw material, it is possible to produce protein hydrolysates with functional and nutritional properties of high quality.
In this study, viscera from the chicken were hydrolysed using four different hydroly- sis conditions; inactivation of endogenous enzymes and addition of commercial enzymes, both endogenous and commercial enzymes, thermal separation of fat from the rest raw material before hydrolysis and hydrolysis without enzymes (Inactivation of endogenous enzymes). The main aim of this study was to investigate how the different conditions and pre-treatments affected protein yield and recovery. The secondary aim of the study was to investigate the functional properties of the hydrolysates to find out if the hydrolysate could be of use as ingredients in food products.
The results showed that different pre-treatments affected the protein yield and the functional properties for the different hydrolysates. Hydrolysate obtained from hydrol- ysis with both endogenous and commercial enzymes, and the hydrolysis with thermal separation of fat produced the highest protein recoveries, with recoveries of 64% and 57%, respectively.
All of the hydrolysates had a similar total amino acid profile, which means they all had most of the essential amino acids. The pre-treatments did not seem to affect the total amino acid profile. However, the pre-treatments did seem to affect the free amino acid concentration in the hydrolysate. The hydrolysis with both endogenous and com- mercial enzymes had the highest amount of free amino acids in the hydrolysate, with a concentration of 490 ±110 mgAA/gCPH after 120 minutes. The lowest concentration of free amino acids was obtained in the hydrolysis with inactivation and no addition of commercial enzymes, with a concentration of 66±17 mgAA/gCPH.
The analysis of functional properties showed that hydrolysate from hydrolysis with both endogenous and commercial enzymes gave the highest emulsion capacity. Further- more, the emulsion capacity was found to be increasing with hydrolysis time for all hydrolysates except the hydrolysate from hydrolysis with both endogenous and commer- cial enzymes. The hydrolysate produced from just viscera and endogenous enzyme from Ingvild Fålunds project gave the highest WHC, at 68%. Hydrolysis of bone fraction produced the hydrolysates with the highest oil-absorbing capacity, with an oil-absorbing capacity of 4.0 gOil/gCPH.
1 SAMMENDRAG
1 Sammendrag
Kyllingproduksjonen i Norge har økt med 138% siden 2000 fra 38 000 tonn til 90 000 tonn i året. Dette har ført til en tilsvarende økning i produsert restmaterialer fra slakteindustrien.
Restmaterialet fra kylling inneholder verdifulle proteiner og lipider som kan brukes til fôr av høy kvalitet til havbruksindustrien eller som ingredienser i matvarer for mennesker. Ved en- zymatisk behandling på resten av restmaterialet er det mulig å produsere proteinhydrolysater med funksjonell egenskaper og høy næringsverdi.
I denne oppgaven ble innvoller fra kylling hydrolysert ved bruk av fire forskjellige forbe- handlinger. En hydrolyse ble gjennomført ved å hydrolysere kyllinginnvoller med endogene enzymer og komersielle enzymer (Endocut02L). De tre andre hydrolysene ble forbehandlet med henholdsvis, 1) termisk inaktivering av de endogene enzymene og kommersielle enzymer, 2) bare inaktivering av de endogene enzymene og 3) separasjon av lipidene fra restmateri- alet ved hjelp av termisk separasjon. Hovedmålet i denne masteroppgaven var å undersøke hvordan de forskjellige forbehandlingene påvirket proteinutbyttet i proteinhydrolysatet. De funksjonelle egenskapene til hydrolysatene ble også undersøkt for å finne ut om hydrolysatet kunne være til nytte som ingredienser i matvarer og fôrprodukter.
Resultatene viste at forskjellige forbehandlinger påvirket proteinutbyttet og de funksjonelle egenskapene til de forskjellige hydrolysatene. Hydrolysat fra termisk separert restmateriale og hydrolysen uten forbehandling produserte CPH med det beste utvinning (recovery) av proteiner, henholdsvis 57% og 64%.
Alle hydrolysatene hadde en lignende total aminosyreprofil,og alle hydrolysatene hadde de fleste essensielle aminosyrene. I motsetning til resulatene i analysen av de totale aminosyrene, virket det som forbehandlingene påvirket konsentrasjonen av frie aminosyrer i hydrolysatet.
Hydrolysen med både endogene og kommersielle enzymer hadde den høyeste mengden frie aminosyrer i hydrolysatet etter 120 minutter, med en konsentrasjon på 490±110 mgAA/gCPH.
Den laveste konsentrasjonen ble oppnådd fra hydrolysen med inaktivering og ingen tilsetning av kommersielle enzymer, med en konsentrasjon etter 120 minutter på 66±17 mgAA/gCPH.
Analysen av de funksjonelle egenskapene i denne masteroppgaven fant at hydrolysatet fra hy- drolysen med både endogene og kommersielle enzymer ga de høyeste emulsjonskapasitetene.
Videre ble det funnet at emulsjonskapasiteten økte med hydrolysetiden for alle hydrolysatene unntatt hydrolysatet fra hydrolysen med både endogene og kommersielle enzymer. Hy- drolysatet produsert fra fra bare involler ga den høyeste vannbindingsevnen, med en vannbind- ingsevne på 67 %. Hydrolysen av beinfraksjon produserte hydrolysatene med den høyeste oljeabsorberende kapasiteten, der den høyest oljeabsorberende kapasiteten var på 4.0 gOl- je/gCPH. Den høye oljeabsorberende kapasiteten i beinfraksjons prøvene kommer mest sannsyn- lig av den høye konsentrasjonen av hydrofobe aminosyrer i bindevev, som for eksempel prolin.
1 SAMMENDRAG
CONTENTS CONTENTS
Contents
1 Sammendrag
2 Background 1
2.1 Animal by-products . . . 2
2.2 Hydrolysis of proteins . . . 4
2.2.1 Enzymatic hydrolysis . . . 4
2.3 Hydrolysis process . . . 6
2.3.1 Lipid phase . . . 6
2.3.2 Emulsion . . . 7
2.3.3 Sediment . . . 7
2.3.4 Hydrolysate . . . 7
2.4 Hydrolysate properties . . . 8
2.4.1 Bioactive properties . . . 8
2.4.2 Nutritional properties . . . 8
2.4.3 Functional properties . . . 9
2.5 Sensory qualities of hydrolysates . . . 11
2.6 Optimization methods . . . 12
2.6.1 Water content in hydrolysis . . . 12
2.6.2 Thermal separation of fat/oil . . . 13
2.6.3 Inactivation of endogenous enzymes . . . 13
2.7 Aim of the study . . . 13
3 Material and methods 15 3.1 Rest raw material . . . 16
3.2 Inactivating endogenous enzymes . . . 16
3.3 Thermal separation of fat . . . 16
3.4 Hydrolysis of chicken rest raw material . . . 16
3.5 Dry matter and ash determination . . . 18
3.6 Lipid determination . . . 19
3.7 Degree of hydrolysis . . . 19
3.8 Analyses of protein hydrolysates . . . 19
3.9 Determination of free amino acids . . . 20
3.10 Determination of total amino acids . . . 20
3.11 Oil absorption capacity . . . 20
3.12 Water holding capacity . . . 21
3.13 Emulsion capacity and emulsion stability . . . 21
3.14 C/N analysis . . . 21
3.15 Colourometri . . . 21
3.16 Statistical analysis . . . 22
CONTENTS CONTENTS
4 Results and discussion 23
4.1 Rest raw material . . . 23
4.2 Dry matter, ash and dry yield . . . 25
4.2.1 Ash content . . . 25
4.2.2 Dry yield and dry matter . . . 26
4.3 Lipid yield and recovery . . . 31
4.4 CPH protein yield and recovery . . . 32
4.5 Degree of hydrolysis . . . 36
4.6 Total and free amino acids . . . 38
4.6.1 Total amino acid results . . . 38
4.6.2 Free amino acid results . . . 41
4.7 Functional properties . . . 46
4.7.1 Emulsion capacity and stability . . . 46
4.7.2 Water holding capacity . . . 50
4.7.3 Oil absorbing capacity . . . 52
4.8 CPH as an ingredient in food or feed products . . . 54
4.9 Conclusion . . . 57
4.10 Future Work . . . 59
5 Appendix A: calculations i 5.1 Calculation of dry matter and ash content . . . i
5.2 Bligh and Dyer lipid content calculations . . . ii
5.3 Calculations of nitrogen content Kjeldahl-method . . . ii
5.4 Phase yield calculations . . . iii
5.5 Protein recovery and protein yield . . . iii
5.6 Total and free amino acid calculations . . . iv
5.7 Calculations of DH . . . iv
6 Appendix B: Values from different analysis v 6.1 Protein concentrations . . . v
6.2 Colour analysis . . . vi
6.3 Degree of hydrolysis raw data . . . vii
6.4 Oil absorbing result . . . viii
6.5 Water holding capacity results . . . x
6.6 Wet fractions . . . xi
6.7 Dry matter and ash percentages . . . xiii
7 Appendix C: Other xiv 7.1 HPLC baseline . . . xiv
7.2 Picture of all hydrolysates colour . . . xv
LIST OF FIGURES LIST OF FIGURES
List of Figures
2.1 Flow sheet describing regulations of rest raw material . . . 3
2.2 Endo and exo peptidase illustration . . . 5
3.1 Hydrolysis conditions . . . 15
3.2 Flowchart describing the hydrolysis process for this project step by step. . . . 18
3.3 Flowchart describing how the CPH obtained from the different hydrolyses are further analysed after being freeze dried. . . 19
4.1 Viscera and bone fraction total amino acid profile . . . 25
4.2 Ash percentage in sediment and CPH. SD calculated based on n = 6 . . . 26
4.3 Dry yield CPH 00, 0, 60 and 120 samples. SD with n = 6. . . 27
4.4 Dry yield sediment 00, 0, 60 and 120 samples. SD with n = 6. . . 28
4.5 Correlation graph of sediment and hydrolysate change. . . 29
4.6 Emulsion dry yield for 00, 0, 60 and 120 samples. SD with n = 6. . . 31
4.7 Lipid yield from separation of lipid fraction after hydrolysis . . . 32
4.8 Degree of hydrolysis for parallel A CPH samples . . . 36
4.9 Total amino acid profile after 60 minutes for CPH with endogenous and com- mercial enzymes . . . 39
4.10 Free amino acids after 60 minutes for CPH from inactivated RRM . . . 40
4.11 Free amino acids after 60 minutes for CPH from inactivated RRM with addi- tion of commercial enzymes . . . 40
4.12 Free amino acids after 60 minutes of CPH from thermal separated RRM . . . 41
4.13 Free amino acids after 60 minutes of hydrolysis with commercial and endoge- nous enzymes . . . 43
4.14 Free amino acids after 60 minutes thermally separated RRM . . . 44
4.15 Free amino acids after 60 minutes from hydrolysis with no enzymes . . . 44
4.16 Free amino acids after 60 minutes from hydrolysis with only commercial enzymes 45 4.17 The change in emulsion capacity for peptides during the hydrolysis . . . 48
4.18 The correlation between protein concentration and emulsion capacity . . . 49
4.19 Water holding capacity for 120 minutes CPH samples . . . 51
4.20 OAC plotted against DH . . . 53
4.21 Picture of 120 CPH samples . . . 56
7.1 HPLC baseline strange . . . xiv
7.2 HPLC baseline correct . . . xiv
7.3 Picture with colour of 0 and 60 CPH samples . . . xv
LIST OF TABLES LIST OF TABLES
List of Tables
2.1 Lipid, protein, ash and moisture composition of chicken rest raw material . . 3
2.2 Common functional, nutritional and bioactive properties . . . 8
4.1 Results lipid, protein, dry matter and ash analysis of rest raw material . . . . 23
4.2 Total lipid recovery from the different hydrolyses . . . 31
4.3 Protein yield in the hydrolysates. SD calculated based on n = 3. . . 33
4.4 Total protein recovery from the different hydrolyses . . . 34
4.5 Free amino groups in the different hydrolysates . . . 37
4.6 Total amino acid concentrations in the hydrolysates . . . 38
4.7 Free amino acid concentrations in the hydrolysates . . . 42
4.8 Emulsion capacity and stability at 25 °C . . . 47
4.9 Oil Absorbing capacity for 120 min hydrolysates. SD calculated based on n = 3 52 4.10 Results colorometri analysis, No SD calculated . . . 55
6.1 Protein concentrations . . . v
6.2 Results colorometri analysis . . . vi
6.3 Degree of hydrolysis values with SD calculated based on n = 3 . . . vii
6.4 OAC at 25 °C, raw data nr. 1 . . . viii
6.5 OAC at 25 °C, raw data nr. 2 . . . ix
6.6 Water holding capacity raw data and p-values . . . x
6.7 Raw data wet fractions one . . . xi
6.8 Raw data wet fractions two . . . xii
6.9 Dry matter raw data . . . xiii
6.10 Ash results raw data . . . xiii
LIST OF ABBREVIATIONS LIST OF ABBREVIATIONS
List of abbreviations
AA Amino Acid
Asn Aspargine Asp Aspartate
BEIN Chicken bone fraction CPH Chicken protein hydrolysate
EC Endocut02L
FPH Fish protein hydrolysate Glu Glutamic acid
Gly Glycine
His Histidine
INAKT Inactivation of endogenous enzymes INV Chicken viscera
Leu Leucine
Lys Lysine
OAC Oil absorption capacity PH protein hydrolysate
Pro Proline
RRM Rest raw material SEP Thermal separation
Trp tryptophan
Tyr Tyrosine
WHC Water holding capacity
2 BACKGROUND
2 Background
The world population is growing faster than before, and with it comes an increase in food consumption. Based on the increasing population, it is estimated that by 2050 the food production might have to increase by 70% to ensure sustainable food production[1]. Studies on socioeconomic changes have found that the decrease in people living below the poverty line will give a rise in demand for protein-rich food[2]. This demand is reflected in food industries, such as the increase in meat industry, especially land-based meat. The Norwegian production of poultry has increased by 138% since 2002 from 38 000 tonnes to 90 000 a year[3]. The increase in meat production has led to a rise in rest raw material from the said industry.
However, the growth in rest raw material from these industries is not only because of the rise in consumed poultry. In the last decade, there has been a change in what the average consumer consider suitable as food. Because of this change in food habits, consumers are buying less whole chicken and more processed products such as fillets or minced meat[4]. The rest of the chicken, viscera, skin, bones and head, is therefore no longer considered suitable for human consumption. A report by Nofima in 2016 suggested that only 49% of the chicken is sold as products for human consumption, and the rest is considered as rest raw material[4]. Today most of this rest raw material is used to produce low-value products such as bio-gas and fertilizers instead of food. However, as the rest raw material contains valuable macromolecules, up-cycling the material into ingredients or for high-quality feed production would be beneficial. Further explanation of the definition of rest raw material is described in Section 2.1.
In the last couple of years, the EU has been trying to establish a bio-economy in order to reach the climate goals set by the Paris agreement and to address the increasing demand in food production[5]. EU defines Bio-economy as "production of renewable biological resources and the conversion of these resources and waste streams into value-added products, such as food, feed, bio-based products and bioenergy"[6]. Up-cycling of by-and co-products from the poultry slaughter industry can be placed in this bracket. In 2016, Norway launched a similar strategy, focusing on how to reuse agriculture products such as rest raw material from poultry, red meat, and cereal industry[4] [7]. The main aim of this strategy was to change our society from "use and dispose" into "reuse and recycle". Utilizing rest raw material, by-products and waste for the production of high and low-value products are necessary to accomplish this goal.
Several different methods have been implemented to extract lipids and proteins from different types of rest raw material; the most common are mechanical processing, chemical hydrolysis and enzymatic hydrolysis. In Norway the aquaculture industry has used enzymatic hydrolysis on rest raw material for decades to produce both low and high-value products[8] [9] [10]. In contrast to the aquaculture industry, few companies in the poultry industry have tried to use and up-cycle the rest raw material produced from the slaughter process by hydrolysis.
2.1 Animal by-products 2 BACKGROUND
SINTEF estimated in 2018 that there is potential to generate 80 million NOK from the valorization of poultry rest raw material using enzymatic hydrolysis[11]. However, to reach the economic potential, further research has to be conducted on how to optimize the process to make it more economically viable. Furthermore, as of today, there are certain restrictions linked with the use of rest raw material for food and feed production that has to be taken into account.
2.1 Animal by-products
In most literature on the rest raw material from livestock, fisheries and poultry are divided into either by-products or co-products. The Norwegian Food Safety Authority (Mattilsynet) defines by-products from slaughter industries as resources that are not suitable for human consumption, while co-products are resources suitable for human consumption[10]. Further- more, EU legislation divides by-products into three categories depending on their origin and potential risk[10].
• Category 1: Materials from animal by-products presenting highest risk of diseases such as TSEs or scrapie, and must be completely disposed of as waste by incineration or landfill after appropriate heat treatment.
• Category 2: Materials including animal by-products presenting a risk of contamination with other animal diseases, animals that die on the farm or are killed in the context of disease control measures. This material may be recycled for uses other than feeds after appropriate treatment, for instance bio-gas or fertilizers
• Category 3: Material that can be further processed into feed or pharmaceutical prod- ucts.
A study from 2020 that investigated the challenges with industries within the bio-economy found that impeding regulations and policies were one of the reasons it was difficult to im- plement a process using bio-materials such as rest raw material[5]. In addition to the EU legislation’s, Norway has their own definitions and regulations in place to ensure the side products from the food industry is safe for further use. The Norwegian Food Safety Au- thority has one set of regulations for food, one for animal by-products and one for waste.
Materials from the slaughter industry that has been treated according to the food regulations can be used as food. However, the moment these regulations are not followed the material has to be treated as either animal by-product or waste, an overview of this can be seen in Table 2.1. Consequently, it is up to the industry to decide how to process the side products produced from the slaughter process, as long as the material is not in category 1 or 2 of the EU regulations. Rest raw material will in this project, be the collective term for the material obtained from side streams in the poultry slaughter industry. Today, most chicken rest raw material is classified as a by-product, and has to be processed as feed or fertilizers to be rein-
2 BACKGROUND 2.1 Animal by-products
Figure 2.1: The figure describes how by-products from slaughter industries has to be processed accord- ing to EU regulations and the regulations from the Norwegian food authorities before being reintroduced into the food chain.[13]. This illustration was also presented in the specialisation project.
Table 2.1: Protein, lipid and ash composition in chicken rest raw material from chicken viscera and bone fraction[16] [17].
Lipid[%] Protein[%] Ash[%] Moisture[%]
Viscera 11 9.85 0.4 77
Bone fraction 7.9 20.9 10.6 66.9
troduced into the food chain, as seen in Figure 2.1[12]. However, with the focus on recycling and reuse of rest raw material more companies are looking at the aquaculture industry, where up-cycling rest raw material is more common[10] [8]. In Norway there are already examples of companies using enzymatic hydrolysis on chicken bone fraction to produce protein products for human consumption, such as the Norwegian company BioCo.
The chicken rest raw material produced from the slaughter industry is in this project divided into two fractions, viscera and bone fraction. The bone fraction is a mixture of heads, skin, wingtips and feet, and contain minimal amounts of endogenous enzymes[14]. The viscera fraction has high amounts of endogenous enzymes, which can hydrolyse the proteins found in the rest raw material. Furthermore, the two fractions both have high amounts of protein and lipid content, as can be seen in Table 2.1. Processing the rest raw material into protein products or lipid products could therefore be beneficial. Chicken muscle has also been shown to contain valuable essential amino acids. This could mean that chicken hydrolysate could be an excellent addition to feed for the aquaculture industry or as supplements for humans[15].
2.2 Hydrolysis of proteins 2 BACKGROUND
2.2 Hydrolysis of proteins
Hydrolysis of the rest raw material can be used to enhance the properties of the proteins and peptides produced. Hydrolysis of proteins is a reaction that breaks down proteins into smaller peptides and amino acids. There are two main methods used to hydrolyse proteins, chemical hydrolysis and enzymatic hydrolysis. Enzymatic hydrolysis is the most common method used in the food industry as it can be used under milder conditions and is easier to control[18] [19]. Additionally, chemical hydrolysis has shown to degrade some of the amino acids found in the rest raw material, which can lower the nutritional value and the functional properties to the product produced. The amino acids Glutamine (Gln) and Aspargine (Asn) are converted into Glutamic acid (Glu) and Aspartate (Asp), while amino acids like Tryptophan (Trp) and sulfur containing amino acids are fully destroyed.
The peptides and amino acids produced from a hydrolysis have a higher solubility than the original protein, which makes it possible to extract a water phase with high peptide concentration[8] [18]. Furthermore, the peptides produced have shown to possess enhanced functional properties, bio-active properties and increased nutritional value, some which are further described in Section 2.4 .
2.2.1 Enzymatic hydrolysis
Enzymatic hydrolysis of proteins can produce protein hydrolysates (PH) with increased bio- activity, functional properties and digestibility compared to the original protein[8]. The enzymes used to break down proteins belong to a group of enzymes called proteases[8]. There are several types of proteases used in the cleavage of proteins, such as trypsin, bromelain, pepsin and chemotrypsin. All of the mentioned enzymes are known as endoproteinases, as they can only break the internal peptide bonds[8] [20]. These are also the most common enzymes found in the viscera of most animals. Enzymes that break the peptide bonds from the terminal ends are called exopeptidases, and are less common. An illustration of how endo- and exoproteases break down the proteins can be seen in Figure 2.2. In a hydrolysis process it is possible to combine the two different enzymes type to obtain a more digested product.
In an enzymatic hydrolysis process of rest raw material it is possible to use either endogenous or commercial (exogenous) proteases. As mentioned earlier, endogenous enzymes are present in the rest raw material. The most common enzymes to find in rest raw material such as viscera are proteases and lipases. In rest raw material from chicken viscera the enzymes are mainly the digestive enzymes mentioned earlier. Exogenous proteases are enzymes that are added to the process after the rest raw material has been transferred to a reactor or con- tainer. Using the endogenous enzymes makes the breakdown of the rest raw material more unpredictable, as it contains a mixture of different proteases and lipases. The Lipases can decrease the quality of the fat phase and sediment phase, as the they produces free fatty acids
2 BACKGROUND 2.2 Hydrolysis of proteins
Figure 2.2: An illustration of how endo and exo protases cleave their respective bonds. The picture was also used in a study on exo and endo proteases[20].
which is an undesirable compound[21]. Because of this, using only commercial enzymes could be favorable, as it gives a more controlled enzymatic hydrolysis[8]. The most common com- mercial proteases used in food industry are bromelain, papain and bacterial enzymes such as alcalase[8]. Using commercial enzymes can make it easier to produce PH with predetermined properties, such as PH with functional properties or specific nutritional properties. However, commercial enzymes are expensive, and this should be taken into account when choosing the enzyme type for the enzymatic hydrolysis. In some cases it is favorable to use both com- mercial and endogenous proteases for short hydrolyses, as this would increase the amount of hydrolysed proteins and increase the degree of hydrolysis. This will in turn increase the yield and recovery of proteins in the hydrolysate produced, which is economical favorable.
However, this could lead to a full digestion, which could lower the functional and nutritional abilities of the PH. Some studies also suggest that if the concentration of proteins are too high in the hydrolysate during the hydrolysis process, a plastein reaction can occur[21]. Plas- tein reaction is not favorable as it can produce aggregates and gelation. Careful planning is therefore important when designing a hydrolysis process, to make the process efficient and economically viable.
Each enzyme has a temperature and pH optimum, where they are most active[8]. If the temperature is too low the reaction rate is slow, but if the temperature is too high the enzymes could denature and they will not be able to catalyze the reaction. The enzymes used in enzymatic hydrolysis of proteins are in most cases highly pH specific, and if the pH is too high or low the enzyme could denature. A project investigating the endogenous enzymes of chicken viscera suggested that lower pH (pH = 3) gave a higher proteolytic activity than at normal pH ( pH = 7)[14]. Furthermore, in the same study, an analysis of the temperature optimum of the endogenous enzymes was performed, and the optimum was determined to be at 57°C. Considering the optimal conditions is essential when designing the hydrolysis process, to make the process efficient.
One of the parameters that are important to know when producing PH is how much of the proteins have been hydrolysed. This is because the amount of hydrolysed proteins often
2.3 Hydrolysis process 2 BACKGROUND
Figure 2.3: An illustration of how the different phases could look after centrifugation of the hydrolysis mixture.
affects the properties of the PH. To determine to what extent the peptides in the solution have been hydrolysed it is possible to calculate the degree of hydrolysis (DH). DH is a control parameter that measures the percentage of broken peptide bonds; the equation (2.1) for DH is shown below[18].
DH = Free amino acids
Total amount of proteins·100 (2.1) 2.3 Hydrolysis process
Hydrolysis processes are in most cases performed in a reactor or a glass container, with or without the addition of exogenous proteases[8]. The protein containing materials, such as rest raw material, are minced before added to the reactor. The mixture is then heated to a prede- termined temperature. Depending on the enzyme(s) used this temperature can vary from 30 -60 °C[22] [21] [8]. To control the pH an electrode measures the pH at all times. Depending on the material hydrolysed the pH can vary between 5-7, in a hydrolysis of chicken viscera a pH of 6 is expected. When desired hydrolysis is attained, the enzymes has to be inactivated to stop the enzymatic reaction. Inactivation can be performed by heating the mixture between 75-100 °C, depending on the enzyme(s)[8]. The resulting mixture after inactivation consists of different phases with solved peptides or amino acids, lipids and the proteins that is not soluble. This mixture can be separated by centrifuging and then separated. An illustration of how a hydrolysis mixture could look after centrifugation is shown in Figure 2.3.
2.3.1 Lipid phase
After hydrolysis of chicken rest raw material, a fat layer is formed at the top of the hydrolysis mixture. This layer contains lipids that can be of nutritional value. Few studies have been performed on the composition of the lipids found in the fat layer after hydrolysis of chicken
2 BACKGROUND 2.3 Hydrolysis process
rest raw material. One study indicates that it contains fatty-acids such as omega 6 and omega 9 fatty acids, which can both be used as a supplement in feed or food[23] [24]. During the hydrolysis, the lipid quality decreases and free fatty acids and peroxides are formed.
There are methods to prevent the decrease in lipid quality obtained from hydrolysis. One method was investigated in a project on hydrolysis of trout heads. Antioxidants was added to the mixture before hydrolysis, and their research indicated that adding the antioxidants gave a lower PV, T-BAR and free fatty acids values[25]. Another method involves separating the lipids out before the hydrolysis, as chicken fat has been reported to have a melting point of 35 °Cwhich could preserve the lipids and produce fat with a higher quality[26]. This method is further discussed in Section 2.6.2.
2.3.2 Emulsion
Between the hydrolysate phase and fat phase an emulsion phase can be formed. The emulsion is mainly formed by mixing amphiphilic peptides or proteins and oil droplets from the fat phase. The peptides form a coat around the droplets which prevents them from coalescing.
Some other compounds can also be found in the emulsion, such as minerals and carbohydrates.
Emulsion formed in a hydrolysis has little commercial value, and it would be favorable to minimize it in the hydrolysis process. However, peptides present in the hydrolysate with emulsifying properties could be of use in various food products, and is further discussed in Section 2.4.
2.3.3 Sediment
Sediment or sludge is the bottom layer after centrifugation. The layer contains larger proteins that were not hydrolysed into soluble peptides, lipids and minerals. Sediment from hydrolysis of fish rest raw material has been used as a protein powder for pet food and feed at Norwegian company named Nutrimar. Work by Guro Tveit on hydrolysis of chicken rest raw material indicated that the sediment could contain up to 65% of the protein from the rest raw material after 60 minutes of hydrolysis[22]. Based on this, sediment from hydrolysis of chicken rest raw material have a high economical value and could be utilized as a protein source.
2.3.4 Hydrolysate
The hydrolysate phase contains the most valuable peptides, as they have been made soluble by the hydrolysis reaction, which enhances their nutritional and functional value. Hydrolysate from fish rest raw material has been used in food, cosmetic and feed industry for some time.
However, few studies have been conducted on the production of chicken protein hydrolysate (CPH). Based on the composition of chicken rest raw material from Table 2.1 in Section 2.1, there is reason to believe that hydrolysing chicken rest raw material is a good method to pro- duce high-quality PH. One study suggested that 32 % of proteins from the rest raw material ended up in the PH after 60 minutes of enzymatic hydrolysis[22]. Another study comparing
2.4 Hydrolysate properties 2 BACKGROUND
PH from salmon and chicken rest raw materials found that hydrolysing the chicken bone frac- tion using the commercial enzyme alcalase, gave a PH with good functional properties and high digestibility[17]. The study further indicated that hydrolysing chicken rest raw material could produce PH with good water holding capacity, foaming abilities and oil binding capac- ity. In a study on the valorisation of different types of meat rest raw materials, both total and free amino acid profile was determined[27]. The results obtained from this study indicated that CPH from chicken rest raw material contained most of the essential amino acids and had good nutritional value. Consequently, CPH produced from the hydrolysis of chicken rest raw material could be used as ingredients in food, feed and pharmaceutical industry. Further research has to be conducted to understand more about this potential.
2.4 Hydrolysate properties
Peptides have been shown to have a wide variety of properties. Enzymatic hydrolysis of rest raw material is used to enhance these properties found in the protein hydrolysate. The most common properties are functional, bioactive and nutritional properties, and can be seen in Table 2.2.
Table 2.2: Common functional and nutritional properties associated with peptides used in food chem- istry[28] [29]
Functional properties Nutritional properties BioActive properties Emulsifying properties Digestibility Antioxidant properties Water holding capacity Complete amino acid profile Anti-microbial
Oil absorbing capacity High nutritional value Growth enhancing
Foaming abilities Antihypertensive
Solubility properties
2.4.1 Bioactive properties
Bioactive peptides are peptides with properties that have health effects, such as antioxidant properties, immunologic properties or can induce growth stimulation. Studies in PH have shown that hydrolysed proteins posses these properties. One study on the bioacitive prop- erties of FPH found evidence for this[30]. There have been a few studies on how PH from chicken rest raw material has bio-active properties. One study suggested that CPH from chicken residues could be used as growth stimulants in animal feed[31].
2.4.2 Nutritional properties
Nutritional properties are dependent on the degree of hydrolysis and the amino acid compo- sition of the hydrolysate. Amino acids are biomolecules that are important in all animals, as they serve as building blocks and various metabolic pathways[32]. Furthermore, amino acids
2 BACKGROUND 2.4 Hydrolysate properties
are classified as either essential or non-essential. The nine essential amino acids are Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Threonine (Thr), Tryptophan (Trp), and Valine (Val). Some of the non-essential amino acids that in later years have been classified as functional amino acids. These are amino acids that are important for regulations and participation in metabolic pathways. The amino acid classified as functional amino acids are; Arginine (Arg), Cystine (Cys), Leu, Met, Trp, Tyro- sine (Tyr), Aspartate (Asp), Glutamic acid (Glu), Glycine (Gly) and Proline (Pro)[32]. High quality protein hydrolysate should be easily digestible. Hydrolysing the proteins present in the rest raw material into smaller peptides increases the digestibility[8].
2.4.3 Functional properties
In addition to their nutritional and bio-active value, protein hydrolysates have been shown to hold several functional properties that make them suitable as ingredients in food, phar- maceutical and cosmetic products[8]. However, to tailor the protein hydrolysate functional properties to the different products, appropriate processing might be needed first[33]. Hy- drolysing the proteins is one processing method that can be used to produce hydrolysate with improved functional properties. This is because the hydrolysis change the properties of the peptides, such as molecular weight, hydrophobicity and surface exposure of polar groups.
These physical properties can directly influence the functional properties, and therefor change the hydrolysate suitability as an additive in food, cosmetic or pharmaceutical products[8]. Studies on FPH and its properties have been conducted and have shown that hydrolysis combined with thermal separation or enzymatic inactivation could improve functional prop- erties such as water holding capacity, emulsifying and foaming abilities[8] [21]. However, few researchers have studied how the functional properties of chicken protein hydrolysate are affected by different pre-treatments, such as thermal separation and enzymatic inactivation.
Emulsifying abilities
Emulsion is the formation of a phase consistent of two immiscible liquids, in most cases oil and water. The ability to create an emulsion is dependent on the ability of a compound to lower the interfacial tension between hydrophilic and hydrophobic molecules in a solution[8]. Hydrolysing proteins produces peptides with surface active groups that promotes oil in water emulsion. This is because proteins and peptides have both hydrophilic and hydrophobic ends, and they are able to adsorb to the surface of hydrophobic droplets in solution and form a membrane that prevents the droplets from coalescing[8]. The emulsifying properties of a PH is therefore dependent on the amino acids composition of the PH. Research on amino acid profiles of chicken muscle indicates that chicken rest raw material could have good emulsifying properties[15]. Because chicken muscle contains a good mixture of hydrophobic and hydrophilic amino acids. The stability of the emulsion phase is highly dependent on the molecular weight distribution of the PH. PH with higher amount of large peptides have
2.4 Hydrolysate properties 2 BACKGROUND
a higher surface hydrophobicity which makes them better emulsion stabilizers. PH from sardines with a low DH showed better emulsion stability than PH from sardines with high DH and molecular weight distribution[8].
When producing protein hydrolysate for feed, peptides with high solubility over a wide range of pH is often favorable[34]. Solubility has been shown to increase with DH in hydrolysate from fish rest raw material[8]. Previous studies on hydrolysis of fish rest raw material have shown that partly hydrolysing proteins increase their emulsifying abilities[8]. Although making the peptides slightly more soluble has a positive effect on emulsion capacity, studies on emulsifying properties of peptides have indicated that hydrolysing over an extensive time period can cause loss of emulsifying capacity and stability[35]. One study on emulsifying properties of whey protein suggested that peptides smaller than 5kDa was not optimal for producing a stable emulsion. However, the study also indicated that peptides smaller than 5 kDa did retain their ability as emulsifying agents as long as there were some larger peptides in the solution[36]. Little research has been performed on the emulsifying abilities of CPH from chicken rest raw material. One study suggested that the emulsifying abilities of peptides from chicken rest raw material have similar properties as the FPH from fish rest raw material[17]. Investigating these abilities could prove important in the commercialization of CPH for both food and feed production.
Water holding capacity
Water holding capacity (WHC) is a measurement of the water contained in a protein system, and often in food such as cod muscle, chicken fillet or different types of minced meat. Adding hydrolysed peptides into a food system can increase the WHC of the system, increasing different sensory properties such as mouthfeel. There are two reasons why water can be retained or bound to a protein system. It could either be trapped in the three-dimensional structure of the protein, or it could be bound to the molecule through weak bonds and can no longer be available as a solvent[33] [37]. The water contained in the muscle can be further divided into three groups.
• Water that binds to secondary bonds and has a lower energy than solvent water.
• Water trapped in capillaries and macro molecular matrices. Water trapped here has a lower freezing point than water found on the outside in solution[38].
• Water that is immobilised in a web of protein structures and cell membranes. These structures are large enough for the water to have approximately normal properties (freezing point, evaporation point), and represent most of the water trapped in the muscle[38].
The structure of the peptides added into the product to increase WHC is considered essential.
A study on FPH from salmon rest raw material indicated that the degree of hydrolysis influ-
2 BACKGROUND 2.5 Sensory qualities of hydrolysates
enced the WHC. As the peptides became smaller the three-dimensional structure changed[8]. Furthermore, the increase of polar groups after hydrolysis such as NH2 and COOH, affected the PH ability to absorb water and moisture to some extent[8]. According to a study on hydrolysis of rest raw material from salmon, FPH exhibited better WHC properties than egg and soy protein. Work on water-holding capacity in proteins indicates that thermal treat- ment on protein from lentil-flour did not decrease the WHC but increases it[33]. Few studies have been conducted on WHC abilities of CPH and if it can be used as an ingredient in food products to increase the WHC. Investigating how CPH from pre-treated rest raw material would therefore be beneficial.
Oil absorbing capacity
Fat or Oil absorbing capacity (OAC) is a measure of how oil and lipid materials interact with proteins[17] [39]. PH with high OAC can be used as an ingredient in meat and pastry products, as it enhances the sensory properties[40]. The mechanism of OAC is primarily due to the entrapment of the lipid molecules, but hydrophobic interactions are also affecting the OAC abilities[41]. The hydrophobic or non-polar groups have an affinity for the non-polar bonds found in most lipids. These groups interact with hydrophobic side groups of the proteins found in the PH. Therefore, it is expected that PH with a high amount of hydrophobic side groups would have a high OAC. Furthermore, PH with smaller particles have been shown to entrap more oil than PH with larger particles[41]. Processing that increases the bulk density of the protein powders has shown to greatly increase the OAC, and it could therefore be beneficial to use to obtain PH with high OAC.
An investigation into OAC of hydrolysed salmon backbone suggested that OAC did not change when the DH changed. The same study found that FPH with a DH of 5% had an OAC between 5 ml Oil/g FPH[8]. In an Iranian study performed by Taheri, it was found that CPH from chicken bone fraction had an OAC of 2.8 g Oil/g CPH[17]. It would be beneficial to investigate further the difference between CPH from bone fraction and CPH from viscera, as there are few other studies on the OAC properties of CPH.
2.5 Sensory qualities of hydrolysates
Hydrolysates that have nutritional or functional properties can be suitable as ingredients in food or feed products. For hydrolysate to be added into food systems other factors also have to be considered. One limiting factor for CPH to be used as an ingredient or an additive in food products for humans are the bitter taste. Several studies on the bitterness of FPH suggest that the hydrophobic side groups are one of the reasons for the bitter taste[18]. Research have suggested that the length of peptides is connected to the bitter taste in some hydrolysates.
However, the reason for a correlation between length and bitterness could be the peptide conformation and how the hydrophobic groups are angled differently in larger peptides than in smaller ones.
2.6 Optimization methods 2 BACKGROUND
Colour is another factor that has to be determined before adding hydrolysate into a food prod- uct. Colour is considered an important quality by most consumers and adding hydrolysate that turn light fish mince brown is often considered not appealing. It is therefore important to have a system that objectively determines the colour of food. Hunters law was established for this exact purpose. Hunters law divides colour into three parameters a, b and L. L mea- sures the light (+) and dark colour (-) of the sample, b measures blue (-) and yellow (-) and a is a measurement of the red (+) and green (-) colour of the sample.
Over the recent years, there have been several studies on the colour of FPH from different types of marine rest raw material. One study on FPH from hydrolysed rainbow trout inves- tigated the colour change after storage[42]. The study obtained an L value between 86 and 90 and an a value of approximately 0 for the FPH before storage. However, few studies have investigated the colour of chicken hydrolysate. Using chicken viscera as rest raw material would most likely produce CPH with some red colour present, which is not always beneficial.
This is because viscera contains a lot of blood. Blood contains hemoglobin which depending on the ligand, can produce a red colour. This was seen in the study on rainbow trout, but first after a storage time of 2 months. One of the few studies on the colour of CPH from chicken rest raw material (heads and bones) obtained the following colour values L = 79, a
= - 5, and b = 11[17]. The values obtained indicates that CPH has some of the same colour features as FPH, although CPH is somewhat darker. Understanding how different hydrolysis conditions affect the colour of CPH could be an essential step in the right direction if the CPH is to be used as an ingredient in food products.
2.6 Optimization methods
To obtain PH with the desired functional properties, nutritional values and at the same time make hydrolysis of chicken rest raw material economical viable, further optimization has to be implemented. There are several methods to optimize the hydrolysis process, such as decreasing amount of water added to the hydrolysis mixture, thermal pre-treatment and inactivation of the endogenous enzymes in the rest raw material, to name a few.
2.6.1 Water content in hydrolysis
Decreasing the amount of water used in the hydrolysis process could be favorable from an economic standpoint, as this would reduce the amount of heating needed for the reaction[21]. Lower amount of added water in hydrolysis of cod rest raw material has also been shown to increase the yield of oil obtained[21]. However, work on hydrolysis of fish rest raw material is inconclusive when it comes to how it affects the yield and quality of the PH and the amount of emulsion produced. A project investigating the effect of decreased (10 %) water content on yield of chicken hydrolysate from chicken rest raw material found that the emulsion was significantly higher than in the hydrolysis with addition of water (50 %)[43]. There are few studies investigating how the functional properties are affected by not adding extra water to
2 BACKGROUND 2.7 Aim of the study
the hydrolysis. Further investigation into the functional properties of CPH from hydrolysis with no or low amount of added water would therefore be beneficial.
2.6.2 Thermal separation of fat/oil
Previous Work on thermal separation of fat/oil from fish rest raw material prior to hydrolysis indicates that it could be a beneficial pre-treatment. In a study performed by Rasa, a two stage thermal separation of oil from fish rest raw material and how affected the yield of FPH was investigated[26]. Their results indicated that it would be of economical value to implement a thermal separation before hydrolysis of the fish rest raw material[26]. Few studies have been conducted on thermal separation of fat from chicken rest raw material. One study on thermal separation of fat from chicken bone fraction showed that it was possible to separate 45 % of fat from chicken rest raw material using a temperature of 40°C[22]. Additionally, a project on characterization of chicken rest raw material investigated the effect of thermal separation of fat from viscera at different temperatures[44]. The study suggested that to maximize fat separation without inactivating the endogenous enzymes a temperature of 65 °C would be optimal. Neither of these studies conducted an analysis of the protein hydrolysate obtained from the thermal separation. Doing so would give valuable information about the possibility of using thermal separation on an industrial scale chicken hydrolysis plant.
2.6.3 Inactivation of endogenous enzymes
As mentioned earlier, the endogenous enzymes found in viscera of chicken are mainly di- gestive enzymes such as trypsin, pepsin and chemotrypsin. They all have different optimal conditions which makes them difficult to control. This is partly because the different endoge- nous enzymes have different binding site, which produces peptides in a wide variety of sizes.
Furthermore, in most rest raw material there will be some lipases present, which can produce unfavorable side reactions such as free fatty acids[21]. Inactivating the endogenous enzymes could therefore lead to a more controlled process[8]. This could help tailor the PH for specific purposes, because the properties of the commercial enzyme is known. However, studies con- ducted on inactivation of endogenous enzymes on fish rest raw material have shown that the yield significantly decreases after inactivation of endogenous enzymes[45] [21]. Furthermore, the process in question is not energy friendly, and heating the material to the inactivation temperature can be costly. Using inactivation as a method to optimize the production of chicken protein hydrolysate is possible. However, further investigation has to be conducted to find out if it is beneficial.
2.7 Aim of the study
The main aim of this master thesis and the specialisation project performed last semester was to investigate how different pre-treatments before hydrolysis of chicken viscera affected the protein and dry matter yield of the CPH produced. The commercial enzyme Endocut02L
2.7 Aim of the study 2 BACKGROUND
was used as the added enzyme in some of the hydrolyses. Four hydrolyses were conducted;
in three of them, a pre-treatment was applied. In the fourth chicken viscera was hydrolysed using endogenous enzymes and commercial enzymes. For the three other hydrolyses, the following pre-treatments applied.
• Thermal separation of fat
• Inactivation of the endogenous enzymes and addition of commercial enzymes
• Inactivation of endogenous enzymes without addition of commercial enzymes
To complete the main aim, the CPH obtained from these hydrolyses was analysed to deter- mine if they had nutritional value that could be useful in either food or feed. The nutritional and physical properties were investigated by analysing protein concentration, degree of hy- drolysis and total amino acid composition.
The secondary aim of the project was to determine if the CPH had any functional properties.
The following functional properties were analysed; OAC, WHC, EC and ES. CPH obtained from two other master students were also analysed, Daniel Forshaug and Ingvild Fålund. The CPH analysed from Daniel Forshaug was produced by hydrolysing chicken bone fraction and chicken viscera, with less water added to the hydrolysis mixture[43]. One CPH from Ingvild Fålund was produced from bone fraction with the addition of Endocut02L, and one CPH was produced from using just chicken viscera (Autolysis)[46].
3 MATERIAL AND METHODS
3 Material and methods
Four hydrolyses with two parallels (A and B) each were performed on chicken viscera, de- scribed in Figure 3.1. They were named based on the pre-treatment used, hydrolysis with both endogenous and commercial enzymes (INV-EC), thermal separation of fat from the rest raw material before hydrolysis (INV-SEP-EC), inactivation of the endogenous enzymes before hydrolysis with addition of commercial enzymes (INV-INAKT-EC) and inactivation of en- dogenous enzymes without addition of commercial enzymes (INV-INAKT). The commercial enzyme used was Endocut02L, and the reason this enzyme was chosen was because Nutrimar wanted to investigate this enzyme. The hydrolyses described in this section was preformed last fall during the specialisation project. All of the other analyses, were performed in the spring during the master project.
All of the CPH produced from the four hydrolyses were analysed. In addition to the CPH from this project, samples from the hydrolyses of chicken rest raw material by Ingvild Fålund and Daniel Forshaugs in their master thesis was also analysed. The samples from Fålund consisted of CPH from hydrolysed viscera with no commercial enzymes (INV) and CPH from hydrolysed bone fraction with addition of Endocut02L (BEIN-EC). The samples analysed from Daniel Forshaug were CPH from hydrolysed viscera with decreased amount of added water with addition of Endocut02L (INV-EC-UV) and CPH from hydrolysed bone fraction with decreased amount of water and addition of Endocut02L (Bein-EC-UV)[43].
Figure 3.1: A flowchart describing parameters of the different hydrolyses performed in this project. This flowchart was also presented in the specialisation project.
3.1 Rest raw material 3 MATERIAL AND METHODS
3.1 Rest raw material
Chicken viscera used in the hydrolyses was transported on ice from Norsk-kylling at Støren on Monday 31 of August. The rest raw material arrived at NTNU Kalvskinnet after approxi- mately an hour. During the next hour the viscera was ground into a homogeneous mass using a meat grinder. Thereafter the ground viscera was placed in bags of 1 kg each. The bags were then stored in a freezer at -24 °C, later that day the bags were moved to a -80 °C freezer at NTNU. When needed for a hydrolysis, the necessary amount of rest raw material was thawed overnight in a cold room at 4 °C. Later on in the semester, another batch with viscera was delivered, and the same procedure was followed. The last batch with rest raw material was used when thermally separating fat from the rest raw material prior to the hydrolysis.
3.2 Inactivating endogenous enzymes
In a separate project performed by Helgeland-Rossavik, the inactivation conditions of the endogenous proteolytic enzymes in chicken viscera was determined to be thermal treatment at 80°C for 10 minutes[44]. To accomplish this, the rest raw material (1000 g) was divided into five 250 ml centrifuge tubes and heated in a microwave. The temperature was kept constant at 80 °C by measuring the temperature every other minute and heating it up if the temperature fell below 80 °C. The inactivated rest raw material was then used in the hydrolysis process described in section 3.4. The hydrolysis mixture obtained after hydrolysis was also inactivated using a microwave. The conditions used for thermal inactivation the hydrolysis mixture were slightly different as they followed another procedure. The method used for inactivation after hydrolysis made by Kathrine Five.
3.3 Thermal separation of fat
To perform the thermal separation, the viscera had to be vacuum packed in bags containing 300gviscera each. Thereafter, the bags were placed in a water bath at 66 °Cfor 15 minutes.
The content of the bags were then transferred into six 250 mL tubes and centrifuged at 9000 xg for 10 minutes (20 °C). The fat phase was then transferred to another container, and stored in a freezer at -80 °C, while the remaining rest raw material was transported to Kalvskinnet for the enzymatic hydrolysis, described in 3.4.
3.4 Hydrolysis of chicken rest raw material
Before the hydrolyses a sample (90 g) of the rest raw material was put aside for lipid, dry matter, ash and protein analysis, from the thawed rest raw material. Pre-treatment was then applied, if a pre-treatment was to be applied.
After this, a sample of rest raw material (300g) was mixed with water at a ratio of 1:1, and then transferred into 250 ml tubes and inactivated in a microwave at 90 °C for 10 min. The sample was named 00. The 00 sample was taken to determine the composition before any
3 MATERIAL AND METHODS 3.4 Hydrolysis of chicken rest raw material
hydrolysis occurred, and to compare the change from 00 to 0 min samples taken after the viscera was added to the reactor. The reactor used in this project could only hold 2 L, which is why the 00 sample was not added into the reactor.
The remaining rest raw material was then used for hydrolysis. Each hydrolysis used 1000 g of chicken viscera and 1000g of pre-heated water (58 °C). These two parts were then mixed 1:1 into a homogeneous solution and then transferred to the reactor. The reactor used for the hydrolysis was a Syrris Atlas Synthesis System. The mixture was stirred at a rotating speed of 100 rpm. The temperature water heating the reactor was heated to 57 °C, the desired temperature of the mixture in the reactor was 55 °C. When the homogeneous solution reached a temperature of 55 °C, a sample was removed from the reactor (600g, sample 0 min). Right after the sample was removed, the commercial enzyme Endocut02 (0.1% of the remaining wet weight of the rest raw material) was added to the reactor. The addition of enzymes marked the beginning of the hydrolysis. Samples (600g) were then taken at 60 min and 120 min. Every sample (00, 0, 60, 120) was inactivated by heating (90 °C) for 15 min. After the inactivation, the samples were transferred into 50mLtubes and centrifuged at 4500xg for 15 minutes (40°C). They were then stored at -80 °C. After the samples were frozen, they were separated into four fractions: fat, hydrolysate, emulsion and sediment. The total wet weight of the fractions was measured and used to calculate the phase yield. Dry matter and the ash content was then determined on the different fractions, this is further described in section 3.5.
All of the samples from the hydrolysate fraction were then freeze dried for further analysis of the composition in these fractions. The process is summarized in Figure 3.2. The hydrolyses analysed in this project was performed during the specialisation project Autumn 2020.
3.5 Dry matter and ash determination 3 MATERIAL AND METHODS
Figure 3.2: Flowchart describing the hydrolysis process for this project step by step.
3.5 Dry matter and ash determination
To determine the dry matter content in the sediment, hydrolysate and emulsion phase, 2 gram of material was weighed out accurately (4 decimals) in triplicates, and placed in ceramic crucibles. The crucibles were then placed in an oven at 105 °Cfor 24 hours, after the 24 hours the crucibles were cooled down in a desiccator. The weight of the crucibles were measured,
3 MATERIAL AND METHODS 3.6 Lipid determination
and the crucibles were placed in an oven at 550 °Covernight. The samples were cooled down, and the final weight was measured[47].
3.6 Lipid determination
Lipid analysis was performed on the protein hydrolysate according to the Bligh and Dyers method[48]. Methanol and chloroform were used as solvents for the extraction. The calcula- tion of the lipid content was performed according to the equations in Appendix 5.2.
3.7 Degree of hydrolysis
The formol titration method was used to determine the degree of hydrolysis of the hy- drolysates. Triplicate samples of hydrolysate (0.5 g) were weighed out in a beaker and 49.5 gram of water was added. The solutions were mixed by using a magnet stirrer until the solution was homogeneous. Thereafter the solutions were pH ( pH =7) adjusted with NaOH (0.1 M), before formaldehyde (10mL) was added. After waiting for 5 minutes the solutions were titrated against 0.1 M NaOH using TITROLINER©7000 (Xylem Analytics, USA). The amount used of NaOH was then used in the calculations of the degree of hydrolysis. This method was based on the method in Tyler (1957)[49].
3.8 Analyses of protein hydrolysates
The different analyses performed on the freeze dried hydrolysate from the different hydrolyses are shown in Figure 3.3.
Figure 3.3: Flowchart describing how the CPH obtained from the different hydrolyses are further analysed after being freeze dried.
3.9 Determination of free amino acids 3 MATERIAL AND METHODS
3.9 Determination of free amino acids
The amount of free amino acids in the hydrolysate was determined according to Osnes &
Mohr[50]. The hydrolysate and rest raw material were analysed using triplicates.
1 mL water soluble protein extract (2%) was mixed with 0.25 mL sulfocyclic acid (10%) in an Eppendorf tube. The solution was then put in a cooling room for 30 minutes, and then they were centrifuged at 11 100xg for 10 minutes in a 415R-centrifuge ( Eppendorf, Ger- many). When there was no more precipitation in the Eppendorf tube, the solution was diluted 1:100 before it was filtrated through a 0.2 µmWHATMAN-filter ( GE Healthcare, UNITED KINGDOM). 0.205mLof the filtrated solution was then transferred in to HPLC-tubes. The HPLC-analysis was performed by department engineer Siri Stavrum using reversed phase liquid chromatography-fi ( SIL-9A Auto Injector, LC-9A Liquid Chromatograph, RF-530 Fluorescence HPLCMonitor, Shimadzu Corporation, Japan).
3.10 Determination of total amino acids
The total amount of amino acids in the hydrolysate and the raw material were determined by using Blackburns method (1978)[51]. Similar to the free amino acid analysis, three parallels were used.
0.1g of hydrolysate was weighed out in flat bottom test tubes for each parallel and 2mLof 6 M HCl was added to the tube. The tubes were then placed in a 105 °Cheating cabinet for 22 hours. Thereafter, the solution in the tubes was neutralized with NaOH(0.1 M and 3M).
The neutralized solution was filtered using a WHATMANGF/C-filter with diameter 25 mm (GE Healthcare, United Kingdom) and a vacuum pump (Heto-Holten A/S, Denmark). 205 µL was then transferred in to HPLC-tubes, and the method described in 3.9 was performed by Siri Stavrum.
3.11 Oil absorption capacity
The method used to determine the OAC was based on the method from Taheri (2011)[17], and a slightly modified version of Lin and Zayas (1987)[52].
Three Eppendorf tubes were weighed and 100 mg of hydrolysate was added in each of the Eppendorf tubes. 1mL vegetable oil was then added to each tube and the Eppendorf tubes were vortexed at maximum speed for 1 minute. The solution was incubated at room tempera- ture for 30 minutes. Thereafter, the Eppendorf tubes were centrifuged (Eppendorf, centrifuge 5415 R) at speed of 13000xG and a temperature of 25 °Cfor 10 minutes. The oil was drained for oil at a 45 degree angle.
3 MATERIAL AND METHODS 3.12 Water holding capacity
3.12 Water holding capacity
Cod fillets were bought, and minced in a food processor. Thereafter, the minced fillets were frozen in portions of 40 g. WHC was determined according to Børresen 1980[53]. Then 2 g of the minced fish material was added into special centrifuge tubes with sample holders with a polyester membrane in the bottom. Centrifugation was performed at 201xG for 5 min in a Sigma 202 centrifuge. The test was performed in quadruplicate. To test if the protein powder influenced the water holding capacity, protein powder was mixed with thawed mince to give a concentration of 3 % (w/w) and the WHC was analysed as described above. The calculation of water holding capacity was calculated according to the equation:
V = V1−∆r
V1 (3.1)
V1 is the percentage of water before centrifugation and∆r is the percentage weight loss due to centrifugation.
3.13 Emulsion capacity and emulsion stability
The procedure to analyse emulsion properties in the hydrolysate was done according to Šližyt˙e procedure from 2009[54]. 0.2 gram of PH was dissolved in 10 mL of ionized water to make a 2% protein extract. Then 4 ml of the protein solutions were mixed with 4 l of rapeseed oil in a VWR 10 ml centrifuge tubes. The mixtures of oil and PH were homogenised at 10 000 RPM using IKA T 10 basic Ultra-Turrax for 90 seconds. The tubes were then centrifuged for 3 min at 2300 xG in an Eppendorf centrifuge 5804R. Determination of the emulsion fraction was carried out by reading the volume of the VWR 10 ml tubes. In order to determine the emulsion stability, the emulsions were left at room temperature for 24 hours. The remaining emulsion after 24 hours was determined after centrifugation at 2300xG for 3 min (McClements, 2004).
The samples were analysed in duplicates.
3.14 C/N analysis
The nitrogen content of the freeze dried hydrolysate from all of the samples was determined using C/N analyser. The samples were accurately weighed out (0.2-1 mg) in small tin contain- ers, in triplicates. The analysis was performed at Biologen by Siv Anina Etter. To calculate the crude protein content from the measured nitrogen content, a factor of 6.25 was used.
3.15 Colourometri
The 120 minutes samples were transferred to weighing boats. The colour of the samples were then measured using KONICA MINOLTA Chroma Meter CR-400. The colorimeter was first calibrated using a standard white calibration plate.
3.16 Statistical analysis 3 MATERIAL AND METHODS
3.16 Statistical analysis
SPSS and Microsoft Excel was used for the statistical analysis of the results in this study.
To find significant differences between the results, student t-test was used. In this study the results were defined as significant if it was outside the 95% confidence level(P<0.05).
Standard sample deviation was used to calculate the standard deviation (SD) in this project.
