application and sensory evaluation of enzymatically texturised vegetable proteins in food models

ORIGINAL PAPER

Application and sensory evaluation of enzymatically texturisedvegetable proteins in food models

Christian Schafer • Sybille Neidhart •

Reinhold Carle

Received: 7 December 2010 / Revised: 23 March 2011 / Accepted: 30 March 2011 / Published online: 21 April 2011

� Springer-Verlag 2011

Abstract Using simplified model systems, the effects of

salts and oil on enzymatic texturisation of protein isolates

from soy (Glycine max (L.) Merr.; SPI), pea (Pisum sati-

vum L.; PPI) and sweet lupine (Lupinus albus L.; LPI) were

evaluated. In aqueous systems, protein cross-linking by

microbial transglutaminase (MTG) was significantly

improved when NaCl (1–2 g hg-1) was added, but

respective doses of CaCl2 reduced gel strengths. As shown

by emulsion model systems of PPI and SPI with oil/protein

ratios of 1 and 2 g g-1, emulsification of corn oil into

aqueous protein suspensions prior to enzymatic cross-

linking enhanced gel formation depending on the emulsi-

fication technique. The impact of NaCl and oil varied

among the protein isolates as to obtainable maximum gel

strengths and optimum doses of these ingredients. The

applicability of MTG to leguminous proteins in complex

plant foodstuffs was finally deduced from their perfor-

mance in complex food models of the liquid (thickened

soup), foamed (mousse) and solid (sausage-like substitute)

type, respectively. The sensory characteristics of the latter

were evaluated by trained panellists relative to their milk-,

gelatin- and meat-based counterparts. Texture was

appealing in the foamed and solid food models, but the

liquid soup model suffered from unfavourable grittiness.

Without masking their beany off-flavour, the food models

containing leguminous proteins deviated from the refer-

ence products. On the whole, MTG-catalysed cross-linking

rendered the leguminous proteins suitable for the food

applications in terms of visual appearance, texture and

colour. Especially, the gels representing mousse-type

foams and cuttable sausage-like vegetarian substitutes were

very promising.

Keywords Cross-linking � Leguminous proteins �Microbial transglutaminase � Sensory profile �Texturisation

Introduction

Price advantage, security of supply and especially, the high

acceptance of plant-derived foodstuffs are reasons for the

growing market of vegetarian products. As the importance

of convenience foods is concomitantly increasing, the

development of highly convenient vegetarian foods with

favourable sensory and nutritionally enhanced properties

opens new perspectives for the food industry. Their texture,

being a key quality attribute with respect to consumers’

acceptance, plays a key role in food process engineering

[1]. Adequate selection of the protein components is mostly

crucial for their techno-functional properties. But replace-

ment of animal proteins by those of plant origin has not

always been successful, since products were rejected by

consumers due to insufficient textural properties [2].

Leguminous protein isolates have gained attention due

to their functional characteristics, including gelling,

foaming, emulsifying, fat-absorbing and water-binding

properties [3]. Generally, vegetable proteins enjoy high

C. Schafer

Fraunhofer Institute for Process Engineering and Packaging,

Giggenhauser Strasse 35, 85354 Freising, Germany

Present Address:C. Schafer

DSM Nutritional Products Ltd, 4002 Basel, Switzerland

S. Neidhart (&) � R. Carle

Institute of Food Science and Biotechnology,

Chair of Plant Foodstuff Technology, Hohenheim University,

Garbenstrasse 25, 70599 Stuttgart, Germany

e-mail: [email protected]

123

Eur Food Res Technol (2011) 232:1043–1056

DOI 10.1007/s00217-011-1474-0

acceptance [4] and nutritional value as regards their amino

acid profile [5]. Applicability of leguminous proteins from

pea [6], soy [3, 7] and lupine [8, 9] was exemplarily shown

in a variety of foods such as drinks, bakery products,

processed meat and desserts [10], but their texturising

capacities were mostly dissatisfying.

Cross-linking of proteins by the use of microbial trans-

glutaminase (MTG) is an innovative tool for the

improvement of techno-functional properties, enabling

texturisation, gelation and water-binding based on inter-

molecular and intramolecular formation of e-(c-glutamyl)-

lysine isopeptides [11, 12]. Calcium-independent MTG

(EC 2.3.2.13) is commercially available for texturisation of

protein-rich foods and food ingredients [13], with fish,

seafood, meat and meat products being the main areas of

food applications [14]. MTG has also been used for textural

improvement of vegetable proteins from soy and wheat in

applications such as tofu, bread, bakery products [14] and

pasta [15, 16]. Unlike the wheat albumins and globulins of

Triticum aestivum and Triticum durum, their gliadins par-

ticipate in MTG cross-linking [17]. As quantified by

HPLC–MS for leguminous proteins from soy, pea and

lupine in a simple aqueous model food system, advanta-

geous modification of gelation properties by MTG-induced

cross-linking involved increasing e-(c-glutamyl)lysine iso-

peptide levels up to *500 lmol hg-1 of protein depending

on the substrate used [18]. Recently, rheological properties

of heat-set pea protein gels have successfully been modi-

fied by MTG-catalysed cross-linking [19], although pea

legumin has been considered as a relatively poor substrate

for transglutaminase [20]. Indeed, the level of e-(c-glut-

amyl)lysine isopeptide cross-links correlated well with gel

strength, but specifically for the leguminous protein,

depending on the availability of reaction sites and their

individual contributions to texturisation [21]. Accordingly,

enzymatic digestion with chymotrypsin prior to cross-

linking with MTG greatly improved the emulsifying

properties of proteins of two sorghum cultivars [22].

Enhancement of amino acid composition by cross-linking

rice flour with pea protein [23] may provide further

application areas of MTG-treated leguminous proteins.

For the successful development of vegetarian products,

texturisation of protein isolates involving MTG should be

predictable for frequently used food ingredients. However,

although gel strength proved to be a reliable command

variable that allows indirect prediction of the cross-linking

potential [21], MTG performance in foods is still empirical

for most vegetable proteins. Besides the accessibility of

reaction sites, interference of proteins with further ingre-

dients, e.g. lipids, carbohydrates and salts, may influence

texture formation and play an essential role in stability and

sensory characteristics of the final product. But so far,

interferences with other food ingredients have insufficiently

been considered because of missing systematic investigations

of textural design and sensory evaluation [24]. Relevant

reports consider gel firming of MTG-treated skimmed milk

due to added NaCl or CaCl2 [25] and optimum conditions

for enzymatic cross-linking of oat globulin after adding

0.2 M NaCl [26]. Activity and thermal stability of MTG

were increased in the presence of NaCl and KCl, whereas

MgCl2 and CaCl2 were indifferent or even led to a decline

of both [27].

Therefore, this study aimed at evaluating the suitability

of leguminous proteins for enzymatic cross-linking in the

presence of selected food ingredients and the impact of the

food matrix on the MTG-catalysed reaction in terms of

resulting gel strengths. For this purpose, the impacts of

salts and food oil on MTG-induced cross-linking of sus-

pended protein isolates from soy, pea and lupine were to be

explored in simplified aqueous and emulsion model sys-

tems, respectively. Subsequently, cross-linking of legumi-

nous proteins via MTG in complex food matrices was to be

exemplarily investigated for a liquid, a foamed and a solid

food model. In this context, the sensory characteristics of

cross-linked leguminous proteins were evaluated in terms

of the suitability as ingredients for vegetarian foodstuffs.

Materials and methods

Enzyme

Microbial transglutaminase (Activa� WM) consisting of

the active enzyme (1 g hg-1) from Streptomyces mobara-

ensis (syn. Streptoverticillium mobaraense) and malto-

dextrin (99 g hg-1) was provided by Ajinomoto (Hamburg,

Germany) for enzymatic cross-linking of vegetable pro-

teins. Following the supplier’s instructions as described

earlier [21], the enzyme activity was 94.8 ± 0.7 units g-1

of commercial enzyme preparation. Throughout, the

enzyme preparation was applied as aqueous solution

(100 g L-1) with an active MTG content of 1 g L-1, thus

being equivalent to 9.48 units mL-1.

Protein samples

Two commercial protein isolates from soy [Glycine

max (L.) Merr.], SUPRO� Ex 33 (SPI; DuPont Protein

Technologies International, Ieper, Belgium), and pea

(Pisum sativum L.), Pisane� HD (PPI; Cosucra, Fontenoy,

Belgium), were used as substrates for enzymatic cross-

linking. A lupine protein isolate (LPI) produced on pilot

plant scale was additionally included in parts of this

study. For the latter purpose, sweet lupine seeds of Lupinus

albus L. (Grain Pool, West Perth, Australia) were selected

because of their low alkaloid levels. Quinolizidine alkaloid

1044 Eur Food Res Technol (2011) 232:1043–1056

123

contents, determined according to Wink et al. [28],

amounted to 100 mg kg-1 in the seeds and \10 mg kg-1

in the protein isolates, thus being far below the critical

value of 200 mg kg-1 for lupine-based foodstuffs. The

lupine protein isolate was manufactured by combined

separation of fatty oil and removal of antinutritive com-

ponents such as alkaloids and indigestible sugars, e.g.

verbascose and stachyose [29]. In brief, LPI was produced

by mild alkaline extraction (pH 7–8) followed by iso-

electric precipitation (pH 4–5) and spray drying. The

protein content of LPI, as analysed by the Dumas method

(conversion factor N 9 6.25) [30], was 94 g hg-1. For

the commercial protein isolates SPI and PPI, the pro-

ducers specified protein contents of 90 ± 2 g hg-1. As

deduced from producers’ data sheets, SPI and PPI

equalled in the total percentages of charged (47 g hg-1,

including asparagine and glutamine), uncharged non-polar

(39 g hg-1) and polar amino acids (AA) (14 g hg-1 of

total AA). But PPI was marginally richer in alanine,

valine, and the basic AA lysine, arginine (Arg) and his-

tidine, whereas SPI had slightly more glutamic acid/glu-

tamine (Glx), aspartic acid/asparagine and proline.

According to Ansynth Service (Rosendaal, The Nether-

lands), who was charged with AA analysis of LPI, total

percentages of charged (52 g hg-1) and uncharged polar

AA (16 g hg-1) were comparatively elevated in LPI,

especially Glx and Arg, to the disadvantage of non-polar

AA (33 g hg-1 of total AA). Percentages of lysine ranged

at 7.5, 6.2 and 4.3 g hg-1 of total AA in PPI, SPI and

LPI, respectively, whereas their Glx percentages amoun-

ted to 17.8, 18.9 and 24.2 g hg-1 and those of sulphur-

containing AA to 2.0, 2.6 and 1.8 g hg-1.

Food ingredients

NaCl (C99.5%, p.a.) and CaCl2 (anhydrous, C97.0%)

added to samples of the protein cross-linking experiments

were purchased from Fluka (Buchs, Switzerland). Refined

and standardised corn oil (DSM Nutritional Products,

Basel, Switzerland) was used for the preparation of oil-

in-water (o/w) emulsions. Liquid nitrogen for the

expansion of mousse products was from Linde Gas

(Unterschleißheim, Germany). For the preparation of

completely vegetarian food models, animal fats such as a

fat concentrate, gelatinised fat and fat-containing meat

were totally replaced by fractionated palm oil (August

Storck, Halle, Germany), hardened soy fat and coconut

fat, respectively. The latter two and all further ingredients

used for the production of the food models were provided

by Wachter Nahrungsmittelwerke (Schwaig, Germany).

Fresh parsley was purchased from a local supermarket,

and sausage casings consisting of cellulose were from a

local butcher.

Model systems for studying effects of selected food

ingredients on protein cross-linking

Enzymatic cross-linking of leguminous proteins in the

presence of salts and oil was systematically explored by

comparing simplified model systems in terms of final gel

strength. Size distribution of oil droplets in the oil-con-

taining model systems was additionally evaluated. The

contents of the protein isolates in the model systems were

syllogised from their solubility and processing properties,

such as the ability to be stirred. Protein contents and MTG/

protein ratios ranged at levels that previously proved most

suitable for fundamental gelation studies with these sub-

strates in aqueous systems [18, 21].

To produce 1,000 g batches of enzymatically texturised

vegetable protein (basis model systems constituting MTG-

containing aqueous control samples), either PPI, SPI or LPI

was suspended in demineralised water under stirring with

an anchor stirrer (IKA, Staufen, Germany) at 200–400 rpm

for 30 min at 20 �C according to final doses of 16, 14 and

20 g hg-1, respectively. An aqueous solution of the MTG

preparation (0.1 g mL-1; 9.48 units mL-1) was subse-

quently added while mixing at 400 rpm for 30 s at 20 �C,

adjusting the ratio of active MTG to protein of the sample

to 0.1 g kg-1 (948 units kg-1). The resulting suspension

was immediately transferred into standard Bloom test

vessels (Schott, Mainz, Germany) and incubated in a water

bath at 40 �C for 120 min. Enzymatic cross-linking was

stopped by heating at 90 �C for 5 min in a water bath,

followed by re-cooling in ice water to a core temperature of

20 �C prior to texture analysis.

Aqueous model systems containing salts

To study enzymatic cross-linking in the presence of salts,

the amounts of NaCl and CaCl2, respectively, necessary for

doses of 1 or 2 g hg-1 of end product, were dissolved in

demineralised water before suspending the protein isolates

for subsequent enzymatic cross-linking as described above.

Saline samples and salt-free formulations were prepared in

duplicate. In addition, control samples of leguminous

proteins containing NaCl and CaCl2, respectively, were

prepared without MTG.

Model systems representing o/w emulsions

To study enzymatic cross-linking in the presence of oil,

aqueous suspensions of PPI (16 g hg-1 of end product) and

SPI (14 g hg-1) were prepared as described above by

suspending the protein isolates in demineralised water,

containing NaCl (2 g hg-1 of end product). Subsequently,

corn oil was added to the protein suspensions during con-

tinuous emulsification until oil/protein ratios of 1 or

Eur Food Res Technol (2011) 232:1043–1056 1045

123

2 g g-1 were attained. While simultaneously stirring with

the anchor stirrer at 200 rpm, samples were homogenised

either with an Ultraturrax T 25 (IKA) or a Bandelin

ultrasonic device Sonoplus HD 2070 (Berlin, Germany)

with a maximum capacity of 70 W at 20 kHz. The Ultra-

turrax was operated at 8,000 rpm for 1 and 10 min and at

20,500 rpm for 1 min, respectively. The sonotrode TT 13

was used at amplitudes of 60% for 1 and 10 min and 100%

for 1 min, respectively. Total lot size of each emulsion was

300 g. Enzymatic cross-linking of the leguminous protein

isolates in emulsions and heat deactivation of MTG were

carried out as described for the aqueous model systems.

Samples of the enzymatically cross-linked protein isolates

in emulsions and respective blanks without MTG were

prepared in duplicate. Since availability of LPI was limited,

emulsion model systems were only produced from SPI and

PPI.

Texture analyses

Gel firmness was measured at a core temperature of 20 �C,

using a TA XT plus/5 texture analyser [Stable Micro

Systems (SMS), Surrey, UK] with the SMS texture analysis

software Exponent. Analyses were performed with the

SMS P/0–5 probe unit according to a modified AOAC

method for determining the gel strength of gelatin [31]. In

contrast to the latter, the penetration depth was set to

20 mm instead of 4 mm, since the samples tended to pro-

duce a superficial skin following heat deactivation of MTG.

Microscopic particle size measurements

Laser diffraction or photon correlation spectroscopy,

requiring dispersing of the sample in water, were not

applicable to the emulsions for the determination of par-

ticle size distribution after enzymatic cross-linking.

Therefore, oil droplets in the emulsion model systems were

sized, using an AX 70 microscope (Olympus, Hamburg,

Germany) at 500-fold magnification.

Composition and preparation of complex food models

MTG-catalysed texturisation of leguminous proteins in

complex plant foodstuffs was explored by means of liquid,

foamed and solid plant-derived food models, which were

exemplarily deduced from non-vegetarian standard prod-

ucts (the latter produced without enzymatic texturisation)

and subjected to sensory evaluation. Unlike the simplified

model systems described above, these food models mim-

icked real protein-based foods and covered a broad diver-

sity of recipes, involving a variety of ingredients other than

proteins. Animal proteins were completely replaced by

enzymatically cross-linked vegetable proteins, using PPI

and SPI as commercial protein isolates for texturisation.

Optimum doses of SPI and PPI were deduced from the

results reported for the simplified model systems described

above and additional preliminary trials (data of the latter

not shown). Pure vegetable products were obtained by

concurrent replacement of all ingredients of animal origin,

such as fats and gelatinised fats, with appropriate ingredi-

ents of plant origin. Gums used as thickeners in the original

recipes were removed, because enzymatically cross-linked

plant proteins should provide the necessary texture and

consistency of the food model. Ingredients contributing to

flavour and colour were added at levels corresponding to

the non-vegetarian counterparts used as reference stan-

dards. The food models were finally heated (85 �C core

temperature, 10 min) to ensure complete deactivation of

MTG. The liquid, foamed and solid light-coloured food

models represented a thickened soup, a mousse and a

cuttable sausage-like substitute, imitating products like an

asparagus cream soup, a vanilla mousse and a Bavarian

veal sausage, respectively. All relevant ingredients com-

prising water, lipids, carbohydrates and proteins (i.e. pro-

teins of animal origin in the standard food product and

MTG-texturised plant proteins in the food models) are

specified in Tables 1, 2, 3, without listing minor compo-

nents, e.g. flavouring and colouring ingredients. Pre-

liminary trials involving identical amounts of ingredients

as used for the manufacture of the non-vegetarian proto-

types (Tables 1, 2, 3) had revealed that flour, starch and

maltodextrin did not affect gel formation during MTG-

induced cross-linking of SPI and PPI (data not shown).

While the liquid food model was prepared with PPI and

SPI, respectively, the foamed and the solid ones were

exclusively produced from SPI, because the pea-like fla-

vour of PPI already known from previous studies [18, 21]

was deemed more acceptable for a soup than for a mousse

or a sausage product. Modified food models were prepared

without adding MTG in order to evaluate MTG activity in

the complex food matrices and its contribution to their

texture. For comparison in terms of sensory appearance,

reference samples of the conventional cream soup and

mousse products were prepared according to their standard

recipes. Original Bavarian veal sausages purchased from a

local butcher served as reference for the sausage-like

substitute by analogy.

Liquid food model (thickened light-coloured soup)

Basic composition of reference and leguminous protein-

based model samples of the thickened-soup type is shown

in Table 1. For the preparation of the reference product

(500 g), all dry ingredients, except the fat concentrate and

the gelatinised fat, were shaken in an appropriate sealed

beaker for 1 min, followed by addition of both fat


123

components and further shaking for 2–3 min. After sus-

pending the dry mixture in water at 40 �C with an egg

whisk, the soup prototype was heated for 10 min at 90 �C

under occasional stirring. The food models were prepared

in batches of 500 g by suspending the vegetable protein

isolate (PPI and SPI, respectively) in demineralised water,

Table 1 Composition of the liquid food models, containing PPI or SPI, and the prototype, representing a light-coloured thickened soup

Product type: thickened soup Prototype Food models

Based on PPI Based on SPI

(g) (g hg-1) (g) (g hg-1) (g) (g hg-1)

Salt 2.98 0.60 2.98 0.60 2.98 0.60

Sucrose 0.50 0.10 0.50 0.10 0.50 0.10

Instant flour 10.90 2.18 2.73 0.55 2.73 0.55

Maize powder 1.49 0.30 0.37 0.07 0.37 0.07

Cream powder (42% fat) 4.96 0.99 – – – –

Skimmed milk powder 4.96 0.99 – – – –

Waxy maize starch 2.98 0.60 0.74 0.15 0.74 0.15

Maltodextrin 3.96 0.79 0.99 0.20 0.99 0.20

Xanthan gum 0.60 0.12 – – – –

Fat concentrate 4.96 0.99 – – – –

Gelatinised fat 4.96 0.99 – – – –

Hardened soy fat – – 9.92 1.98 9.92 1.98

Pea protein isolate (PPI) – – 45.00 9.00 – –

Soy protein isolate (SPI) – – – – 35.00 7.00

Aqueous MTG solutiona – – 13.50 2.70 10.50 2.10

Aroma, colouring, extracts, etc. 6.79 1.36 6.79 1.36 6.79 1.36

Waterb 449.96 89.99 416.48 83.30 429.48 85.90

Total 500.00 100.00 500.00 100.00 500.00 100.00

a 9.48 units mL-1 (active MTG: 1 g L-1); prepared by dissolving 10 g of enzyme preparation (Activa� WM) in 100 mL of deionised water.

By the calculated MTG dose, the enzyme/protein ratio was adjusted to 0.3 g kg-1 for both PPI and SPI (2,844 units kg-1)b Without water included in ingredients (residual moisture)

Table 2 Composition of the foamed food model, containing SPI, and its prototype, representing a light-coloured mousse

Product type: mousse Prototype Food model based on SPI

(g) (g hg-1) (g) (g hg-1)

Gelatinised fat 61.25 12.25 – –

Fractionated palm oil – – 65.00 13.00

Sucrose 42.41 8.48 25.00 5.00

Saccharin – – 0.17 0.03

Dextrose 11.78 2.36 – –

Fat replacer (starch & cellulose) 4.71 0.94 4.71 0.94

Gelatin 9.42 1.88 – –

Rasped white chocolate 18.84 3.77 18.84 3.77

Soy protein isolate (SPI) – – 35.00 7.00

Aqueous MTG solutiona – – 10.50 2.10

Aroma, colouring 1.60 0.32 1.60 0.32

Waterb 349.99 70.00 339.18 67.84

Total 500.00 100.00 500.00 100.00

a 9.48 units mL-1 (active MTG: 1 g L-1); prepared by dissolving 10 g of enzyme preparation (Activa� WM) in 100 mL of deionised water.

By the calculated MTG dose, the enzyme/protein ratio was adjusted to 0.3 g kg-1 for SPI (2,844 units kg-1)b Without water included in ingredients (residual moisture)


123

containing NaCl, while stirring with a propeller stirrer

(IKA) at 600–700 rpm and 40 �C for 5 min. All other

ingredients, except fat and the aqueous MTG solution,

were subsequently added and heated to 60 �C under con-

tinuous stirring. The hardened soy fat was added and dis-

persed with the IKA Ultraturrax T 25 at 20,500 rpm for

2 min. After cooling to 40 �C, the aqueous MTG solution

(9.48 units mL-1) was added and the mixture was stirred

with the propeller stirrer at 400 rpm for 180 min in a

covered beaker. Enzymatic cross-linking at 40 �C was

terminated by heating the soup to 85 �C in a water bath for

a holding time of 10 min.

Foamed food model (light-coloured mousse)

For the preparation of the mousse prototype, the ready-

to-use mixture containing all dry ingredients (Table 2) was

vigorously stirred in water at 40 �C for 5 min with an egg

whisk and subsequently stored at 4 �C for 120 min. To

ensure similar dry matter contents of food model and ref-

erence product despite the necessary dose of leguminous

protein isolate (7 g hg-1), the total sugar content of the

prototype had to be reduced by 46% in the food model

(Table 2). For this purpose, sucrose and dextrose were

lowered to 5 and 0 g hg-1, respectively, in favour of the

additional use of saccharin for sweetening of the foamed

food model, taking into account the superior sweetness of

the latter relative to sucrose (450:1). The standard mousse

product according to the reference recipe and the foamed

food model based on SPI (Table 2) were prepared in bat-

ches of 500 g. For the latter purpose, SPI was suspended in

demineralised water under stirring with the propeller stirrer

at 600–700 rpm and 40 �C for 5 min. While the tempera-

ture of 40 �C was maintained, all other ingredients, except

palm oil and aqueous MTG solution, were subsequently

added under continuous stirring. The fractionated palm oil

was heated to 90 �C, added to the mixture and dispersed

with the Ultraturrax at 20,500 rpm for 2 min. Subsequently,

the aqueous MTG solution (9.48 units mL-1) was added,

and the mixture was stirred at 400 rpm for 1 min. Foam was

produced by feeding nitrogen at 1.5 bar through a rotating

porous ceramic tube (pore size 5–100 lm) into the semi-

liquid suspension, until stable foam was obtained. The

resulting product was incubated in a water bath at 40 �C for

120 min. Enzymatic cross-linking was terminated by heat-

ing to 85 �C in a water bath for a holding time of 10 min.

Solid food model (light-coloured sausage-like substitute)

The composition of the solid food model, which was based

on SPI, and the average composition of an original

Bavarian veal sausage [32] representing the reference for

the former are listed in Table 3. For the solid food model,

batches of 1.345 kg were prepared by mixing 240 g of SPI,

300 mL of a NaCl solution (10 g hg-1) and 696 mL of

demineralised water with the anchor stirrer at 200 rpm and

20 �C for 60 min. This suspension was simultaneously

homogenised with the Ultraturrax at 8,000 rpm. Finally,

24 mL of a MTG solution (9.48 units mL-1), 75 g of

rasped coconut fat and 10 g of finely chopped parsley were

added to the protein suspension under continuous stirring

(200 rpm) for 10 min at 20 �C. The sausage-like substitute

was filled into casings, using a Dick filling press CNS 9 L

with a 22-mm filling pipe (Lugama, Mauterndorf, Austria),

and subsequently heated in a water bath at 40 �C for

120 min. The MTG reaction was terminated by heating at

85–90 �C for 30 min in a water bath to ensure a core

temperature of 85 �C for 10 min.

Sensory evaluation of food models

The liquid, foamed and solid food models produced from

leguminous protein isolates and the respective reference

products were evaluated by 12 trained panellists, using an

extended directional difference test [33] based on a 4-point

rating. The liquid and solid food models as well as their

Table 3 Composition of the solid food model, containing SPI, and

the light-coloured reference product of the type Bavarian veal sausage

Product type: Bavarian

veal sausage/respective

sausage-like substitute

Prototypea Food model based on SPI

(g hg-1) (g) (g hg-1)

Veal meat 37.50 – –

Bacon (pork neck) 26.40 – –

Calf’s head (cooked) 8.00 – –

Salt 1.90 30.00 2.23

Coconut fatb – 75.00 5.30

Soy protein isolate (SPI) – 240.00 17.84

Aqueous MTG solutionc – 24.00 1.78

Spices 2.80 – –

Parsley 0.40 10.00 0.74

Waterd 23.00 966.00 72.10

Total 100.00 1,345.00 100.00

a As the proportion of ingredients varies among producers, average

composition in accordance with German food law [32] is given. Data

originate from personal communication of meat producers and recipes

open to the publicb Residual moisture of the commercial coconut fat (5 g hg-1 as

analysed) was considered in the calculation of product compositionc 9.48 units mL-1 (active MTG: 1 g L-1); prepared by dissolving

10 g of enzyme preparation (Activa� WM) in 100 mL of deionised

water. By the calculated MTG dose, the enzyme/protein ratio was

adjusted to 0.1 g kg-1 for SPI (948 units kg-1)d Without water included in ingredients (residual moisture, especially

that of original meat ingredients in the reference product), except that

of coconut fat


123

counterparts were tasted at a serving temperature of 45 �C.

The foamed food model and the reference mousse were

evaluated at room temperature. Visual appearance, aroma,

colour, texture and flavour of the food models were assessed

in terms of the relative deviation from the respective refer-

ence product. For each attribute, a score on a continuous

scale between 0 and 4 was recorded, distinguishing indis-

cernible (0), barely perceptible (1), slight (2), marked (3) and

striking (4) differences between food model and reference

product. Data acquisition was performed using the FIZZ

Sensory Analysis and Consumer Test Management Soft-

ware, version 2.10 A (Biosystemes, Couternon, France).

All results of the sensory tests were expressed as the

arithmetic means (n = 12 per food model and attribute).

Using the GLM procedure of SAS 9.1 (SAS Institute, Cary,

NC, USA), the data obtained for each of the three product

types were subjected to two types of multiple comparison

tests. Significant differences (P B 0.05) among the ratings

of the attributes evaluated for the respective product type

were identified by means of the Tukey–Kramer method

(option TUKEY). Furthermore, the means were explored

for significant deviations (P B 0.05) of the food model

from its reference product in individual attributes, using

Dunnett’s test (control level = 0).

Results and discussion

Interaction of selected food ingredients with enzymatic

cross-linking in model systems

As previously shown for simplified model systems [21], gel

strength can serve as a reliable and rapidly measurable

command variable for managing MTG-induced gelation

due to substrate-specific correlation between the levels of

isopeptide cross-links and the gel strength of the texturised

proteins. Consequently, simplified model systems have

concurrently been instrumental in identifying suitable

concentration ranges of MTG and the substrate protein

regarding desired textural properties, because viscosity and

texture of the final product are largely determined by both,

besides the incubation conditions (time, temperature, pH).

Likewise, interferences of further food ingredients were

explored in this study via the same approach, but now

involving specifically adapted model systems based on a

set of fixed basic parameters (temperature, contents of

MTG and substrate protein).

Impact of salts on enzymatic cross-linking

of leguminous proteins

Without addition of MTG, no gels were formed by each of

the three leguminous protein isolates in samples with NaCl

or CaCl2 added at doses of 1–2 g hg-1. Texturisation

required MTG-catalysed cross-linking of the proteins,

yielding measurable gel strengths even in absence of salts

(control samples in Table 4). As shown earlier [21], gel

strength is the overall effect caused by MTG-induced

cross-linking and thermal gelation of the proteins, with

the latter resulting from subsequent heat inactivation of

the enzyme. This equally applied to this study, where the

leguminous protein isolates were thermally treated (90 �C,

5 min) after enzymatic cross-linking (40 �C, 120 min). In

the presence of added NaCl, enzymatic cross-linking of

each protein isolate resulted in increased gel strength,

whereas the addition of CaCl2 weakened gel formation

(Table 4). This effect was observed for all protein isolates

irrespective of their contents in the model systems. At a

NaCl dose of 2 g hg-1, gel strength of the model system

with SPI (14 g hg-1) increased by 69% relative to its

Table 4 Impact of NaCl and CaCl2 doses added to an aqueous model system (MTG: 948 units kg-1 of leguminous protein) on enzymatic cross-

linking of leguminous protein isolates from soy (SPI), pea (PPI) and lupine (LPI) and resulting gel strengths

Sample A B C D E F G

Added salt dose (g hg-1) 0a 1 (NaCl) 2 (NaCl) 1 (CaCl2) 2 (CaCl2) 1 (CaCl2), 1 (NaCl) 1 (CaCl2), 2 (NaCl)

cadded (mmol kg-1)b 0 171 342 90 180 261 432

Iadded (mmol kg-1)c 0 171 342 270 540 441 613

Gel strength relative to the respective control sample A (F/Fo) (%)d

SPI (14 g hg-1) 100 ± 2 140 ± 4 169 ± 4 59 ± 6 40 ± 8 65 ± 11 61 ± 9

PPI (16 g hg-1) 100 ± 3 106 ± 3 119 ± 4 15 ± 33 49 ± 7 55 ± 12 51 ± 12

LPI (20 g hg-1) 100 ± 5 130 ± 3 128 ± 9 66 ± 7 46 ± 5 75 ± 6 73 ± 5

a Control without addition of saltb Molar content of the added salt dose in the model systemc Mass-specific ionic strength contributed by the added salt dose to the model system, assuming complete dissociation: Iadded ¼ 0:5

Pci � z2

i ; ci,

molar content of the ion type i; zi, valence of the ion type id Mean ± average of absolute deviation from the mean


123

salt-free control, whereas gel strengths of samples with LPI

and PPI at contents of 20 and 16 g hg-1, respectively, only

rose by 28 and 19%. As regards the specific gel strength of

the different leguminous proteins, this was in agreement

with results published earlier [21].

Since the activity of the MTG used in this study has

been reported to be independent of activation by Ca2? ions

[34], their inhibition of gel formation (Table 4) may be

unexpected at first, but was in accordance with an earlier

report [27], indicating significant inhibition of MTG

activity by CaCl2 and an indifferent effect of MgCl2. The

same study and the findings by Siu et al. [26] gave evi-

dence of synergistic effects of NaCl on MTG activity and

gel formation of non-hydrolysed proteins, respectively.

However, NaCl addition did not affect MTG activity dur-

ing cross-linking of wheat gluten hydrolysates [35]. The

beneficial effects of NaCl doses on MTG- and heat-induced

protein gels were ascribed to enhanced protein–protein

interactions due to suppression of ionic repulsion at

appropriate ionic strength [26]. Activation of MTG by

NaCl and KCl added (5–20%) to the enzyme solution was

attributed to improved solubility of the enzyme [27].

Owing to improved protein solubility, accessibility of

reaction sites and interactions between enzyme and sub-

strate protein, ionic strength may also affect the number of

isopeptide bonds formed enzymatically.

Due to similar total percentages of charged, uncharged

polar and non-polar amino acids, SPI and PPI were expected

to be equal in their water-binding capacities. But obviously

influenced by the individual ionic strength resulting from

both the content and the surface charge of the suspended

protein isolate (zeta potential data unavailable), the relative

increase in gel strength induced by each NaCl dose greatly

varied among the three substrates (Table 4). The largest

increment of 40% was recorded for the SPI-based model

system at a NaCl dose of 1 g hg-1, but the same NaCl dose

only led to an increase of 30% and even merely 6% for LPI

and PPI, respectively. Gelation was further enhanced by

29% for SPI and 13% for PPI, when the NaCl dose was

raised from 1 to 2 g hg-1, whereas the higher salt concen-

tration was irrelevant in case of LPI. Hence, PPI, which had

the most basic amino acids, overall presented the highest

demand for NaCl. LPI, which was characterised by the

lowest content of non-polar amino acids, profited least by

NaCl addition. By contrast, the relative decline of gel

strength at each CaCl2 dose was rather uniform among the

three leguminous proteins (Table 4). In view of the findings

reported by Kutemeyer et al. [27], reduced gel strength

primarily had to be ascribed to inhibition of MTG by CaCl2.

Assuming total dissociation, CaCl2 doses of 2 g hg-1 and

NaCl addition of 1 g hg-1 roughly provided the same molar

content of salt (Table 4, sample groups B and E). The ionic

strengths caused by the salt doses rose relative to that after

adding NaCl (1 g hg-1) in the order CaCl2 (1 g hg-1)

\NaCl (2 g hg-1)\CaCl2 (2 g hg-1) by the factors 1.6, 2.0

and 3.2, respectively (Table 4, sample groups B–E). Hence,

it was a specific inhibitory effect of the Ca2? ions rather than

an effect of the ionic strength. Simultaneous addition of

CaCl2 (1 g hg-1) and NaCl (1 and 2 g hg-1) thus likewise

lowered the gel strengths of the model systems (sample

groups F–G in Table 4) relative to the controls (sample

group A in Table 4); the inhibitory effect of Ca2? ions was

not compensated by the synergistic effect of Na? ions. The

increase in ionic strength from saline sample group F to G

due to the elevated NaCl dose was even irrelevant regarding

gel strength. As a consequence, Ca2? minimisation is a

prerequisite for MTG-catalysed protein texturisation.

Therefore, the use of softened or even deionised water is

conducive. In contrast, NaCl addition in the range of

1–2 g hg-1 has been demonstrated to be advantageous.

Consequently, NaCl addition to deionised water at a dose

of 2 g hg-1 was the preferred solvent in subsequent

experiments.

Influence of lipids on enzymatic cross-linking

of leguminous proteins

To study the effect of lipid interaction with enzymatic cross-

linking, o/w emulsions based on leguminous proteins and

corn oil were prepared by ultrasonication or shearing with a

rotor–stator system. In general, emulsions of control sam-

ples without added MTG already displayed measurable

gelling properties (Figs. 1, 2). Compared with the control

samples, gel formation was clearly enhanced after MTG-

catalysed cross-linking. Under the chosen conditions, max-

imum gel strengths were obtained for SPI and PPI, when

ultrasonication was applied at an oil/protein (o/p) ratio of

1 g g-1 (Fig. 1b, d). However, when the same emulsifica-

tion technique was used at the elevated oil/protein ratio of

2 g g-1, firmness of the gels produced from cross-linked SPI

and PPI was still comparable or even weaker (Fig. 2b, d).

After emulsifying the model systems with the Ultraturrax,

increased gel strengths were observed at higher doses of oil

in case of SPI (Figs. 2a vs. 1a), but gel formation of PPI was

not affected by higher o/p ratios (Fig. 2c vs. 1c). Irrespective

of the o/p ratio, enhancement of gel formation due to MTG-

catalysed cross-linking was more pronounced for PPI than

for SPI, provided that ultrasonication was used instead of the

rotor–stator system (Figs. 1, 2).

Textural improvement after ultrasonication may be

supported by intensified dispersion of the oil phase com-

pared with the other technique. A larger number of dis-

tinctly smaller oil droplets in the range of 1–5 lm

occurred, when the emulsions were prepared by ultrasoni-

cation (Fig. 3). In contrast, particle sizes exceeding 10 lm

were observed after rotor–stator dispersion. With


123

ultrasound emulsification, optimum oil dispersion and gel

strength were achieved at an o/p ratio of 1 g g-1, when the

maximum amplitude (100%) was applied to the SPI model

system for 1 min (Fig. 1b), but a lower amplitude (60%)

for the extended time of 10 min was clearly conducive for

the PPI model system (Fig. 1d). Hence, MTG-catalysed

cross-linking of SPI primarily enhanced the stability of the

emulsions (Figs. 1b, 2b), whereas higher gel strengths were

attainable after extended sonication of PPI-based emul-

sions (Figs. 1d, 2d). Consistently, the comparatively poor

emulsifying potency of rice flour blends with protein iso-

lates from pea and soy, respectively, was reported to sig-

nificantly increase after MTG-catalysed cross-linking [36].

On the whole, cross-linking of the leguminous protein

isolates SPI and PPI by MTG in emulsions resulted in a

broad range of gel strengths depending on the emulsifica-

tion technique and time as well as the percentage of oil

emulsified. Compared with our findings for SPI and PPI in

merely aqueous systems under the same incubation con-

ditions [21], MTG-induced gel formation in emulsions

based on these protein isolates was enhanced. As a con-

sequence, desired gels could partly be produced at reduced

protein contents, when MTG-catalysed cross-linking is

applied to emulsion-based foodstuffs.

As revealed by the comparison of achievable gel

strengths in the presence of NaCl (2 g hg-1) in aqueous

and emulsion systems, the superior cross-linking behaviour

of SPI in aqueous systems (Table 4) complied with the

enhanced gel formation of PPI in emulsions (Fig. 1b, d).

Although SPI and PPI came quite close in their amino acid

profile, the latter protein isolate presumably had a more

appropriate distribution of hydrophobic areas, leading to a

greater ability of the protein to unfold at the oil–water

interfaces and thus to an improved accessibility of the

reaction sites for MTG in emulsions.

Applicability of enzymatic cross-linking of leguminous

proteins in complex food models

Compared with the model systems described above,

MTG-catalysed cross-linking was performed in the three

light-coloured food models at o/p ratios of either

0.22–0.28 g g-1 (thickened-soup type, Table 1),[1.8 g g-1

(mousse type, Table 2) or 0.31 g g-1 (sausage-like type,

o/p ratio: 1 g g-1

0

2

4

6

8

10

Ultraturrax8,000 rpm, 1 min

Ultraturrax 8,000 rpm, 10 min


Gel

str

engt

h (N

cm

-2)

a

0

2

4

6

8

10

Ultrasonication,60%, 1 min



Gel

str

engt

h (N

cm

-2)

b

0

2

4

6

8

10




Gel

str

engt

h (N

cm

-2)

c

0

2

4

6

8

10




Gel

str

engt

h (N

cm

-2)

d

Fig. 1 Gel strengths of o/w emulsions containing NaCl (2 g hg-1)

and protein isolates without (open columns) and with (grey shadedcolumns) enzymatic cross-linking (MTG: 948 units kg-1 of legumi-

nous protein) at an oil/protein (o/p) ratio of 1 g g-1 after application

of different emulsification techniques. Emulsification via rotor–stator

system (Ultraturrax): a, SPI (14 g hg-1, open column/light grey

column); c, PPI (16 g hg-1, open column/dark grey column).

Emulsification via ultrasonication: b, SPI (14 g hg-1, open column/

light grey column); d, PPI (16 g hg-1, open column/dark greycolumn). Emulsification techniques: cf. ‘‘Materials and methods’’

section. Error bars: average of absolute deviation from the mean


123

Table 3), while the SPI content was reduced by 50% to

7 g hg-1 in the former two food models, but slightly raised

to 17.84 g hg-1 in the latter. Accordingly, the MTG/

protein ratio of 0.1 g kg-1 (948 units kg-1) was retained

for the solid food model, but increased to 0.3 g kg-1

(2,844 units kg-1) for the liquid and the mousse type. By

supporting gel formation in the food models of the foamed

(mousse) and solid types (sausage-like substitute), MTG

enabled their production. Due to permanent stirring during

manufacture of the liquid food model (thickened-soup

type), increased viscosity was noted instead of the forma-

tion of a settled gel. In contrast, respective control samples

of each food model without active MTG did not form

measurable gels in case of the mousse and the sausage-like

substitute, while a markedly lower viscosity was perceived

for the MTG-free soup model compared with the prototype.

Comparison of sensory characteristics between texturised

food models and reference products

The sensory profiles of all texturised food models signifi-

cantly differed from those of the prototypes in terms of

texture, flavour and aroma (Table 5). Unlike the liquid and

foamed food models, the solid one was comparable with

its reference product as regards visual appearance and

colour.

The greatest relative deviations, especially in terms of

textural appearance, flavour and aroma, were observed for

the liquid food model, irrespective of type and dose of the

protein isolate used (SPI: 7 g hg-1; PPI: 9 g hg-1). Both

thickened model soups were characterised by a typical

legume-like note as well as a granular and sandy mouth

feel. Compared with the reference soup, colour was more

greyish, thus differing slightly and even markedly, when

the thickened model soup was produced from SPI and PPI,

respectively. Likewise, the model soup based on SPI

showed less divergence from the prototype in flavour and

aroma than the one containing PPI (Table 5). Whereas the

relative differences in both attributes were marked to

striking, when PPI had been used, they were just noticeable

after application of SPI. On the whole, MTG-induced

texturisation of SPI clearly proved to be advantageous

compared with the use of PPI regarding the sensory

properties that were to be achieved.

o/p ratio: 2 g g-1

0

2

4

6

8

10




Gel

str

engt

h (N

cm

-2)

a

0

2

4

6

8

10

Ultrasonication60%, 1 min



Gel

str

engt

h (N

cm

-2)

b

0

2

4

6

8

10




Gel

str

engt

h (N

cm

-2)

c

0

2

4

6

8

10




Gel

str

engt

h (N

cm

-2)

d

Fig. 2 Gel strengths of o/w emulsions containing NaCl (2 g hg-1)

and protein isolates without (open columns) and with (grey shadedcolumns) enzymatic cross-linking (MTG: 948 units kg-1 of legumi-

nous protein) at an oil/protein (o/p) ratio of 2 g g-1 after application

of different emulsification techniques. Emulsification via rotor–stator

system (Ultraturrax): a, SPI (14 g hg-1, open column/light grey

column); c, PPI (16 g hg-1, open column/dark grey column).

Emulsification via ultrasonication: b, SPI (14 g hg-1, open column/

light grey column); d, PPI (16 g hg-1, open column/dark greycolumn). Emulsification techniques: cf. ‘‘Materials and methods’’

section. Error bars: cf. Fig. 1


123

Compared with the gelatin-containing reference mousse

(Table 2), the SPI-based formulation uniformly showed

slight to marked differences for all sensory attributes

(Table 5). To avoid undesirable saltiness, the foamed

mousse-type food model was prepared by cross-linking of

the protein without adding NaCl. Despite successful MTG-

induced texturisation, marked adverse deviations from the

mousse prototype were recorded for flavour, texture, aroma

and visual appearance. A legume-like note, watery and less

intensive taste as well as less creaminess were found. Foam

formation and stabilisation by proteins is based on flexible,

cohesive films surrounding the gas bubbles after protein

adsorption on the interfaces via hydrophobic areas.

MTG-induced cross-linking of SPI substantially contributed

to stabilisation of the foam by generating a firm protein film,

but without the melting properties of gelatin gels. Compared

Ultraturrax Ultrasonication

SPI (14 g hg-1) SPI (14 g hg-1)

o/p: 1 g g-1

UTX: 20,500 rpm, 1 min o/p: 2 g g-1

UTX: 20,500 rpm, 1 min o/p: 1 g g-1

USC: 100%, 1 min o/p: 2 g g-1

USC: 100%, 1 min

PPI (16 g hg-1) PPI (16 g hg-1)

o/p: 1 g g-1

UTX: 20,500 rpm, 1 min o/p: 2 g g-1

UTX: 20,500 rpm, 1 min o/p: 1 g g-1

USC: 60%, 10 mino/p: 2 g g-1

USC: 60%, 10 min

Fig. 3 Light micrographs of oil droplets in o/w emulsions containing

cross-linked protein isolates (SPI: 14 g hg-1; PPI: 16 g hg-1) after

application of different emulsification techniques at oil/protein (o/p)

ratios of 1 and 2 g g-1. Left: rotor–stator system (Ultraturrax, UTX).

Right: ultrasoncation (USC). Emulsification techniques: cf. ‘‘Materi-

als and methods’’ section. Scale indicates 10 lm

Table 5 Deviation of the food models relative to the respective light-coloured prototypes in sensory attributes on a scale from 0 to 4 including

no (0), barely perceptible (1), slight (2), marked (3) and striking (4) differences

Food model Average deviation in sensory attributes relative to the prototypea ( )

Visual appearance Texture Flavour Aroma Colour

Thickened-soup type

PPI 2.0 ± 0.4E*** 3.3 ± 0.4AB*** 3.6 ± 0.4A*** 3.2 ± 0.4ABC*** 2.7 ± 0.6BC***

SPI 1.9 ± 0.4E*** 3.2 ± 0.4ABC*** 2.8 ± 0.5BC*** 2.6 ± 0.7CD*** 2.0 ± 0.5DE***

Mousse type

SPI 2.5 ± 0.7AB*** 2.7 ± 0.5A*** 3.0 ± 0.5A*** 2.7 ± 0.8AB*** 2.0 ± 0.5B***

Sausage-like type

SPI 0.7 ± 0.6B 2.3 ± 0.6A*** 1.9 ± 0.8A*** 2.1 ± 0.6A*** 0.5 ± 0.4B

a Mean ± standard deviation, resulting from 12 sensory ratings

Within each product type, means followed by different letters (A–E) were significantly different (P B 0.05) according to Tukey–Kramer’s

multiple comparison test. Means significantly (P B 0.05) deviating from the respective reference are indicated by asterisks (***), as shown by

Dunnett’s test for multiple comparison to a control


123

with the other attributes, colour was least affected, although

the model mousse, containing cross-linked SPI, appeared to

be more pale and greyish than the prototype.

Overall minimal deviations from its reference product

were displayed by the solid food model (Table 5). Barely

perceptible divergence between the sausage-like substitute

based on cross-linked SPI and its reference as to visual

appearance and colour was insignificant, while texture,

aroma and flavour differed only slightly. Although fat is

generally considered as an ingredient strongly influencing

taste, texture and mouth feel, reduction in the fat content

from 25 to 5 g hg-1 in the sausage-like model (Table 6)

resulted in an overall acceptable sensory perception.

Perception of enzymatically texturised vegetarian food

models

Deviations of the soup model from its reference product

became particularly evident, when the panellists were

asked about their general perception (Fig. 4). Acceptance

of the liquid food model was very poor because of the

unfavourable grittiness. For the mousse-type food model

and the sausage-like substitute, acceptance rates of 60 and

80%, respectively, were obtained, thus indicating the high

potential of MTG cross-linked leguminous protein isolates.

In particular, the texturising properties of SPI in the solid

food model were appreciated.

However, aroma and flavour of the liquid and foamed

food models were negatively rated when PPI and SPI were

used. Therefore, masking of characteristic leguminous off-

flavour is a prerequisite for the successful commercialisa-

tion of products based on texturised leguminous proteins,

except for the application e.g. in pulse-based products.

Production of protein isolates with improved sensory

appearance by prevention of rancidity and enzymatic

degradation of flavour compounds through the removal of

residual lipids and associated substances, in particular

phospholipids, with a lipase during protein extraction has

therefore been proposed [38]. Slight to marked colour

deviations as recorded for the liquid and the foamed food

model might be overcome by adding suitable food colou-

rants or colouring foodstuffs.

In summary, the present study provided evidence of the

applicability of leguminous protein isolates cross-linked by

MTG in the production of texturised solid and foamed

foods. Based on exemplary food models, the most crucial

components of basic recipes have been identified.

Conclusions

Previous monitoring of e-(c-glutamyl)lysine isopeptide

formation via HPLC–MS [18] and evidence of the protein-

specific correlation of isopeptide cross-link levels with gel

strength [21] were completed by this application study of

leguminous proteins. The use of simplified models was

shown to be a valuable tool for the systematic development

of vegetarian foods based on enzymatically texturised

leguminous protein isolates. Successful gel formation of

soy, pea and lupine proteins in simplified models was

proven, identifying optimum process conditions without

tedious adaptation of textural properties. Best results were

obtained when deionised/softened water was used with the

addition of NaCl (1–2 g hg-1) and when preparing emul-

sions with corn oil.

Table 6 Main constituents of original Bavarian veal sausage (aver-

age composition) and the respective solid food model based on SPI

Bavarian veal

sausageaSolid food model

based on SPIb

(g hg-1) (g hg-1)

Water 61.70 75.34c,d

Protein 11.60 6.06

Fat 24.70 5.30

Minerals 2.00 1.49e

Othere – 1.81

Total 100.00 100.00

a Composition according to Food Composition and Nutrition Tables

[37]b Composition calculated based on the protein content of SPI

(90 g hg-1), dry matter content of SPI (95 g hg-1) and residual

moisture of palm fat (5 g hg-1)c Including water content of ingredients (residual moisture)d Including water content of aqueous MTG solutione Only known salt content of the recipe (without additional minerals

e.g. of SPI, see otherf)f Calculated as residual difference by subtracting the total of known

constituents from 100%; represents e.g. polysaccharides, fibres in SPI,

maltodextrin in the MTG preparation, minerals

0%

20%

40%

60%

80%

100%

A B C D

Fig. 4 Perception of food models based on enzymatically cross-

linked plant protein isolates. A: liquid model (thickened soup), PPI; B:

liquid model (thickened soup), SPI; C: foamed model (mousse), SPI;

D: solid model (sausage-like substitute), SPI. Percentage of accep-

tance, as deduced from a yes–no question type: (open column) yes,

(light grey shaded column) undecided, (dark grey shaded column) no


123

Using complex solid, foamed and liquid food models,

MTG-induced texturisation of leguminous proteins, such as

PPI and particularly SPI, in real food formulations was finally

demonstrated, evaluating the contribution of the cross-linked

proteins to the sensory profiles of cuttable sausage-like sub-

stitutes, mousse-type and thickened-soup type products.

Textural properties, i.e. mouth feel of settled gels in the sau-

sage-like and the mousse-type model, were overall accepted,

whereas textural impression of the soup model, which needed

stirring during preparation, was unfavourable. Most impor-

tant, successful product development of low-fat texturised

meat substitutes based on plant-derived ingredients was fea-

sible when gel formation was induced by MTG cross-linking

of SPI, as shown for the sausage-like model.

Acknowledgments This research project was supported by the

German Ministry of Economics and Technology (via AiF) and the

FEI (Forschungskreis der Ernahrungsindustrie e. V., Bonn, Germany).

Project AiF 13177N.

Provision of food ingredients for the production of the food models

and their liquid and foamed reference counterparts by Horst Ganzer

(Wachter Nahrungsmittelwerke, Schwaig, Germany) is gratefully

acknowledged. The authors thank Manuel Uhrmeister and Miguel

Amaro for their contributions to cross-linking studies in model sys-

tems and Sylvia Hini for her excellent technical assistance in pre-

paring the complex food models.

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