Partial substitution of fat with rye bran fibre in Frankfurter sausages – Bridging technological and sensory attributes through inclusion of collagenous protein

Accepted Manuscript Partial substitution of fat with rye bran fibre in Frankfurter sausages – Bridging technological and sensory attributes through inclusion of collagenous protein Line Hjelm, Line Ahm Mielby, Sandra Gregersen, Nina Eggers, Hanne Christine Bertram PII:

S0023-6438(18)31006-5

DOI:

https://doi.org/10.1016/j.lwt.2018.11.055

Reference:

YFSTL 7619

To appear in:

LWT - Food Science and Technology

Received Date: 5 June 2018 Revised Date:

9 November 2018

Accepted Date: 16 November 2018

Please cite this article as: Hjelm, L., Mielby, L.A., Gregersen, S., Eggers, N., Bertram, H.C., Partial substitution of fat with rye bran fibre in Frankfurter sausages – Bridging technological and sensory attributes through inclusion of collagenous protein, LWT - Food Science and Technology (2018), doi: https://doi.org/10.1016/j.lwt.2018.11.055. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Partial substitution of fat with rye bran fibre in Frankfurter sausages – bridging technological and

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sensory attributes through inclusion of collagenous protein

3 Line Hjelm, Line Ahm Mielby, Sandra Gregersen, Nina Eggers, Hanne Christine Bertram*

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Department of Food Science, Aarhus University, Kirstinebjergvej 10, 5792 Årslev, Denmark

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*Corresponding author: Hanne Christine Bertram, email: [email protected]

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Abstract

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This study investigated the effects of rye bran and collagen addition to a Frankfurter-like sausage as

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partial fat replacement. Sensory descriptive analysis revealed that sausages where 6 g/100 g rye bran

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was added alone was associated with an undesired stored flavour in both cooked (P<0.05) and fried

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(P<0.01) sausages. Combining rye bran addition (3-5 g/100 g) with collagen addition (1-3 g/100 g ) had

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significant effects on the textural attributes firmness (P<0.01) and hardness (P<0.05) of both cooked and

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fried sausages. Texture profiling analysis corroborated these findings as collagen inclusion had

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significant effects on texture attributes measured instrumentally. Flavour attributes described as

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spiciness (P<0.05) and fried sausage (P<0.05) were also enhanced in the fried sausages manufactured

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with collagen. NMR T2 relaxation measurements revealed that addition of collagen to the recipe resulted

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in an additional and distinct T2 water population and thus different intrinsic water-protein interactions in

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the protein network when collagen protein was added, whereas the collagen addition did not impact

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the intrinsic microstructure of the sausages when examined by confocal laser scanning microscopy.

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Industrial relevance: Collagen inclusion modified technological and sensory attributes and appears as a

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promising ingredient for development of fat replacement strategies in the manufacturing of processed

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meat.

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Keywords: nuclear magnetic resonance relaxometry; dietary fibre enrichment; processed meat;

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functional meat ingredients; healthier meat products

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1. Introduction Consumers’ demands for healthier foods are increasing and meat industry is therefore facing a massive

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interest in the development of meat products with an improved nutritional profile. One of the concerns

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with processed meat products is that they have a relatively high content of saturated fat. Thus, attempts

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to enhance the nutritional profile of processed meat have largely focused on fat reduction or

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replacement of fat with healthier alternatives including various dietary fibres (Mehta, Ahlawat, Sharma,

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& Dabur, 2015). Peterson, Godard, Eliasson, & Tornberg (2014) investigated the addition of rye bran, oat

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bran, and barley fibres to low-fat sausages and found that fibre addition generally increased frying loss

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and barley while rye bran addition also reduced firmness of the sausages. Thus, fibre addition resulted in

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challenges in relation to technological traits of the final sausages. Studies emphasizing how fibre

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addition influences the sensory attributes of processed meat have also been reported. A study where a

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trained sensory panel evaluated the effect of rye bran and pea fibre addition to meat balls, concluded

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that the addition of rye bran to the meatballs increased the grainy odour, grainy texture and grainy

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flavour while pea fibre addition resulted in a more crumbly, firm and gritty texture (Kehlet, Pagter,

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Aaslyng, & Raben, 2017). Also other studies have reported adverse effects on the sensory and textural

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profile of the fibre-enriched meat products (Garcia-Garcia & Totasaus, 2008; Gibis, Schuh, & Weiss,

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2015). Thus, fibre addition can result in challenges in relation to both technological traits and sensory

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attributes.

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Many processed meat products include ingredients to enhance the functional properties of the product.

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Thus, various salts and phosphates are commonly used as ingredients to improve emulsion formation

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and functionality through solubilisation of myofibrillar proteins. Proteins, especially milk-derived

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proteins, are also frequently included in comminuted meat recipes where they may improve stability by

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enhancing fat and water binding (Youssef & Barbut, 2010). Collagen represents another category of

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proteins that may improve functionality of comminuted meat products. Recent studies have elucidated

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the inclusion of collagen for partial fat replacement in chicken sausages (Schmidt et al., 2016) and in a

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pork-based Frankfurter sausage (Sousa et al., 2017) with promising results. In addition, Ham et al. (2016)

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examined fat replacement with mixtures of collagen and dietary fibres in a fermented sausage product

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with focus on the effects on lipid oxidation and storage stability. However, to the best of our knowledge,

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the use of collagen protein and dietary fibre mixtures for fat replacement in comminuted meat products

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has not been comprehensively studied. Based on the hypothesis that the inclusion of collagenous

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proteins may improve the technological and sensory traits in comminuted meat products subjected to

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fat replacement with dietary fibre, the present study aimed to investigate and characterize the

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technological and sensory attributes of meat products with different mixtures of rye bran and

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collagenous protein as ingredients added for partial fat replacement. For this purpose, a pork-based

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Frankfurter-like sausages including different levels of rye bran (0, 3 and 6 g/100 g) and collagenous

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protein (0, 1 and 3 g/100 g) were examined using a multiple analytical approach including low-field

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nuclear magnetic resonance (NMR) relaxometry, texture profile analysis (TPA), confocal laser scanning

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calorimetry (CLSM) and sensory descriptive analysis to comprehensively describe intrinsic water

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attributes, textural and colour attributes, microstructure as well as sensory attributes. The levels of fiber

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and collagen addition were chosen based on experience from former studies on similar product types

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(Jakobsen, Vuholm, Aaslyng, Sorensen, Raben, & Kehlet, 2014). In addition, a model meat system was

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introduced for mechanistic studies over a wider range of collagen concentrations applied (0-10 g/100 g).

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2.

Materials and Methods

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2.1 Frankfurter sausage formulations

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The ingredients of the Frankfurters are given in Table 1. A control high fat sausage batter without any

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rye bran/collagen added and five different batters with varying rye bran (Skaertoft moelle, Denmark)

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and two types of collagen products (scanpro 1015-1 and scanpro 1015-3, Essentia Protein Solutions,

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Denmark) were prepared. The two collagen types have similar composition but vary in particle size;

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scanpro 1015-1 (fine collagen) has a particle size of <1 mm and scanpro 1015-3 (coarse collagen) has an

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particle size of <3 mm. Rye bran has a total dietary fibre content of 45 g/100 g, and the fibre

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composition of rye bran has previously been found to arabinoxylans (60%), cellulose (17%), Klason lignin

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(10%) and β-glucan (5%) (Nilsson et al., 1997). For the manufacturing, pork and pork fat were minced

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through a 3 mm perforated disc prior to preparation. The six sausage batters were prepared with a bowl

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cutter using a standard procedure for Frankfurters. Casings (22/24 lamb casing) were filled with approx.

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80 g sausage batter in order to reach a final weight of 75 g after heat treatment. Heat treatment was

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carried out in a smoking and cooking cabinet (Doleschal, Wien, Austria) and consisted of the following

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steps: drying at 60 °C for 15 min, smoking at 60 °C for 20 min, heating at 80 °C for 20 min, chilling with

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water sprinkling in 15 minutes. The sausages were stored at 4 °C until further analyses (TPA, NMR

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relaxometry and CLSM) and sensory profiling, which was conducted within one week.

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96 2.2 Differential scanning calorimetry (DSC) of collagen ingredients

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The thermal behaviour of the collagen types was examined by differential scanning calorimetry (DSC).

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Approx. 20-30 mg of the collagen ingredient product was loaded in aluminium pans and analysis was

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performed on a Q 2000 DSC (TA Instruments, UK) by performing a fast cooling to 3 °C followed by

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heating to 90 °C at a rate of 1 °C/min. An empty pan was used as reference. From the resulting

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thermograms, the denaturation point (°C) (minimum of the peak) and enthalpy (J/g) (integral of the

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peak) was determined. The two collagen ingredients were analysed in quintuplicate.

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104 2.3 Studies on model meat systems

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Minced pork (8-10% fat content) and minced pork back fat were purchased from a local meat

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distributor. Seven different formulations of a model meat system was prepared for each collagen

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ingredient as shown in Table 2. Minced pork, fat, ice, NaCl , collagen (either scanpro 1015-1 or scanpro

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1015-3, Essentia Protein Solutions, Denmark) and rye bran (Skaertoft moelle, Denmark) included the

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ingredients added to the model systems. All ingredients were added to a LB20E Waring variable speed

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laboratory blender (Waring Commercial Blender, New Hartford, CT, USA), and blended 2 minutes with a

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speed of 10,000 rpm. During blending, there was 10 s rest in every 30 s interval. The meat emulsion was

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transferred to a 50 mL caped plastic test tube, centrifuged at 5000 rpm for 6 min at 4°C using a Sorvall

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RC 5B Plus Centrifuge (Sorvall Products, LP Newton, CT, USA) to remove air bubbles in the samples.

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Thereafter the samples were heated for 30 min at 80 °C in a water bath (Lauda Ecoline RE 306; Lauda-

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Königshofen, Germany). The samples were cooled at room temperature, and the processing yield

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(water-binding capacity) was determined by weighing. Subsequently, TPA and NMR relaxation

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measurements were performed (section 2.4 and 2.5).

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2.4 Low-field NMR relaxation measurements

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NMR relaxation measurements were performed on a Maran Benchtop Pulsed NMR Analyser (Resonance

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Instruments, Witney, UK) with a magnetic field strength of 0.47 Tesla, and with a corresponding

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resonance frequency for protons of 23.2 MHz. The NMR instrument was equipped with an 18 mm

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variable temperature probe. Transverse relaxation, T2, was measured at 25 °C using the Carr–Purcell–

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Meiboom–Gill sequence. For the model meat systems, T2 measurements were performed with a τ-value

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(time between 90 pulse and 180° pulse) of 500 µs, and for the Frankfurter sausage samples, the

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measurements were performed with a τ-value of 150 µs, and the lengths of the pulses were 8.25 and

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16.5 µs, respectively. Data were acquired from 4096 echoes as 16 scan repetitions. The repetition time

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between two succeeding scans was 2 s, which allowed the longitudinal relaxation to return to

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equilibrium (T1=0.5 s at 25 °C). Upon acquisition, data were phase rotated for phase correction, and only

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even echoes were included in further analyses. The NMR T2 relaxation measurements were carried out

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on three replicates (about 5 g sample material each) from each of the meat model batches produced

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and three replicates of each of the Frankfurter sausages. The obtained T2 relaxation decays were

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analysed by distributed exponential fitting analysis by means of in-house-made scripts in Matlab

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(software release version 6.5, The Mathworks Inc., Natick, MA, USA). This analysis results in a plot of

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relaxation amplitude for individual relaxation populations versus relaxation time. Areas reflecting

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relative proportions and mean values of the T2 relaxation populations found were calculated.

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2.5 Texture profile analysis (TPA)

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Instrumental texture profile analysis (TPA) of the samples was carried at RT using a Brookfield CT3

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texture analyzer (Brookfield Engineering Laboratories, INC. Middleboro, Massachusetts, USA) with a load

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cell of 25 kg. Three cylinders (approximate thickness of 2 cm and a diameter of 3 cm) per recipe/batch

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were used to evaluate the texture. The samples were axially compressed into two consecutive cycles of

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50% compression with a 40 mm square probe. The instrument settings were as follows: pre-test speed

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of 120 mm/min, test speed of 300 mm/min, posttest speed of 300 mm/min, trigger force of 5 g and hold

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time after downward movement of the probe of 2 s. TPA curves were constructed by the Texture Pro CT

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V1.6 software provided by the Brookfield CT3 texture analyzer (Brookfield Engineering Laboratories,

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INC. Middleboro, Massachusetts, USA). The parameters hardness (g), fracturability (g), springiness (mm),

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resilience, cohesiveness, and chewiness (g∗cm) were calculated.

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2.6 Confocal laser scanning microscopy (CLSM)

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The microstructure of thin slices of the Frankfurter sausages (two from each recipe) cut out with a

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scalpel, was studied by confocal laser scanning microscopy (CLSM). Nile red and FITC were used as

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florescence strain agents for liquid fat and proteins, respectively. Both Nile red and FITC were dissolved

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in acetone to a final concentration of 0.01% (w/w). A small droplet of the dye solutions were placed on a

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glass slide, allowing the acetone to evaporate before adding the sample. Analysis of the microstructure

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was performed after approx. 20 min with a Nikon C2 confocal laser scanning microscope (Nikon

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Instrument Inc, Tokyo, Japan) using a 10x objective. Laser lines of 488 nm and 561 nm were used for

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excitation to induce fluorescence emission.

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159 2.7 Sensory descriptive analysis

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Two descriptive analyses, one on cooked sausages and one on fried sausages were conducted according

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to “generic sensory descriptive analysis” (Murray, Delahunty, & Baxter, 2001).The sensory descriptive

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analyses were for both the cooked and the fried sausages conducted on all recipes except the high fat

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control recipe. The reason for excluding the high fat control sausage was that these sausages were, from

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a sensory perspective, markedly different from the remaining recipes. The procedure included the

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following steps: 1) panel selection and sample preparation, 2) vocabulary development and training, and

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3) evaluation similar to Mielby, Kildegaard, Gabrielsen, Edelenbos, & Thybo (2012). The panel consisted

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of 9 assessors (all women, age 21–60 years). In terms of sample preparation, the cooked sausages were

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heated at 75-80 °C in water for 10 min, and the fried sausages were cooked on a pan for 40 min at 150

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°C and turned every 5th min. All sausage samples were continuously distributed in plastic containers

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suited to contain warm food materials and closed with a lid prior to being distributed to the panelists in

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order to keep them warm and contain the volatiles. One sample serving corresponded to 1/3 sausage

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omitting the ends. Prior to evaluation of the sausages the panel went through a vocabulary

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development session and a training session on two separate days. During the vocabulary development

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and training sessions the panel was presented with the two sausage recipes with 6 g/100 g rye bran and

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3 g/100 g rye bran/3 g/100 g coarse collagen samples and developed and agreed on a list of sensory

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attributes (including a definition of the attributes), that could describe and discriminate the sausages.

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Further, they trained and agreed on scale use. The final vocabulary and keywords for interpretation of

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attributes can be found in Table 3. The sensory profiling of the sausages was carried out in one session.

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The sausages were evaluated in triplicate according to a block design with two blocks of eight and seven

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sausages, respectively, with small breaks in between. The sausages were presented in a balanced order

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to account for sample order and carry-over effects (Macfie & Bratchell,1989). The panel evaluated the

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sausages on 15 cm unstructured continuous scales (Compusense Inc., Guelph, Canada). To cleanse

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palates, water, weak lukewarm white tea and thin crackers were used between samples. The sensory

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evaluation was performed in a sensory evaluation laboratory compliant with international standards

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(ASTM, 1986).

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2.8 Statistical analyses

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The descriptive sensory data were analysed using a three-way mixed model ANOVA including sausage

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recipe and cooking method (cooked versus fried) as the fixed effects and with assessor/panellist as a

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random effect by using the PanelCheck software (V1.4.2, Nofima Mat, Ås, Norway). Least squares

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difference (LSD) values were used to determine significance (p < 0.05). Principal Component Analyses

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(PCA) were performed using the software LatentiX (Version 2.13, LatentiX Aps, Frederikberg, Denmark).

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The PCA models for the sensory analyses and the distributed T2 relaxation data from the model system

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were mean centred prior to the analyses.

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Data from the low field NMR populations and texture analyses were analysed using the program RStudio

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(Version 1.1.419, RStudio Inc., Boston, MA, USA). A two-way ANOVA model was performed that included

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the effects of recipe, cooking time and the interaction between recipe and cooking time. DSC results

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were analysed using one-way ANOVA. For parameters with significant differences (p < 0.05) the package

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‘agricolae’ (Mendiburu, 2017) was used to perform a Tukey multiple comparison, which also operated

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with a significance level of p < 0.05, whereas graphs were made in the RStudio software using the

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package ‘ggplot2’ (Wickham and Chang, 2016). Pearson correlation analyses were also performed in

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RStudio using the package ‘Hmisc’ (Harrell, 2018).

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3.1 DSC characterization of collagen ingredients

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DSC thermograms obtained on the two collagen ingredients are displayed in Figure 1. Two peaks were

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visible in the thermograms. The first peak, appearing in the range between 20 to 30 °C, was assigned to

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melting of fat present in the collagen ingredients. The second peak was assigned to the denaturation of

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the proteins. Integral analyses of this peak resulted in a denaturation temperature of 54.2 ± 1.6 °C for

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the fine collagen and a denaturation temperature of 55.1 ± 0.6 °C for the coarse collagen. A one-way

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ANOVA showed no significant difference between these temperatures. The enthalpy of the protein

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denaturation was found to be 0.8 ± 0.1 J/g for the fine collagen, and 1.5 ± 0.2 J/g for the coarse collagen,

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which was significantly different (P< 0.001).

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Results

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215 3.2 Sensory profiling of Frankfurter sausages

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To explore the differences in the overall sensory properties according to inclusion of varying levels of rye

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bran and collagen, a PCA was performed on the sensory data obtained for the five Frankfurter sausages

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(Figure 2). Both for cooked and fried sausages, the sausages with rye bran only (RB) were associated

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with a stale flavour. Replacing some of the rye bran with collagen enhanced the textural attributes

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firmness and hardness of the sausages. Flavour attributes including spiciness, and fried sausage were

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also enhanced in sausages with collagen. Effects of collagen type was also observed; coarse collagen

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enhanced juiciness most, and fine collagen had higher impact on firmness and hardness (Figure 2).

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Univariate statistical analyses confirmed significant effects of collagen addition on both textural, taste

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and mouthfeel attributes (Table 4).

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3.3 Microscopic and functional properties of Frankfurter sausages

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The microstructure of the Frankfurter sausages was visualized by application of CLSM (Figure 3).

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Independent of recipe, the sausages exhibited an amorphous and inhomogeneous protein network with

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fat integrated. An effect of collagen addition to the recipes was not apparent from the CLSM images.

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Distributed exponential fitting analysis of the T2 relaxation decay data was performed in order to

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investigate how the water was distributed in the sausages and impacted by collagen inclusion (Figure 4).

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Distributed analysis of the T2 relaxation decay data revealed the presence of two partly overlapping

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relaxation populations in the region between 0 and 10 ms, a major water population centred around 50

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ms, and a more slow relaxing T2 population in the region between 300 and 700 ms. These results are

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consistent with previous work where the relaxation population populations in the region between 0 and

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10 ms (T2B) have been assigned to water closely associated with macromolecules, the major population

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(T21) has been assigned to water located intrinsically in the myofibrillar protein structures, and the slow-

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relaxing population (T22) has been assigned to water outside the highly dense protein network

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constituted by the myofibrillar proteins (Bertram et al., 2001; Andersen, Andersen, & Bertram, 2007;

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Møller et al., 2011). Intriguingly, inclusion of collagen was clearly evident in the distributed T2 relaxation

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data as it gave rise to the manifestation of an additional T2 population characterized by a relaxation time

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in the region between 150 and 400 ms and appearing as a shoulder on the myofibrillar water

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population, T21.

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Textural attributes of the sausages were determined by texture profiling analysis (Figure 5). A significant

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effect of recipe on the parameters hardness (P<0.0001), fracturability (P<0.0001), resilience (P<0.0001),

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cohesiveness (P<0.0001), and chewiness (P=0.001) was observed. Collagen inclusion in the sausages incr

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eased hardness and fracturability. For resilience, cohesiveness and chewiness, a reduction was observed

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with increasing content of the fine collagen type. For the coarse collagen type, the effect was dependent

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on cooking time; a reduction in resilience, cohesiveness and chewiness was observed with increasing co

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arse collagen content when sausages were cooked for 10 min whereas an increase in resilience, cohesiv

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eness and chewiness was observed with increasing collagen content when sausages were cooked for 60

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min (Figure 5).

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3.4 Model meat systems

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In order to elucidate the effect of collagen addition to a meat emulsion system over a larger

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concentration range, a meat model system was introduced where collagen and rye bran were added in

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varying levels from 0 to 10 g/100 g . Intrinsic water distribution and water-ingredient interactions were

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studied by low-field NMR relaxometry, and Figure 6 shows results from PCA of the distributed T2

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relaxation data. A strong effect of collagen concentration is evident along principal component 1.

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Differences in the distributed T2 relaxation data as function of collagen type (coarse versus fine) appears

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to be explained by principal component 2. Inspection of the distributed T2 relaxation data revealed that

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the differences in the distributed T2 relaxation data as function of collagen type could be attributed to a

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longer T2 relaxation time for the most slow-relaxing T2 population for model systems containing coarse

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collagen as ingredient as compared with fine collagen (Figure 7a). To further elucidate the specific effect

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of collagen inclusion on the distributed T2 relaxation data, the area of this slow-relaxing T2 population

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characterized by a relaxation time in the region between 150 and 400 ms was determined. A correlation

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analysis revealed a strong and significant linear correlation between collagen concentration and area of

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this T2 population with a relaxation time in the region between 150 and 400 ms (R=0.99, P<0.001)

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(Figure 7b).

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Textural attributes of the model meat systems were determined after heat treatment for 30 min,

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respectively. Similar as for the Frankfurter sausages significant effects of recipe on hardness,

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fracturability, resilience and cohesiveness were observed. Hardness, fracturability and resilience

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generally increased with increasing levels of collagen inclusion (data not shown). Correlation analyses

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were performed between textural attributes and NMR T2 relaxation parameters (Table 5). For the

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textural attributes cohesiveness, springiness and chewiness, no correlations were found to the area of

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slow-relaxing NMR T2 population. However, for the textural attributes hardness, fracturability and

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resilience positive correlations were seen, and for resilience a significant correlation was found to the

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area of the slow-relaxing T2 population characterized by a relaxation time in the region between 150 and

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400 ms (Table 5).

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4 Discussion

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Recently the urge for developing and introducing healthier meat products has been intensified. For this

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purpose a common approach has been to partially replace fat in comminuted meat products with

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healthier alternative ingredients. Thus, the inclusion of a variety of dietary fibre sources such as oat

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fibre, cashew apple fibre, carrot fibre and rye bran has been reported to mention few examples (Møller

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et al., 2011; Peterson et al., 2014; Guedes-Oliveira, Salgado, Costa-Lima, Guedes-Oliveira, & Conte-

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Junior, 2016; Kehlet et al., 2017). However, replacing fat in a comminuted meat product is extremely

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challenging and poses difficulties in terms of appearance, flavor, and texture (Weiss, Gibis, Schuh, &

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Salminen, 2010). This was also evident in the present study where sensory descriptive analysis revealed

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that addition of 6% rye bran to a Frankfurter-like sausage resulted in the development of an undesired

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flavour characterized as a stale flavour. The development of this stale flavour occurred in both boiled

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and fried sausages. Several other studies have also reported that inclusion of dietary fibres in a meat

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product may lead to the introduction of flavour changes (Pinero et al., 2008; Sanchez-Zapata et al., 2010),

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and Kehlet et al. (2017) reported that inclusion of rye bran in meat balls resulted in a grainy flavour.

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Intriguingly, in the present study replacement of 1 or 3 g/100 g of the rye bran with collagen in the

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sausages resulted in a significant reduction in the stale flavour and significantly enhanced the attributes

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sausage taste, spiciness and mouthfeel (Figure 2, Table 3). These findings are consistent with results

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reported by Sousa et al. (2017) who found that Frankfurter-sausages undergoing replacement of fat

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with collagen up to 75% received higher taste evaluation during sensory analysis than their full-fat

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counterparts. This flavour-enhancing effect can likely be attributed to the fact that the collagen

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ingredient used is derived from pork skin and thus also contains compounds that act as substrates to

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generate a typical meaty flavour.

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Texture of meat products is a vital attribute in terms of consumer acceptability. In comminuted meat

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products, fat also contributes to how texture is perceived, and thus formulating a fat-reduced product

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with texture attributes equal to its full-fat counterpart is challenging. We therefore investigated how

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inclusion of collagen in the manufacturing of frankfurter-like sausages could facilitate partial fat

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replacement. Sensory descriptive analysis revealed significant effects of collagen on texture of the

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product as collagen inclusion increased the perception of hardness and firmness (Table 4). Texture

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profile analysis confirmed significant effects of collagen inclusion on several texture attributes in both

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sausages (Figure 4) and model systems (data not shown). Thus, the inclusion of collagen supported the

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development of an attractive texture of the Frankfurter-like sausage product, which has also been

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reported previously (Sousa et al., 2017). In the vivo situation, one of the physiological functions of

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collagen as a structural component is to provide elasticity and extensibility, which corroborates that

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collagen influences the textural attribute resilience when used an ingredient in a meat product.

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possesses to create a network and bind water is anticipated to be advantageous in fat replacement

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(Brewer, 2012). In the present study, microscopic examinations with CLSM did not reveal any impact of

322

collagen addition on the microstructure of the Frankfurter sausages, implying a very efficient

323

assimilation into the meat protein network formed during emulsification (Figure 3). While it has been

324

found that collagen inclusion holds a potential to improve water-binding capacity (Sousa et al., 2017), to

325

the best of our knowledge, no previous studies have elucidated how inclusion of collagen as an

326

ingredient in comminuted meat influences the intrinsic water mobility and distribution. Low-field NMR

327

relaxometry is a useful tool for exploring intrinsic water characteristics. Transverse relaxation, T2, will

328

depict the local environment that the water molecule experience, which is influenced by structural

329

constraints within the matrix and interactions with inherent macromolecules acting as relaxation sinks

330

(Bertram, Purslow, & Andersen, 2002). Intriguingly, distributed exponential fitting analysis of T2

331

relaxation data on the Frankfurter-like sausages revealed a pronounced effect of collagen on the

332

intrinsic water mobility and distribution as the inclusion of collagen resulted in the presence of a distinct

333

water population located in the range between 150 and 400 ms (Figure 4). The complementary studies

334

on a model system where collagen concentration was varied in the range between 0 and 10% (w/w)

335

confirmed a strong linear correlation between collagen concentration and size of this T2 population

336

(Figure 7b). Thus, the T2 relaxation data revealed differences in the water-protein interactions for

337

collagen and myofibrillar proteins, respectively. Intrinsic myowater constituted in myofibrillar protein

338

structures is characterized by a T2 relaxation time in the range between 30 and 100 ms, and thus

339

appears to be more constrained than the water confined in the collagenous structures that are

340

introduced to the meat system with inclusion of collagen. Correlation analysis revealed a strong and

341

significant correlation (R=0.62-0.70) between the collagen-associated slow-relaxing T2 relaxation

342

population located in the range between 150 and 400 ms and the textural attribute resilience (Table 5).

343

This finding emphasizes that unique water-protein interactions created from collagens helix structures

344

contribute to the effects that collagen exerts on product texture.

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Understanding ingredients’ functional properties are important in the development of meat product

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reformulation strategies. Collagen-rich ingredients are commonly extracted from animal skin and hides

348

in a process that involves defattening, drying and a mechanical milling that results in a pulverization that

349

can be adjusted to determine the particle size of the final product. Maximo & Cunha (2010) have

350

formerly investigated the rheological properties of intact collagen fibre and powdered collagen,

351

respectively and found that gel network strength increased with decreasing particle size. In the present

352

study two different collagen-rich ingredients that were identical in composition but varying in particle

353

size were investigated. Sensory analysis revealed that particle size influenced how homogenous the

354

sausages appeared; the larger particle size resulted in a less homogenous appearance of the sausages

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than the fine particle sized collagen. In addition, particle size of the collagen ingredient also impacted

356

how juicy the sausages was evaluated by the sensory panel as inclusion of the coarse collagen resulted

357

in a higher juiciness evaluation than the collagen ingredient with the lower particle size. In the model

358

systems PCA of the low-field NMR relaxometry data also revealed an effect of collagen particle size on

359

the intrinsic water mobility and distribution (Figure 6). Inspection of distributed T2 relaxation times

360

revealed that the collagen particle size influenced the relaxation time of the slow-relaxing population

361

around 150-400 ms accompanying collagen addition; the coarse collagen ingredient resulted in a longer

362

T2 relaxation time for this water population compared with the fine collagen ingredient (Figure 7a). A

363

possible explanation is that the finer collagen particles with a smaller diameter creates a more dense

364

network and thus gives rise to a system comprised of smaller pore sizes than the coarse collagen

365

particles characterized by a larger diameter, where the larger particles could be anticipated to result in a

366

less dense packing with larger pore sizes. This interpretation would be in association with the

367

Brownstein-Tarr model proposing that the T2 relaxation rate will be proportional with surface-to-volume

368

ration of the structures that confines the water protons (Brownstein & Tarr, 1979). This relation has

369

previously been demonstrated to apply to muscle-based structures (Bertram et al., 2002). DSC analyses

370

of the two collagen ingredients revealed identical denaturation temperatures but higher enthalpy values

371

for the coarse collagen product than the fine collagen product. This finding can possibly be ascribed to

372

stronger electrostatic interactions that may result in that additional energy is required to denature

373

collagen in the coarse particle size. Such a higher resistance to temperature-induced structural changes

374

for the collagen ingredient with a coarse particle size may explain the different behaviours observed for

375

the textural attributes resilience, cohesiveness and chewiness after heat treatment for 10 and 60 min,

376

respectively (Figure 5). Thus, the findings corroborate that the coarse collagen ingredient can be

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anticipated to be more resistant to structural changes during a 60 min heat exposure as compared with

378

the fine collagen ingredient.

379 380 Conclusions

382

The present study showed that the technological and sensory attributes of Frankfurter-like sausages

383

could be modified by inclusion of different ratios of rye bran and collagenous protein as ingredients

384

added for partial fat replacement. Both texture profiling and sensory descriptive analyses revealed that

385

collagen inclusion resulted in a firmer texture of the sausages. NMR T2 relaxation measurements

386

revealed a strong and distinct effect of collagen inclusion on intrinsic water mobility and distribution

387

that likely can explain that sausages manufactured with collagen obtained different textural attributes.

388

Sausages manufactured with rye bran alone resulted in a stale flavour, which was reduced with inclusion

389

of a collagen-rich ingredient. Inclusion of collagen in the recipe resulted in high evaluations of the

390

flavour attributes including spiciness and fried sausage flavour. Collectively the present study points at

391

collagen protein as a promising ingredient to preserve attractive technological and sensory attributes

392

during fat replacement in the manufacturing of processed meat products.

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Acknowledgement

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The authors wish to thank Ms. Rita Cold, Mr. Per Madsen and Mr. Joan Planiol, Essentia Protein

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Solutions A/S for manufacturing of the sausages and for valuable discussions on the project. In addition,

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Skærtoft Mølle is thanked for providing the rye bran used for the project.

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Bertram, H.C., Purslow, P.P., & Andersen, H.J. (2002). Relationship between Meat Structure, Water

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Guedes-Oliveira, J.M., Salgado, R. L., Costa-Lima, B. R. C., Guedes-Oliveira, J., & Conte-Junior, C. A.

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characteristics and consumer liking of sausages with 10% fat and added rye or wheat bran. Food Science

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Kehlet, U., Pagter, M., Aaslyng, M. D., & Raben, A. (2017). Meatballs with 3% and 6% dietary fibre from

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Maximo, G.J., & Cunha, R.L. (2010). Mechanical properties of collagen fiber and powder gels. Journal of

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Mehta, N., Ahlawat, S. S., Sharma, D. P., & Dabur, R. S. (2015). Novel trends in development of dietary

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Araújo, I.B.S. (2017). Quality parameters of frankfurter-type sausages with partial

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https://github.com/tidyverse/ggplot2.

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Figure captions

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Figure 1. DSC thermograms for the two collagen ingredients. Black lines represent data for the coarse

476

collagen type (particle size <3 mm) (n=5) and dotted lines represent data for the fine collagen type

477

(particle size <1 mm).

478

Figure 2. Bi-plots from principal component analysis (PCA) of sensory profiling data for (A) cooked

479

sausages, and (B) fried sausages using a trained sensory panel (n=9). The bi-plots presents the sausages

480

with 6 g/100 g rye bran (RB), 5 g/100 g rye bran/1 g/100 g fine collagen (CF_1), 3 g/100 g rye bran/3

481

g/100 g fine collagen (CF_3), 5 g/100 g rye bran/1 g/100 g coarse collagen (CC_1) and 3 g/100 g rye

482

bran/3 g/100 g coarse collagen (CC_3).

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Figure 3. Representative confocal laser scanning microscopy images. For each recipe three images

484

obtained at different places in the same sausage are visualized. (A) High fat control sausage (HF), (B)

485

sausage with 6 g/100 g rye bran (RB), (C) sausage with 5 g/100 g rye bran and 1 g/100 g fine collagen

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(CF_1), (D) sausage with 5 g/100 g rye bran and 1 g/100 g coarse collagen (CC_1), (E) sausage with 3

487

g/100 g rye bran and 3 g/100 g fine collagen (CF_3), and (F) sausage with 3 g/100 g rye bran and 3 g/100

488

g coarse collagen (CC_3). Red areas represent liquid fat and the green areas represent protein.

489

Figure 4. Distributed NMR T2 relaxation times measured on sausage samples cooked in a water bath at

490

80 °C for 10 min. The black curve represents the high fat control sausage, dotted curve represents recipe

491

with 6 g/100g rye bran, green curve represents recipe with 5 g/100 g rye bran and 1 g/100 g fine

492

collagen, and dotted green curve represents recipe with 3 g/100 g rye bran and 3 g/100 g fine collagen.

493

Figure 5. Means of textural attributes obtained from texture profile analysis of high fat control sausages

494

(HF) and sausages with 6 g/100 g rye bran (RB), 5 g/100 g rye bran/1 g/100 g fine collagen (CF_1), 3

495

g/100 g rye bran/3 g/100 g fine collagen (CF_3), 5 g/100 g rye bran/1 g/100 g coarse collagen (CC_1) and

496

3 g/100 g rye bran/3 g/100 g coarse collagen (CC_3). Cooking refer to minutes of heat-treatment in a

497

water bath at 80 ° C prior to the analyses, which was performed after tempering of the samples at room

498

temperature (23 ° C).

499

Figure 6. Principal component analysis (PCA) score plots of distributed NMR T2 relaxation data obtained

500

for model meat system with varying levels of rye bran and collagen added and cooked in water bath at

501

80 °C for 30 min. Fine and Coarse refer to the type of collagen used as ingredient. C0/R10: 0 g/100 g

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collagen and 10 g/100 g rye bran, C2/R8: 2 g/100 g collagen and 8 g/100 g rye bran, C4/R6: 4 g/100 g

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collagen and 6 g/100 g rye bran, C6/R4: 6 g/100 g collagen and 4 g/100 g rye bran, C8/R2: 8 g/100 g

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collagen and 2 g/100 g rye bran, and C10/R0: 10 g/100 g collagen and 0 g/100 g rye bran.

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Figure 7.

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(A) Distributed NMR T2 relaxation times measured in samples of meat model system with 6 g/100 g

507

collagen and 4 g/100 g rye bran added after heat treatment for 30 min in a water bath at 80 °C. The

508

black curve represents the fine collagen ingredient (scanpro 1015-1) and the dotted green curve

509

rrepsents the coarse collagen ingredient (scanpro 1015-3). (B) Correlation between amount of collagen

510

added (% w/w) to a meat model system and NMR T22 area, which represents a T2 population with a

511

relaxation time in the region 150-400 ms. Black symbols represent data from samples added fine

512

collagen (particle size <1 mm) and green symbols represent data from samples added coarse collagen

513

(particle size <3 mm). For both collagen types R2=0.99***

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Table 1. Ingredients (% w/w) of the Frankfurter sausage recipes. High-fat Control (HF)

6g/100g rye bran (RB)

5 g/100g rye bran/1 g/100g fine collagen (CF_1)

3 g/100g rye bran/3 g/100g fine collagen (CF_3)

5 g/100g rye bran/1 g/100g coarse collagen (CC_1)

3 g/100g rye bran/3 g/100g coarse collagen (CC_3)

60.0

60.0

5.0

5.0

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Recipe

51.0

60.0

60.0

60.0

Pork fat

19.0

5.0

5.0

5.0

Rye bran

-

6.0

5.0

3.0

5.0

3.0

Water/ice

26.35

25.35

25.35

25.35

25.35

25.35

Nitrite salt (0.6 % nitrite)

1.5

1.5

1.5

1.5

1.5

1.5

Spice mixture

1.2

1.2

1.2

1.2

1.2

1.2

Ascorbic acid

0.05

0.05

0.05

0.05

0.05

0.05

Water bouillon

0.6

Phosphate (E451)

0.3

Fine collagen (ScanPro 1015/1)

-

Coarse collagen (ScanPro 1015/3) Total

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Pork meat (5 % fat)

0.6

0.6

0.6

0.6

0.3

0.3

0.3

0.3

0.3

-

1.0

3.0

-

-

-

-

-

-

1.0

3.0

100.0 %

100.0 %

100.0 %

100.0 %

100.0 %

100.0 %

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10 g/100g rye bran (C0/R10)

120.0

120.0

30.0

10.0

Rye bran (g)

-

Collagen (g)

-

Salt (g)

3.0

Total (g)

200

2 g/100g collagen/ 8 g/100g rye bran (C2/R8)

4 g/100g collagen/ 6 g/100g rye bran (C4/R6)

6 g/100g collagen/ 4 g/100g rye bran (C6/R4)

8 g/100g collagen/ 2 g/100g rye bran (C8/R2)

10 g/100g collagen (C10/R0)

RI PT

High fat control (HF)

120.0

120.0

120.0

120.0

10.0

10.0

10.0

10.0

10.0

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120.0

47.0

47.0

47.0

47.0

47.0

47.0

20.0

16.0

12.0

8.0

4.0

-

-

4.0

8.0

12.0

16.0

20.0

3.0

3.0

3.0

3.0

3.0

3.0

200

200

200

200

200

200

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Table 2. Formulations for meat model systems. The 7 formulations were prepared with both the fine (scanpro 1015-1) and the coarse (scanpro 1015-3) collagen ingredients in separate experiments. Ingredient/Reci pe Minced pork meat (10% fat) (g) Minced pork back fat (g) Ice water (g)

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Table 3. Attributes developed for the descriptive sensory analysis. The attributes are divided in 4 different categories; Aroma and Appearance (AA), Texture (T), Taste and Flavour (TF), and Mouth Feel (MF). Description*

Scale anchors*

Cooked/Fried Sausage (AA)

Intensity of cooked/fried sausage aroma – the cooked attribute for the cooked samples and the fried attribute for the fried samples

Absent/Very intense

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Attribute

Homogeneous (AA)

Degree of uniformity at the ends of the cut sample

Low/High

Crispiness (T)

The intensity of the sound made when biting through the sample using the incisors

Absent/Very intense

Hardness (T)

Force necessary to bit through the sample** (using the incisors)

Low/High

Firmness (T)

Firmness perceived during

Low/High

ACCEPTED MANUSCRIPT

chewing (using the molars) Perceived water content during chewing*

Low/High

Saltiness (TF)

Intensity of salty basic taste

Absent/Very intense

Cooked/Fried Sausage (TF)

Intensity of cooked/fried flavour – the cooked attribute for the cooked samples and the fried attribute for the fried samples

Absent/Very intense

Stored (TF)

Intensity of stored flavour

Absent/Very intense

Spiciness (TF)

Intensity of spiciness*

Absent/Very intense

Piquantness (MF)*

Intensity of stinging sensation in mouth and throat – the one that ‘follows you home’

Absent/Very intense

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Juiciness (T)

* = inspiration from Tomaschunas et al., 2013; ** = inspiration from Guàrdia et al., 2008.

6 g/100g rye bran (RB)

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Cooked sausages

5 g/100g rye bran/1 g/100g fine collagen (CF_1)

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Table 4. Mean intensity ratings of the sensory attributes for cooked and fried sausages 3 g/100g rye bran/3 g/100g fine collagen (CF_3)

5 g/100g rye bran/1 g/100g coarse collagen (CC_1)

3 g/100g rye bran/3 g/100g coarse collagen (CC_3)

P-value

Cooked sausage (AA)

5.5 ± 5.3

7.7 ± 4.9

7.8 ± 4.8

6.9 ± 5.1

8.0 ± 4.8

-

Homogeneous (AA)*

8.6 ± 4.6bc

9.7 ± 4.0c

9.6 ± 3.38bc

6.4 ± 4.1ab

4.5 ± 3.6a

P < 0.05

Crispiness (T)*

6.9 ± 4.8a

7.8 ± 4.4ab

10.0 ± 4.1b

8.3 ± 4.7ab

8.6 ± 4.5ab

P < 0.05

Hardness (T)*

7.5 ± 4.8ab

9.4 ± 3.9b

7.2 ± 5.0ab

7.3 ± 4.4ab

6.2 ± 4.4a

P < 0.05

ACCEPTED MANUSCRIPT

4.9 ± 3.8a

10.0 ± 3.5c

9.7 ± 4.3bc

6.9 ± 4.3ab

6.5 ± 4.2a

P < 0.01

Juiciness (T)

11.5 ± 2.9

10.1 ± 3.7

9.3 ± 4.5

10.3 ± 4.1

10.8 ± 2.9

-

Saltiness (TF)

7.8 ± 5.0

6.6 ± 4.0

7.1 ± 4.6

7.2 ± 4.6

7.3 ± 4.4

-

Cooked Sausage (TF)

6.3 ± 4.9

7.5 ± 4.1

7.8 ± 4.6

7.1 ± 4.9

8.4 ± 4.3

-

Stored (TF)*

9.8 ± 4.8b

6.2 ± 4.3a

5.5 ± 4.4a

5.9 ± 5.2a

Spiciness (TF)

6.1 ± 5.1

6.9 ± 4.7

6.5 ± 4.7

6.6 ± 5.1

Piquantness (MF)

5.3 ± 4.2

6.6 ± 4.3

6.6 ± 4.6

6.4 ± 4.6

Fried Sausage (AA)

6.7 ± 5.0

6.5 ± 4.4

7.2 ± 4.8

8.7 ± 4.8

7.4 ± 4.3

-

Homogeneous (AA)*

10.3 ± 4.0b

9.4 ± 4.6b

9.5 ± 4.5b

5.8 ± 4.5a

4.7 ± 3.6a

P < 0.01

Crispiness (T)

7.9 ± 5.2

7.8 ± 4.4

6.7 ± 5.0

7.6 ± 4.9

10.3 ± 4.3

-

Hardness (T)*

7.7 ± 5.0ab

8.7 ± 4.2ab

10.5 ± 4.3b

8.2 ± 4.8ab

5.4 ± 4.4a

P < 0.05

Firmness (T)*

4.8 ± 4.0a

Juiciness (T)*

9.8 ± 4.2bc

Saltiness (TF)

7.0 ± 5.0

Fried Sausage (TF)*

4.9 ± 4.5a

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4.8 ± 4.6a

P < 0.05

7.5 ± 4.6

-

6.8 ± 5.0

-

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Fried sausages

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Firmness (T)*

10.4 ± 4.3b

10.2 ± 4.4b

7.4 ± 5.0ab

P < 0.01

7.6 ± 5.0ab

6.6 ± 4.9a

11.0 ± 3.4c

9.0 ± 4.6abc

P < 0.01

8.3 ± 4.9

8.4 ± 4.7

9.2 ± 4.7

10.0 ± 4.1

-

6.6 ± 4.5ab

7.5 ± 4.3ab

8.9 ± 4.7b

8.6 ± 4.7b

P < 0.05

EP

9.4 ± 4.0b

10.6 ± 5.0b

7.1 ± 5.0a

5.4 ± 4.8a

4.2 ± 4.5a

4.6 ± 4.9a

P < 0.01

Spiciness (TF)*

5.3 ± 4.8a

7.9 ± 4.9ab

8.9 ± 5.0b

8.9 ± 4.3b

9.3 ± 3.8b

P < 0.05

Piquantness (MF)*

4.8 ± 4.4a

7.5 ± 5.0a

8.2 ± 4.7b

7.7 ± 4.6a

9.3 ± 4.3b

P < 0.05

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Stored (TF)*

(AA)=appearance, (T)=texture, (TF)=taste and flavour, (MF)=mouthfeel. Different letters in the same row indicate significant differences (P<0.05).

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Table 5. Pearson correlation coefficients (R) for correlations between textural attributes and area of the slow-relaxing NMR T2 population characterized by a relaxation time in the region between 150 and 400 ms.

0.38 0.53

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Fine collagen Coarse collagen

Fracturability Resilience (N)

EP

Hardness (N)

Cohesiveness

Springiness (mm)

Chewiness (N*cm)

0.45

0.62*

-0.35

0.27

0.32

0.51

0.70*

0.2

-0.04

0.24

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Figure 1. DSC thermograms for the two collagen ingredients. Black lines represent data for the coarse collagen type (particle size <3 mm) (n=5) and dotted lines represent data for the fine collagen type (particle size <1 mm).

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A 2 CC_3

Juiciness (T)

-2.5

-2

-1.5

-1

RB

-0.5

Stale (TF)

0.5

CC_1

Liking_Panel Cooked sausage (TF) (AA) Spiciness (TF) Cooked sausage Piquantness (MF) Crispiness (T) Saltiness (TF) 0 0 0.5 1 1.5 2 -0.5 CF_3 Hardness (T) Firmness (T) -1 CF_1

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PC#2 (40.1 %)

1

RI PT

1.5

-1.5

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Homogeneous (AA)

-2

-2.5

PC#1 (52.6 %)

B

2

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CC_3 Juiciness (T) Crispiness (T)

1 0.5

Stale (TF)

-2

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-2.5

EP

PC#2 (28.3%)

RB

-3

1.5

-1.5

-1

0 -0.5

0

CC_1 Saltiness (TF) Fried Sausage (AA) Fried Sausage (TF) Piquantness (MF) 0.5 1 Spiciness1.5 (TF)

-0.5

Homogeneous (AA)

Liking_Panel

CF_1 -1 Firmness (T)

Hardness (T) -1.5 CF_3

PC#1 (61.0 %)

-2

Figure 2. Bi-plots from principal component analysis (PCA) of sensory profiling data for (A) cooked sausages, and (B) fried sausages using a trained sensory panel (n=9). The bi-plots presents the sausages with 6 g/100 g rye bran (RB), 5 g/100 g rye bran/1 g/100 g fine collagen (CF_1), 3 g/100 g rye bran/3 g/100 g fine collagen (CF_3), 5 g/100 g rye bran/1 g/100 g coarse collagen (CC_1) and 3 g/100 g rye bran/3 g/100 g coarse collagen (CC_3).

2

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Figure 3. Representative confocal laser scanning microscopy images. For each recipe three images obtained at different places in the same sausage are visualized. (A) High fat control sausage (HF), (B) sausage with 6 g/100 g rye bran (RB), (C) sausage with 5 g/100 g rye bran and 1 g/100 g fine collagen (CF_1), (D) sausage with 5 g/100 g rye bran and 1 g/100 g coarse collagen (CC_1), (E) sausage with 3

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g/100 g rye bran and 3 g/100 g fine collagen (CF_3), and (F) sausage with 3 g/100 g rye bran and 3 g/100

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g coarse collagen (CC_3). Red areas represent liquid fat and the green areas represent protein.

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3.5

RI PT

3

Relative intensity

2.5

SC

2

1.5

0.5

0 0.5

5

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1

50

500

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Relaxation time (ms)

AC C

EP

Figure 4. Distributed NMR T2 relaxation times measured on sausage samples cooked in a water bath at 80 °C for 10 min. The black curve represents the high fat control sausage, dotted curve represents recipe with 6 g/100g rye bran, green curve represents recipe with 5 g/100 g rye bran and 1 g/100 g fine collagen, and dotted green curve represents recipe with 3 g/100 g rye bran and 3 g/100 g fine collagen.

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Figure 5. Means of textural attributes obtained from texture profile analysis of high fat control sausages (HF) and sausages with 6 g/100 g rye bran (RB), 5g/100 g rye bran and 1 g/100 g fine collagen (CF_1), 3 g/100 g rye bran and 3 g/100 g fine collagen (CF_3), 5 g/100 g rye bran and 1 g/100 g coarse collagen (CC_1) and 3 g/100 g rye bran and 3 g/100 g coarse collagen (CC_3). Cooking refer to minutes of heattreatment in a water bath at 80 ° C prior to the analyses, which was performed after tempering of the samples at room temperature (23 ° C).

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2.5 2 C0/R10_Fine 1.5

C0/R10_Coarse

C2/R8_Coarse

0.5

C4/R6_Fine

0 -4

-2

0

2

-0.5

6

C4/R6_Coarse

C6/R4_Coarse

-1.5

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-2

-3

4

C6/R4_Fine

-1

-2.5

RI PT

-6

C2/R8_Fine

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Scores PC#2 (11.9 %)

1

C8/R2_Fine

C8/R2_Coarse C10/R0_Fine C10/R0_Coarse

Scores PC#1 (75.5 %)

Figure 6. Principal component analysis (PCA) score plots of distributed NMR T2 relaxation data obtained for model meat system with varying levels of rye bran and collagen added and cooked in water bath at 80 °C for 30 min. Fine and Coarse refer to the type of collagen used as ingredient. C0/R10: 0 g/100 g

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collagen and 10 g/100 g rye bran, C2/R8: 2 g/100 g collagen and 8 g/100 g rye bran, C4/R6: 4 g/100 g collagen and 6 g/100 g rye bran, C6/R4: 6 g/100 g collagen and 4 g/100 g rye bran, C8/R2: 8 g/100 g

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collagen and 2 g/100 g rye bran, and C10/R0: 10 g/100 g collagen and 0 g/100 g rye bran.

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2

A

1.8 1.6

RI PT

Relative intensity

1.4 1.2 1 0.8 0.6

SC

0.4 0.2

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0 0.5

5

50

500

Relaxation time (ms)

B

60 50

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T22 area

40 30 20

0

2

AC C

0

EP

10

4

6

8

10

12

Collagen %

Figure 7. (A) Distributed NMR T2 relaxation times measured in samples of meat model system with 6 g/100 g collagen and 4 g/100 g rye bran added after heat treatment for 30 min in a water bath at 80 °C. The black curve represents the fine collagen ingredient (scanpro 1015-1) and the dotted green curve rrepsents the coarse collagen ingredient (scanpro 1015-3). (B) Correlation between amount of collagen added (% w/w) to a meat model system and NMR T22 area, which represents a T2 population with a relaxation time in the region 150-400 ms. Black symbols represent data from samples added fine collagen (particle size <1 mm) and green symbols represent data from samples added coarse collagen (particle size <3 mm). For both collagen types R2=0.99***

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Highlights

•

Partial substitution of fat with rye bran and collagen was investigated in Frankfurter sausages Collagen inclusion impacted texture and sensory attributes

•

Collagen can assist as a functional ingredient during fat replacement in processed meat

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