Nfc Application.pdf

Nanocellulose as an additive in foodstuff Innventia Report No.: 403

Nanocellulose as an additive in foodstuff Göran Ström, Camilla Öhgren and Mikael Ankerfors

Innventia Report No.: 403 June 2013

Nanocellulose as an additive in foodstuff Innventia Report No.: 403

Acknowledgements The authors wish to thank RISE Research Institutes of Sweden for financial support.

Nanocellulose as an additive in foodstuff Innventia Report No.: 403 1

Table of contents Page

1

Summary ................................................................................................................. 2

Sammanfattning.............................................................................................................. 2 2

Introduction .............................................................................................................. 2

3

Materials and methods ............................................................................................ 3

4

3.1

Preparation of MFC ...................................................................................................... 3

3.2

Characterization of structure of MFC gels using microscopy ...................................... 5

3.3

MFC in food emulsions ................................................................................................ 5

3.4

MFC in food foams ....................................................................................................... 6

3.5

MFC as an additive in bread ........................................................................................ 6

3.6

MFC as an additive in hamburger ................................................................................ 8

Results and discussion ............................................................................................ 9 4.1

Structure of MFC gels as characterized by microscopy .............................................. 9

4.2

Impact of MFC on food emulsions ............................................................................. 13

4.3

Impact of MFC on food foams.................................................................................... 16

4.4

Impact of MFC on properties of baked bread ............................................................ 19

4.5

4.5 Impact of MFC water retention during frying of hamburger ................................. 21

5

Conclusions ........................................................................................................... 23

6

References ............................................................................................................ 24

7

Innventia Database information ............................................................................. 25

Nanocellulose as an additive in foodstuff Innventia Report No.: 403 2

1

Summary

The impact of microfibrillated (MFC) as an additive in food stuff has been studied in a cooperation between the Swedish Institute for Food and Biotechnology (SIK) and Innventia AB. The work included microscopy studies of MFC, the effect of MFC on stability of oil in water emulsions and foams containing high amounts of dissolved sugar. Also studied was the impact of MFC as an additive in bread and hamburger. The work showed that MFC has a strong potential to stabilize oil in water emulsions and foams. Very stable foams were obtained at low additions of MFC. An addition of MFC in dough gave the bread better appearance like higher volume and more even form. The bread also became smoother. As an additive in hamburger MFC gave no offflavour and the same texture and mouthfeel as hamburger without MFC. Moreover, hamburger with MFC could hold more water during frying without negative side effects.

Sammanfattning Mikrofibrillerad cellulose (MFC) har utvärderats som tillsats i livsmedel i ett samarbete mellan Institutet för Livsmedel och Bioteknik (SIK) och Innventia AB. Arbetet omfattade mikroskopistudier av MFC, inverkan av MFC på stabiliteten av olja i vattenemulsioner och skum innehållande höga halter av löst socker. Vidare studerades inverkan av MFC som tillsatsmedel i bröd och hamburgare. Arbetet visade att MFC har en god förmåga att stabilisera olja i vatten emulsioner och skum. Mycket stabila skum erhölls vid låga tillsatser av MFC. En tillsats av MFC till deg resulterade i att brödet fick högre volym, jämnare form och blev slätare. En tillsats av MFC till hamburgare gav ingen bismak och samma textur och munkänsla erhölls som för hamburgare utan MFC. Vidare kunde hamburgare med MFC behålla vattnet i större utsträckning vid stekning än hamburgare utan MFC.

2

Introduction

Microfibrillated cellulose (MFC) also referred to as nanofibrillated cellulose (NFC) or just nanocellulose is microfibrills released from the cell wall of cellulose, see figure 1. The material can be obtained after thorough homogenization using a high pressure homogenizer (Pääkkö et al. 2007; Wågberg et al. 2008; Aulin et al. 2011). The microfibrills have a diameter in the nano size range and a length in the micro size range. It is obtained as a highly viscous gel with a solid content of a few percent. This material was pioneered by Turback and co-workers (Turback et al. 1983) about 30 years ago and the area was recently reviewed (Klemm et al. 2012). The process to prepare MFC has been developed during the last decade. One of the most important discoveries is the fact the various pre-treatments of the pulp may drastically reduce the energy consumption during the homogenization step (Ankerfors 2012), and also generate a more uniform material. An example of a MFC gel is shown in Figure 2.

Nanoce ellulose as an additive in foodstuff Innvenntia Report No o.: 403 3

Figuree 1. Microfibrills in the cellulosee cell wall. (Couurtesy Geoff Daniels, SLU, Sweden). S

Figuure 2. MFC gel with a solid conntent of 2%. (C Courtesy Innvventia AB, Swe eden) Pre-treatm ment according to genneration 2 (see below).

MFC C has the pootential of becoming b ann important componentt in producttion of papeer and boardd. It also has h the pottential of bbeing incorp porated in the bio-bassed materiaals of tomoorrow. The M MFC produuced by Turrback withouut pre-treatm ment was in nvestigated as an additive in food and severaal patents were w filed at that tim me. One of those (Turbbak et al. 1984) descrribes the positive p imp pact of MF FC on the stability of suspensioons, oil-in-water emullsions, dressing mixturres, meat eemulsions for fo production of sausaages and dessert toppiings made from whipp ped foams. When MFC C was used d in hamburrgers they found f that a hamburger containin ng MFC loost less watter on fryin ng, was juiccier and a better b taste. However,, due to th he high ennergy consu umption and cost of pproducing MFC withoout pre-treaatment, the product p wass never com mmercialized d as a food aadditive. The ssituation todday is comp pletely diffeerent since the t pre-treatments makke the produ uction of M MFC cost eff fficient. Thee work in thhe present report r is a short surveyy of the potential of M MFC in variious food applications a like emulssions, foam ms, bread annd hamburg ger. It was shown the MFC has interesting i ppotentials to stabilize food emulssions and fo oams. ng potentiaal to increasse the moissture of ham mburger wiithout The product alsso had stron deterriorating tasste, texture and a mouthffeel.

3

Materia als and methods m

3.1 Preparattion of MFC Geneeration 1 microfibrilllated cellullose was produced p using u a sooftwood sullphite dissoolving pulpp (Dissolvin ng Plus, D Domsjö Faabriker AB, Sweden).. The cell wall delam mination waas carried out by treatinng the pulp in five step ps with enzyymatic treattment beforre homogennization (Pääkkö et al. 22007):

Nanocellulose as an additive in foodstuff Innventia Report No.: 403 4

1. First, a 4% cellulose suspension was mechanically refined using a Voith refiner with 90 kWh/tonne at a specific edge load of 1.5 Ws/m to 50 SR. 2. Next, the enzyme was added. The pulp was dispersed in 2.5 L of a phosphate buffer (pH 7, final pulp concentration 4% w/w) with 0.17 µL mono-component endoglucanase per gram fibre (5 ECU/µL) and was incubated at 50 C for 2 h. The samples were mixed manually every 30 min. 3. Then, the samples were washed with deionized water and the monocomponent endoglucanase was denaturated at 80 C for 30 min. At the end, the pulp sample was washed with deionized water. 4. The pre-refined and enzyme-treated pulp was refined once again with the Voith refiner, this time, to 90 SR (average refining energy 152 kWh/tonne, specific edge load 1.5 Ws/m). 5. Finally the pulp was passed through 3 large and 5 small slits one time in a highpressure homogenizer (Microfludizer M-110EH, Microfluidics, USA). In Innventia’s terminology, this MFC corresponds to generation 1. The total charge density of this MFC is normally 0.04-0.05 meq/g as determined by conductometric titration (Katz et al. 1984). Generation 2. In order to prepare the anionic MFC, a carboxymethylation pre-treatment method was used (Walecka 1956a; Walecka 1956b). In this procedure 110 grams of fibres (Dissolving Plus, Domsjö Fabriker AB, Sweden) were pre-treated per batch. The procedure can be divided into five steps (Wågberg 2008): 1. Firstly, the never dried fibres were dispersed in deionised water at 10000 revolutions using an ordinary laboratory pulper. This was conducted in smaller batches of 30 g of fibres in two litres of deionised water. 2. The fibres were then liquid exchanged to ethanol by washing the fibres in one litre of ethanol four times with a filtering step in between. 3. The fibres were then impregnated for 30 minutes with a solution of monochloroacetic acid amounts in 500 mL of iso-propanol. The fibres were then added in portions to a solution of 16.2 g of NaOH in 500 mL methanol and mixed with two litres of iso-propanol that had been heated just below its boiling temperature in a five litre reaction vessel fitted with a condenser. The carboxymethylation reaction was allowed to continue for one hour. 4. Following the carboxymethylation step, the fibres were filtrated and washed in three steps. Firstly, the fibres were washed with 20 L of deionised water. Secondly, they were washed with two litres of acetic acid (0.1 M) and finally with ten litres of deionised water. The fibres were then impregnated with a two litre NaHCO3-solution (4% w/w solution) for 60 minutes in order to convert the carboxyl groups to their sodium form. Then the fibres were washed with 15 litres of deionised water and drained on a Büchner funnel. 5. Finally, the pulp was passed 3 large and 5 small slits one time in a high-pressure homogenizer (Microfludizer M-110EH, Microfluidics, USA) in the same fashion as was described by (Pääkkö et al. 2007) for enzymatically treated pulps.

Nanocellulose as an additive in foodstuff Innventia Report No.: 403 5

In Innventia’s terminology, this MFC corresponds to generation 2. The total charge density of this MFC is normally 0.5-0.6 meq/g which corresponds to a degree of substation of roughly 0.1%. Seven different samples of MFC were used in the studies, see Table 1. Table 1. MFC samples used in the present work. DS are given for MFC generation 2 and it refers to the degree of substitution of the pulp after the carboxymethylation.

Sample

MFC type

1

Gen 2 (lab)

2 3

Gen 1 (lab) Gen 1 (pilot) Gen 1 (pilot)

1.0 2.4

5

Gen 2 (lab)

2.25

6 7

Gen 1 (lab) Gen 2 (lab)

2.1 1.95

4

Dry substance (%) 1.8

1.0

Comment

Used in CLSM, TEM CLSM CLSM

Sample 3 pressed to 30% dryness and then dispersed to 1%

CLSM, TEM

Hamburger, bread Food emulsions Foams

3.2 Characterization of structure of MFC gels using microscopy CLSM. The samples 1-4 were analysed under the confocal laser scanning microscope, CLSM, Leica TCS SP5 II (Heidelberg, Germany). The staining, akriflavine or acridineorange dissolved in water was dried on a cover glass. The samples were places in the cavity of the objects slide, and the stained cover glass was sealed on top. The light source was an Argon laser with an emission maximum at 488nm and the signal emitted in the wave length interval 500-580 nm was recorded. A 10x air objective and a 63x water objective was used. Computer zooming was done at 1x, 5x and 10x and images were recorded with formats of 1024x1024 pixels. TEM. A small amount of sample 1 and 4 was placed in a gold cup and cryofixed in liquid propane. The frozen samples were fractured in vacuum and etched before coating. After sublimation of the water, replicas were formed by rotary shadowing of platinum/carbon on top. The replicas were cleaned in solvent before examination. Micrographs were taken in a TEM, LEO 906e, (LEO Electron Microscopy Ltd, Germany) at an accelerating voltage of 80 kV. 3.3 MFC in food emulsions Emulsion preparation. Sample 6 of MFC was mixed with a stick mixer, Ultra Turrax T25 (JANKE & KUNKEL IKA® -labortechnik, Germany) together with rapeseed oil (ICA) and water in four different combinations with a an MFC content between 0.5-1% and 10-50% of rapeseed oil for two minutes. The emulsions formed were stored in refrigerator for three days where after the stability was documented before and after centrifugation (3000 rpm for 30 min). Emulsions containing 0.5-1% MFC (sample 6) and 10-20% rapeseed oil was made in a high pressure homogenizer Panda 2k (Niro Soavi, Italy) at two different pressures, 100

Nanocellulose as an additive in foodstuff Innventia Report No.: 403 6

and 1000 bar. Thereafter the stability was checked after storage in refrigerator in three days before and after centrifugation. CLSM. The high pressure homogenized emulsions were studied in confocal laser scanning microscope, CLSM, Leica TCS SP5 II (Germany). Akriflavine and Nile red, staining the MFC and the fat droplets respectively, was dissolved in water, mixed and dried on a cover glass. The emulsions were places in the cavity of the objects slide, and the stained cover glass was sealed on top. The light source was an Argon laser and a HeNe-laser with an emission maximum at 488 nm and 594 nm, respectively. The signals were emitted in the wave length interval of 500-570 nm and 605-670 nm. A 20x water objective was used. Computer zooming was done at 1x, 2x and 4x and images were recorded with formats of 1024x1024 pixels. 3.4 MFC in food foams Foams were prepared from solutions containing a high amount of sugar, MFC generation 2 (sample 7) emulsifier (Colco 2282-00 alpha-gel, delivered by Aromatic AB) and water. The composition of the liquids used is given in Table 2. The foams were generated from a 200 g solution using an OBH kitchen hand mixer at highest speed for 90 seconds. Table 2. Composition of the liquids used for foam generation.

Sample A

Sugar (wt.-%) 48

Emulsifier (wt.-%) 1.0

MFC (wt.-%) 0

Water (wt.-%) 51.0

Volume of 200 g solution (mL) 168

B C D E F G

48 42 42 48 48 48

1.0 1.0 1.0 0.5 0.5 0.5

0.2 0 0.4 0 0.1 0.2

50.8 57,0 56.6 51.5 51.4 51.3

168 185 185 168 168 168

The foams were poured into measuring cylinders. The initial foam volume was determined and is given in Table 2. The drainage of liquid out from the foam column was read from the bottom of the measuring cylinder at different time intervals. The density gradient of the foam column formed from sample D was measured 52 hours after foam generation by sucking out 50 mL foam and take the weight of the rest of the foam and the cylinder, and subtract it from the weight before the foam was sucked out from the cylinder. 3.5 MFC as an additive in bread Bread was produced with and without MFC (sample 5) and with and without different enzymes. The recipe is shown in Table 3.

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Table 3. Recipe of bread.

Ingredients Flour MFC (sample 5) Extra water Yeast Sugar Salt Emulsifier Enzymes  

Relative amount (%)

Amount (g)

100 55.6 (1.35) 7.11 4.16 2.78 1.67 0.5

182,73 g 101.52 (2.54 ) 13 7.62 5.07 3.03 0.9 0-100 ppm

Mixing of the ingredients was performed in a Kitchen Aid (St. Joseph, Michigan, USA) model KSM90. The mixing was carried out as 2:30 min at medium speed and 2:30 min at the highest speed. Thereafter, the dough was removed, briefly kneaded on a lightly floured table and shaped into a round loaf. Covered with kitchen cloth, the dough was left to rest 10 min on the table. After resting, the dough was divided into pieces of 45 g. Pieces were then kneaded, hand shaped into round balls and let to rest 5 min. Using a special board, pieces of dough were then moulded until forming a perfectly even round ball. After that, pieces of dough were placed on grills covered by baking paper and fairly shared into two batches: one for freezing and the second one immediately baked and analyzed. Pieces of dough for freezing were transferred in a freezing room to be frozen at -20 °C. Two hours later, frozen dough pieces were transferred in plastic bags and stored in the same freezing room at -20 °C. After 7 days freezing storage pieces of dough were withdrawn from the freezing room, put on trays with baking paper and covered with a plastic sheet. When placed on tray, dough pieces were thawed 2 hours in a thawing chamber calibrated at 25 °C and 65% relative humidity (RH). Thereafter the plastic cover was removed and the samples were transferred in a proofer from the brand Sveba Dahlen, model Fermatic. The proofing lasted 36 minutes at 38 °C and 80% RH. Then the trays were placed for 10 minutes in an oven from Sveba Dahlen, model S8, previously set at 210 °C and 20% RH. Afterwards trays were put on a table and let cooling one hour at room temperature. Then the buns were transferred into plastic bags. The experimental design is shown in Figure 3. Totally 12 different types of buns were analyzed; with or without MFC, no enzyme or α-amylase or hemicellulase and freeze stored or not (indicated by the ´prefix).

Nanocellulose as an additive in foodstuff Innventia Report No.: 403 8

Emulsifier

MFC

Enzyme No

No (0) 

‐amylase Hemicellulas

E472 (2) No Yes (1)

‐amylase Hemicellulas

Sample 2.0.0 2.0.0´ 2.0.1 2.0.1´ 2.0.2 2.0.2´ 2.1.0 2.1.0´ 2.1.1 2.1.1´ 2.1.2 2.1.2´

Figure 3. Experimental design for evaluating MFC as additive in bread.

A macroscopic investigation was performed on the buns by taking photos of the cross section and the surface of the buns using a Nikon Digital Camera D90 (Nikon Corporation, Japan) equipped with a lens Micro-Nikkor 55 mm 1:2.8. The aim was to observe crust aspect, bread shape and crumb aspects. The slices cut for texture analyzes were also photographed with the same camera, to observe the crumb aspect and control its homogeneity. The texture was anlaysed by measuring the compressive stress using a modified version of the AACC method 74-09 (AACC, 2001) using an Instron Universal Testing Machine 5542 (Instron, USA) two hours after baking. For each sample three breads were used for texture measurements. Two vertical slices of 2 cm thickness were cut out from each bread. A cylindrical metal probe of 20 mm diameter was pushed into the crumb in the middle of the slices with a constant speed of 1.7 mm/s. The compressive stress at 25% compression was used as a measure of the bread firmness. The measurements were performed 2 h after baking. 3.6 MFC as an additive in hamburger Hamburgers were performed with and without different additives; water, MFC (sample 5) and potato starch. All hamburgers contained 100 g of minced meat, and water, MFC and potato starch were added in different amounts. MFC was added to hamburgers in form of a 2.25% gel. The additives were gently kneaded in the minced meat. The hamburgers were formed to a circle with a diameter of 10 cm before frying. The hamburgers were weighted before and after frying and the juiciness and taste was judged after cooling.

Nanocellulose as an additive in foodstuff Innventia Report No.: 403 9

4

Results and discussion

4.1 Structure of MFC gels as characterized by microscopy CLSM. The microstructure of the four gel types of MFC was studied in CLSM using florescent staining. MFC was shown to be distributed differently in the different samples dependent on generations and manufacturing, see Figure 4. Sample 1, generation 2 made in lab scale is shown in Figure 4a-c; sample 2, generation 1 made in lab scale in Figure 4d-f; sample 3, generation 1 made in pilot scale in Figure 4g-I and sample 4, generation1 made in pilot scale (i.e. sample 3) but pressed and re-dispersed in Figure 4j-l. Sample 1, which is a generation 2 sample made in lab scale, contain some very large fibres with a thickness of about 50 m and more than 100 m in length, in contrast to the samples containing generation 1 that contain no such large fibre pieces. However, there are few thin fibres with a thickness of about 0.5 m and length around 100 m in sample 1, which there is a lot of in sample 2-4, compare Figure 4c, f, i and j. In sample 2 there is few large fibres compared to the other samples containing generation 1, sample 3 and 4, see Figure 4d, g and j. Sample 3 and 4 show very similar microstructure at both low and high magnifications in CLSM. However, sample 4 which is the pressed and re-dispersed pilot made generation 1, appears to be somewhat more inhomogeneous at low compared to sample 3, which is the same material but before pressing and re-dispersion.

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Figuree 4. CLSM imagges of MFC sam mples. Fig. a, b and c are imagges of sample 1 (gen. 2) Fig. dd, e and f are im mages of sam mple 2 (lab made gen. 1). Fig. g, g h and i are im mages of sample 3 (pilot made e gen. 1). Fig. j,, k and l are imaages made from sample 4 (sample 3, presssed and re-disspersed). The MFC M is shown in n bright and thee scale bar is 1000 m (a, d, g and j), 25 m m (b, e and h and k) and 5 m ((c, f, I and l).

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TEM M. In order to t get inform mation on a higher mag gnification and the fibrrillar structu ure of the M MFC:s two of the sam mples (samplle 1 and 4) were freezze-etched annd studied under u mages (Figure 5a and d) of the tw the T TEM. Figuree 3 shows the CLSM im wo samples at the higheest magnificcation possible using C CLSM and the images of the sam me microstru ucture show wn by usingg TEM on frreeze-etchedd samples at a a much higher magnnification (F Figure 5b-c,, e-f). The microstructu m ure that wass not possib ble to resolv ve in CLSM M is now posssible to reesolve in TE EM. Sample 1, containning MFC generation 2, is built up by a fib brillar struccture with a thickness of o the fibrilss of 20-30nm m, which co orrespond w well to whatt have beenn seen previiously (Wåg gberg 20088; Siro 2010 0) see Figurre 3b-c. Thhe black spo ots in Figurre 3c are prrobably the cross sectioons of the fibrils. f The fibrillar stru ructure in saample 4, coontaining a generation g 1 MFC, is nnot that easy y to analyzee in TEM aas sample 1 since it conntain a lot of o thin fibrees (around 00.5 m in th hickness wh hich was ressolved in CL LSM) whicch will be veery large in TEM. Theyy are appeaaring in the TEM T imagees as large layers l abovve each otheer, see Figure 5e. Betw ween these layers l the fiine fibrillar structure can be seen,, see Figurre 3f. Thee fibrillar sstructure iss more fine-meched iin generatiion 1 comppared to genneration 2.

Figuree 5. CLSM and TEM T images off MFC samples 1(a-c) and 4(d-f). The MFC iss shown in brighht in the CLSM images and in grey inn the TEM imagges. The scale bbar is 5µm (a, d), d 200 nm (b, e,) e and 50 nm (c (c, f).

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The ffew thin fibbres which were w resolvved using CL LSM in the size of 0.5 m thickneess in sampple 1, was also a found in i TEM. Fiigure 6a sho ows an imaage of the fi fibre and fib brillar microostructure in TEM on n an overaall level off structure and a CLSSM image on a correesponding magnificatio m on of samplle 1. To thee right in th he TEM-imaage in Figu ure 6a anothher type of structure iss revealed w which probably is und delaminated pulp fibre.. This piecee of fibre is built up p by denseely packed fibrils sho ow in Figur ure 6b at higher h magnnification. As A a comparison, the fibrillar strructure wheen not packked in a fib bre is show wn in Figuree 6c.

Figuree 6. TEM imagees of MFC sampple 1. Overview w (a), focusing in a fibre (b) and in the fibrillarr part. The scalee bar is 5 µm ((a) and 100 nm m (b, c,).

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Characterization of MFC using microscopy techniques shows that merging of fibrils into large fibres with different thickness varies between the different generations and to some extent between lab-scale and pilot-scale manufactured MFC. Generation 2 contain the largest fibres (50 m in thickness and >100 m in length). Generation 1 contain a lot of thin fibres (0.5 m in thickness and 100 m in length) which not is found to a large extent in generation 2. On a high magnification in TEM both types are characterized by thin fibrils with a thickness of 20-30 m. The fibres are built up of fibrils densely packed targeted in the same direction. 4.2 Impact of MFC on food emulsions A first test of the potential for MFC (sample 6) as an oil-in-water emulsion stabilizer was made with a simple stick mixer with mixtures of 0.5-1% MFC, 10-50% rape seed oil and water. The results from the stability studies of the emulsions are shown in Table 3. The emulsions containing the highest amount of MFC and oil was the most stable ones after three days storage. After centrifugation it was possible to separate both an oil and a water phase from the emulsion, except in emulsion 2 with 50% oil that was very stable. Homogenizing in Panda was thereafter performed at two different pressures for emulsion 1, 3 and 4. (Emulsion 2 was already stable by simple mixing with Ultra Turrax). This homogenizing showed that it was possible to make emulsion in all the combinations tested. Figure 7 shows the phase separation of the emulsions after three days of storage and following centrifugation. Only some of the water phase was possible to press out of the emulsion after centrifugation. The higher the amount of MFC and oil the less water is possible to press out of the emulsion. Table 4. MFC as stabilizing agent in emulsions. Emulsion

MFC sample 6 (%)

Oil (%)

Mixing in Ultra Turrax and 3 days storage

Centrifugation after Ultra Turrax and 3 days storage

Pressure in homogenizer, (bar)

Homogenizing and 3 days storage

Centrifugation after homogenizing and 3 days storage

1a

1

20

stable

100

stable

1b

1

20

stable

1000

stable

separates in 2 phases separates in 2 phases

2

1

50

stable

3a

0.5

20

separates

100

stable

3b

0.5

20

separates

1000

stable

4a

0.5

10

separates

100

stable

4b

0.5

10

separates

separates in 3 phases separates in 3 phases separates in 2 phases separates in 3 phases separates in 3 phases separates in 3 phases separates in 3 phases

1000

stable

separates in 2 phases separates in 2 phases separates in 2 phases separates in 2 phases

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Figuree 7. Emulsion coontaining MFC, rape seed oil aand water after storage and ce entrifugation.

The eemulsifiers containing 1% MFC w were firmer,, with a con nsistency ressembling off sour cream m, than the emulsions containing 0.5% MFC that instead d resembledd of soured milk. A haarder homoggenizing at a higher ppressure gav ve also a so omewhat firrmer consisstency comppared to thee lower pressure. Imagges on the emulsions e are shown inn Figure 8 and 9 togetther with thhe CLSM-im mages on thhe microstru ucture of th he emulsionns. In the CLSM C imagges the oil droplets d are shown in ggreen colour and the MFC M in red. The oil dro oplets are iincorporatedd in a netw work of MF FC fibres making m a steric stabilizzation of th he oil dropllets. As seeen in the im mages the oiil droplet siize and distrributions vaary dependeent of oil ccontent andd pressure. The higheer pressuree the smalller dropletss and the more homoogeneous siize distributtion of dropplets. At low w pressure there t are booth large dro oplets and very small ones wherreas at highh pressure the larger ones are noon-existing. The a somewh hat smaller at 10% oill content compared to 220% oil content, dropllet size is also but thhe MFC and the oil dro oplets are ddistributed in n aggregatees at the low wer concentrration of oiil. The MFC C is more in ncorporatedd between the t oil droplets/well diistributed arround the ooil droplets when w the hiigher pressuure was used d, see Figurre 9.

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Figuree 8. CLSM imagges of emulsions containing M FC (sample 6). The MFC is sh hown in red andd the oil in green. The sccale bar is 50 m. 

Figuree 9. CLSM imagges of emulsions containing M FC (sample 6). The MFC is sh hown in red andd the oil in green. The sccale bar is 10 m. 

Nanoce ellulose as an additive in foodstuff Innvenntia Report No o.: 403 16

To suummaries; MFC has a very goodd potential as a stabilizin ng agent forr emulsions. It is possiible to makke very firrm emulsioons by incrreasing the content off MFC, oil and increeasing the pressure at homogenizin h ng. The firm m emulsionss is characteerized by sm maller oil ddroplets withh a smaller size distribbution (incrreased homo ogenizing ppressure), a more well--distributed MFC arou und the sepparate oil droplets d (higher MFC content), and a a moree homogeneeous distrib bution of thhe oil drop plets and th he MFC inn the bulk phase p (incrreased oil coontent) com mpared to em mulsions witth a more liquid texturee. 4.3 Impact of o MFC on food foam ms Foam ms generateed from liqu uid fluids coontaining su ugar and em mulsifier diissolved in water with a minor adddition of MFC M dispersiion were geenerated witth a kitchenn hand mixeer and poureed into meaasuring cyliinders. The experimenttal set up iss shown in FFigure 10 which w also shows the drained d liqu uid 23 and 447 hours afteer foam gen neration.

Figuree 10. Experimenntal set up for studies of foam stability. The situations 23 and d 47 hours afterr foam generatiion are shownn. The liquid column beneath thhe foam is obseerved and measured.

The ffoams weree characteriized with reespect to:  Initial fooam volumee. 

Time to drained liqu uid became visible (T1). )



Time to 30 vol.-% of o the liquidd had becom me visible beeneath the ffoam colum mn (T2). Thuus, roughly 50 mL of thhe liquid haad drained out o from thee foam.

No fo foam could be b generateed without eemulsifier. The T initial foam f volum me increased d with increeasing amouunt of emu ulsifier but decreased with increaasing conteent of sugar and MFC C (see Tablee 5).

Nanocellulose as an additive in foodstuff Innventia Report No.: 403 17

Table 5. Composition and foam characteristics of the investigated samples.

Sample Emulsifier (wt.-%) 1 A 1 1 1 0.5 0.5 0.5

B C D E F G

E

F

Sugar (wt.-%) 48

Initial foam volume (mL) 540

T1 (h) ~3

T2 (h) ~5

0.2 0 0.4 0 0.1 0.2

48 42 42 48 48 48

450 620 370 440 380 350

24 2 >52 0,1 0,2 0,3

58 3,5 >52 0,3 1,2 2,0

160

G

Volume of drained liquid, ml

Volume of drained liquid, ml

160

MFC (wt.-%) 0

140 120 100 80 60 40 20 0 0

10

20

30 40 Time, h

50

60

E

F

G

140 120 100 80 60 40 20 0 0

1

2

3 4 Time, h

5

6

Figure 11. Foam stability given as volume of the liquid column beneath the foam column as a function of time after foam generation for samples E, F and G. The right diagram is an enlargement of the time interval up to 7 hours.

Foams generated from sample E, F and G had low stability as can be seen in Figure 11 These samples contained only 0.5% emulsifier and 0, 0.1 and 0.2% MFC, respectively. Even though the foam stability was low, MFC had a significant positive impact on the foam stability When the content of emulsifier was increased to 1% the initial foam volume increased significantly and very high foam stability was obtained in the presence of MFC. The stability parameters T1 and T2 increased roughly 10 times when 0.2% MFC was present (B) compared to the reference without MFC (A). When the content of MFC was raised to 0.4%, the foam showed no tendency to drain liquid within the 52 hours the experiment lasted. Instead of extending the time for the foam stability characterization, this foam was subjected to a determination of its density gradient (see Figure 13). The foam column was divided into 7 volume units, each with a volume of 50 mL. The bottom volume unit had a slightly higher density than the other units, which were very constant in density.

7

Volume of drained liquid, ml

Nanoce ellulose as an additive in foodstuff Innvenntia Report No o.: 403 18

160 140 120 100 80 60 40 20 0

A

0

0 20

B

C

40

D

60 Time, h

80

100

Figuree 12. Foam stabbility given as voolume of the liqquid column benneath the foaam as a function of time after foam f generationn for samples A, A B, C and D.

f Figurre 13. Density ggradient of the foam colum mn of sample C C, 52 hours afteer foam gene eration.

MFC C also appeaared to redu uce foam brrakeage at the t top of the foam. T This phenom menon was observed affter two day ys for foam ms without MFC. M No sign s of this phenomenaa was obserrved withinn 3 days for the system containing 0.2% MFC C as can be sseen in Figu ure 14 and 115

Figuree 14. Observatioons of foam collapsse on top of thee foam column of o samplee C, 23, 27 andd 71 hours afterr foam ggeneration.

Figure 115. Foam collappse did not occur with sample B which contained 0.2% M FC.

Nanoce ellulose as an additive in foodstuff Innvenntia Report No o.: 403 19

4.4 Impact of o MFC on propertie es of baked d bread The effect of adddition of MFC M (samplle 5) in com mbinations with w two diifferent enzzymes on w wheat bread was studied d on bread bbaked from fresh dough h and doughh that was freeze f storeed for 7 days. The quality of the t bread was w evaluateed by measu uring the tex xture by coompression stress in the Instron teesting mach of the bunss. The hine and by taking photos of the appearance a comppression strress of bread d baked froom fresh do ough contain ning MFC w was significcantly softeer than breaad without MFC, see figure 8 an nd comparee the blues stacks sho owing breadd with no additives, a “n no add” witth bread con ntaining MF FC, “MFC””. The effecct was evenn more proonounced in n presence of the en nzymes -aamylase andd hemicellu ulase. (Com mpare the red r and grreen stacks showing breads b con ntaining enzzymes with h and withoout MFC inn Figure 16..) However,, the effect of softening g the bread was absentt after freezze storage of the dough h, see the staacks with prrefix ´in Fig gure 16.

Figuree 16. Compresssion stress at 255% stress for brreads containing different addiitives; no additivves, MFC, enzyymes (αa=α--amylase, he=hhemicellulase) Prefix P ´means ffreeze stored saamples.

In Fiigures 17 and a 18 the surface andd cross secctions of thee buns are shown for buns baked from fressh dough (F Figure 17) aand buns baaked from freeze f storeed dough (F Figure 18). T The buns coontaining MFC M resulte d generally in bread with a more ssmooth crusst and evenn form and also a a higherr bun, whicch is shown in the imag ges in Figurees 17 and 18. To suummarize; MFC has a clear favouurable effecct on bread baked from m fresh doug gh by makiing the bunns softer and giving thhem a betterr appearancce in form oof smooth crust, high volume andd even form m. Howeverr, the effect of MFC on n freeze stoored dough needs n to bee further evaaluated.

Nanoce ellulose as an additive in foodstuff Innvenntia Report No o.: 403 20

Figuree 17. Buns bakeed from fresh doough containingg different addittives; no additivves, MFC, enzyymes (αa=α-am mylase, he=heemicellulase) Prrefix ´means freeeze stored sam mples.

Nanoce ellulose as an additive in foodstuff Innvenntia Report No o.: 403 21

Figuree 18. Buns bakeed from freeze stored s dough coontaining differeent additives; no additives, MFFC, enzymes (αa=α--amylase, he=hhemicellulase) Prefix P ´means ffreeze stored saamples.

4.5 4.5 Im mpact of MFC M water retention during fry ying of haamburger C as a mooisture reten ntion agentt in hambu urgers wass studied bby adding MFC MFC (sam mple 5) to minced m meaat before fryying. Sincee MFC is in n form of a gel with a dry substtance of 2.225% the haamburgers w will be dilu uted with a lot of wateer when MF FC is added. A hambuurger contaaining 100gg meat was diluted witth 0, 10, 166 and 27% water respeectively whhen 0, 0.24, 0.47 and 00.63% MFC C was added d to the meeat. The ressult in weigght loss is seen in Figure 19. IIrrespectivee of additio onal amounnt of MFC C and attenndant water the hambu urgers lost 330±1% wateer during frrying, but ssince more water was present in the hambu urgers contaaining MFC C the end weight w becoome higher after MFC C addition. The end weight w was increased by b 10, 23 and a 41% affter the diffferent addittions of waater, which is a substaantial increease. The highest h wateer addition gave indeeed a more juuice but also o a more waatery taste and a lost tastte of hambuurger. The lo owest addittion of watter (10%) gave a moore juice hamburger h compared c tto the refeerence withoout any adddition of water or MFC C, but otherw wise it was similar in taaste comparred to the hhamburger containing 100% meaat. Photos of o these tw wo types off hamburgerrs are show wn in Figuree 20. The haamburger coontaining ad ddition of MFC M and waater is someewhat largeer than the one o without.

Nanoce ellulose as an additive in foodstuff Innvenntia Report No o.: 403 22

 

160

140

weight (g)

120

startv ikt Start  weight w End w weight slutvik kt

100

‐ ‐30%

‐29% ‐31%

‐29 9%

80

60

40

20

0 ref2 100% meat

MFFC 0,3% MFC  0.24% 10% water

MFC 0,5% MFC 0.47% 16% water

MFC 0 0,9% MFC 0 0.63% 27% w water

Figuree 19. Weight of hamburgers beefore and after ffrying containing different amo ount of MFC andd water.

Figuree 20. A hamburgger containing 100% 1 meat beffore and after frrying (a and c) and a a hamburgeer containing 0.24% MFC aand 10% water before and afteer frying (b and d).

Potatto starch was w used as a referencee to the MF FC. High am mount of ppotato starch h was requiired to loweer the waterr lost to thee same levell as for MFC. Additionn of 1.4% potato p starch was requuired to receeive the sam me water lo ost as for 0.63% 0 MFC C, see Figurre 21. This high amouunt of potato starch ggave the haamburger a bad consisstency and taste. Loweer addition of potato sttarch (0.75% %) gave no o bad taste or o texture off the hambu urger, but nnot either anny effect on the water hholding capaacity.

Nanocellulose as an additive in foodstuff Innventia Report No.: 403 23

To summarize; MFC (i) gave no off-flavor, (ii) gave the same texture and mouthfeel as the hamburger without additions, (iii) hold more water without side effects as watery taste, (iv) is easy to mix with the meat, but (v) due to its low dry weight it is difficult to add the right amount of MFC without adding to much water.  

40

35%

Loss of water (%)

35

35% 29%

30

27%

25 20 15 10 5 0 ref 4

25% water

MFC 0,9% 0.63% MFC 27% water

Potatis stärkelse 1% 0.75% pot. St.

24% water

Potatis stärkelse 2% 1.4% pot. St.

26% water

Figure 21. The loss of water during frying of hamburgers containing different amounts of additives (water, MFC and potato starch).

5

Conclusions

Characterization of MFC using microscopy techniques shows that merging of fibrils into large fibres with different thickness varies between the different generations and to some extent between lab-scale and pilot-scale manufactured MFC. Generation 2 contain the largest fibres (50 m in thickness and >100m in length). Generation 1 contain a lot of thin fibres (0.5 m in thickness and 100m in length) which not is found to a large extent in generation 2. On a high magnification in TEM both types are characterized by thin fibrils with a thickness of 20-30 m. The fibrillar structure is more fine-meched in generation1 compared to generation 2. The fibres are built up of fibrils densely packed targeted in the same direction. MFC has a very good potential as stabilizing agent for emulsions. It is possible to make very firm emulsions by increasing the content of MFC, oil and increasing the pressure at homogenizing. The firm emulsions is characterized by smaller oil droplets with a smaller size distribution (increased homogenizing pressure), a more well-distributed MFC around the separate oil droplets (higher MFC content), and a more homogeneous distribution of the oil droplets and the MFC in the bulk phase (increased oil content) compared to emulsions with a more liquid texture. MFC also has a very good potential as stabilizing agent for food foams already at low additions (0.2%). Higher additions gave extremely stable foams.

Nanocellulose as an additive in foodstuff Innventia Report No.: 403 24

MFC has a clear favorable effect on bread baked from fresh dough by making the buns softer and giving them a better appearance in form of smooth crust, high volume and even form. However, the effect of MFC on freeze stored dough needs to be further evaluated. As an additive in hamburgers MFC gave no off-flavor, gave the same texture and mouthfeel as the hamburger without additions, hold more water without side effects as watery taste, is easy to mix with the meat, but due to its low dry weight it is difficult to add the right amount of MFC without adding to much water.

6

References

Ankerfors, M. (1012) Microfibrillated cellulose: Energy-efficient preparation techniques and key properties. Licentiate thesis, KTH Royal Institute of Technology, School of Chemical Science and Engineering, Department of Fibre and Polymer Technology, Division of Fibre Technology, TRITA-CHE-Report 2012:38, ISSN 1654-1081, ISBN 978-91-7501-4647. Aulin, C., Johansson, E., Wågberg, L. and Lindström, T. (2010) Adsorption behavior and structural properties of microfibrillated cellulose-based multilayers. Biomacromolecules 11: 872-882. Pääkkö, M., Ankerfors, M., Kosonen, H., Nykänen, A., Ahola, S., Östberg, M., Ruokolainen, J., Laine, J., Larsson, P. T., Ikkala, O. and Lindström, T. (2007) Enzymatic hydrolosis combined with mechanical shearing and high-pressure homogenization for nanoscale cellulose fibrils and strong gels Biomacromolecules 8: 1934-1941. Siró, I. and Plackett, D. (2010) Microfibrillated cellulose and new nanocellulose materials: a review. Cellulose 17:459-494. Turbak, A. F., Snyder, F. W. and Sandberg K. R. (1984) Suspensions containing microfibrillated cellulose US Patent 4,487,634 Dec 11, 1984 Wågberg, L., Decher, G., Norgren, M., Lindfors, T., Ankerfors, M. and Axnäs, K. (2008) The build-up of polyelectrolyte multilayers of microfibrillated cellulose and cationic polyelectrolytes. Langmuir 24:784-795

Nanocellulose as an additive in foodstuff Innventia Report No.: 403 25

7

Innventia Database information

Title

Nanocellulose as an additive in foodstuff Author

Göran Ström, Camilla Öhgren and Mikael Ankerfors Abstract

The impact of microfibrillated (MFC) as an additive in food stuff has been studied in a cooperation between the Swedish Institute for Food and Biotechnology (SIK) and Innventia AB. The work included microscopy studies of MFC, the effect of MFC on stability of oil in water emulsions and foams containing high amounts of dissolved sugar. Also studied was the impact of MFC as an additive in bread and hamburger. The work showed that MFC has a strong potential to stabilize oil in water emulsions and foams. Very stable foams were obtained at low additions of MFC. An addition of MFC in dough gave the bread better appearance like higher volume and more even form. The bread also became smoother. As an additive in hamburger MFC gave no offflavour and the same texture and mouthfeel as hamburger without MFC. Moreover, hamburger with MFC could hold more water during frying without negative side effects. Keywords

Microfibrillated cellulose, food, foam, emulsion, microscopy Classification

1320, 1153 Type of publication

Innventia report Report number

403 Publication year

April 2013 Language

English

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