Accepted Manuscript

Optimisation of protein-fortified beef patties targeted to the needs

of older adults: a mixture design approach

Sephora Baugreet, Joseph P. Kerry, Paul Allen, Ruth M. Hamill

PII: S0309-1740(17)30252-8
DOI: doi: 10.1016/j.meatsci.2017.07.023
Reference: MESC 7329
To appear in: Meat Science
Received date: 21 February 2017
Revised date: 27 July 2017
Accepted date: 28 July 2017

Please cite this article as: Sephora Baugreet, Joseph P. Kerry, Paul Allen, Ruth M. Hamill
, Optimisation of protein-fortified beef patties targeted to the needs of older adults: a
mixture design approach, Meat Science (2017), doi: 10.1016/j.meatsci.2017.07.023

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Optimisation of protein-fortified beef patties targeted to the needs of older adults: a

mixture design approach

Sephora. Baugreet1, 2,Joseph P. Kerry2, Paul Allen1 and Ruth M. Hamill1

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Food Quality and Sensory Science Department, Teagasc Ashtown Food Research Centre,

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Ashtown, Dublin 15, Ireland

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Food Packaging group, School of Food and Nutritional Sciences, Food Science Building,

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University College Cork, Ireland
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Email: Ruth.Hamill@teagasc.ie
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Abstract

Mixture design was used to technologically optimise inclusions of protein ingredients [rice
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protein (RP) 0-10%, and lentil flour (LF) 0-10%] in fortified beef patties [meat (M) 90-100%] to
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ensure acceptable technological and sensorial properties. 17 formulations were generated.

Composition, texture parameters, colour, lipid oxidation, microbiological and sensorial

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parameters were assessed. Maximal predicted protein content was 28.7% (P < 0.01) which

positively correlated with RP, but not LF. Models showed that LF inclusion correlated with

improved texture and also reduced cook loss. Two optimised formulations (OF1 and OF2), for

protein content and technological performance, were experimentally validated. Sensory

panellists scored the optimised formulations higher than controls for tenderness and beef aroma

(P < 0.05). This presents an opportunity to produce protein fortified beef patties with softer
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texture and acceptable technological characteristics. The consumption of such protein fortified

beef patties could assist older adults reach their targeted protein requirements.

Keywords

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Elderly; Beef patty; Mixture design; Optimisation; Sarcopenia

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1. Introduction

The proportion of the world population exceeding 60 years of age is predicted to double to 22%

by 2050 (WHO, 2014) and the prevalence of age-related health conditions is expected to

concomitantly increase. Sarcopenia is a condition associated with the gradual loss of muscle

mass and strength (Cruz-Jentoft, Baeyens, Bauer, Boirie, Cederholm, Landi, & Zamboni, 2010),

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leading to an increased susceptibility to fractures, especially in the frail elderly. A decline of 1-

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2% muscle mass is reported in individual aged 50+ (Young, Therkildsen, Ekstrand, Che, Larsen,

Oksbjerg, & Stagsted, 2013) and this increase further for individuals age 80 years and older

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(Bijlsma, Meskers, Westendorp, & Maier, 2012). As an adult advances in age, their body begins

to function less efficiently and their requirement for several nutrients increases. However, factors
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such as loss of appetite, reduced chemosensory function, mobility, dexterity and oral health
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problems contribute to decline in actual food intakes, which can increase susceptibility to

nutritional deficiencies (Baugreet, Hamill, Kerry, & McCarthy, 2017). Nutrient-dense foods, in
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which essential nutrients can be delivered in a relatively small portion size, are therefore
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desirable for this group. In particular, high quality protein foods, that are rich in essential amino

acids, could help mitigate the risk of conditions like sarcopenia in the elderly (Millward,
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Layman, Tomé, & Schaafsma, 2008).

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The predicted growth in the elderly cohort presents an opportunity to develop optimised, high-

protein foods that could help reduce the incidence of age-related conditions, such as sarcopenia.

Consumption of at least 1.0 to 1.2g protein/kg/bodyweight/day is recommended for healthy older

adults aged 65+ in Europe (Bauer, Cederholm, Cesari, Cruz-Jentoft, Morley & Boirie, 2013),
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while older adults with sarcopenia may need between 1.2 to 1.5g/kg of body weight/day. Red

meat is a traditional staple food product, commonly consumed in elderly populations in Europe

(Bernstein & Munoz, 2014). It is a rich source of high biological value protein (17g - 23g/100g),

as well as micronutrients such as iron, selenium, zinc and vitamin B12 (Pereira & Vicente, 2013).

Leucine, which is abundant in beef, helps preserve muscle mass (Pereira & Vicente, 2013).

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Besides their intrinsic protein content, meat products offer further potential as a platform for

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fortification. Achieving consumption intakes of at least 25-30g/100g serving per day could

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stimulate muscle protein synthesis in the elderly, thus reducing their risk of sarcopenia. Legume

flour and plant proteins have been widely used in the processed meat industry, mainly for their

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functional role as extenders/ binders in comminuted meat products. Legumes and plant proteins
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are however, also a nutritionally-dense source of essential amino acids and protein (Rizzello,

Calasso, Campanella, De Angelis, & Gobbetti, 2014). Other benefits include dietary fibres,
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vitamins and minerals, good technological and sensorial properties and low cost (Ma, Boye,
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Fortin, Simpson, & Prasher, 2013). Therefore, fortification of a meat product which is

intrinsically rich in dietary protein, especially, leucine, with additional plant proteins could result
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in a protein-dense product that could help older adults increase their protein.
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Functional plant-based protein ingredients have been widely used for their technological benefits
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in processed meat systems. However, they can also provide nutritional benefits by increasing the

protein content and balancing the amino acid profile of fortified products (Jimenez Colmenero,

2004). Globally the most commonly used plant proteins are derived from peas, soybeans and

wheat (Egbert & Payne, 2009), while rice protein and lentil flour have had little use in meat

applications to date. We previously undertook a preliminary screening study to determine the

impact of plant-based protein ingredients; pea protein isolate (PPI), rice protein (RP) and lentil
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flour (LF) on protein content and technological properties of beef patties (Baugreet, Kerry,

Botinestean, Allen, & Hamill, 2016). Results indicated that RP and LF plant proteins imparted

benefits on protein content and technological performance, respectively, while PPI, on the other

hand had negative effects on technological performance. A mixture design experiment allows the

determination of the ideal composition of each component in an optimised mixture, with the

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purpose of achieving a product with specified quality and technological parameters (Bezerra,

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Santelli, Oliveira, Villar, & Escaleira, 2008). Mixture designs have previously been used in

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formulation optimisation, e.g. inulin as a fat substitute in pork sausages (Keenan, Resconi, Kerry,

& Hamill, 2014) and β-glucan and resistant starch in a prebiotic sausage (Amini Sarteshnizi,

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Hosseini, Bondarianzadeh, Colmenero, & khaksar, 2015). Here, a mixture design experiment
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was conceived to optimise LF and RP for protein fortification in beef patties.
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2. Materials and methods

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2.1 Beef patty preparation

Lean beef meat (95% visual lean) was obtained from a local supplier Kepak (Clonee, Ireland).
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Excess fat and connective tissue was removed. Diced beef was minced (La Minerva Model No.
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CIE701, Bologna, Italy) in a 5 mm sieve plate, followed by mixing for 8 min before finally

manually mixing ingredients (LF and RP) in for 3 min, then passed through a 3.5 mm sieve plate

to obtain a homogeneous mixture. Table 1 represents the experimental design of the three

components (A1 = meat, B2 = Lentil flour, C3 = Rice protein). Proximate analyses on these

ingredients are presented in Supplementary Table 1A. Beef patties (~75 g) were pressed into

shape using a patty former. They were packaged in black amorphous polyethylene terephalate
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(APET/PE) padded retail trays, (Versatile Packaging, Ireland), gas flushed (Ilpra Foodpack VG

400 Packaging Machine, Italy) using a modified atmosphere packaging (MAP) of 80% O2: 20%

CO2 (Air products and Chemical Inc., Dublin), sealed with a low oxygen permeable barrier film

(3cm3/m2/24h at STP) polyvinyl-chloride film (Versatile Packaging, Ireland) and stored for 12

days at 4°C.

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2.2 Proximate composition

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Each beef patty was blended in a Robot Coupe Blender before analysis. Fat, moisture, protein (N

x 6.25) and ash for raw patties were determined as described by Baugreet, Kerry, Botineştean,
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Allen, & Hamill (2016).
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2.3 Physicochemical parameters

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The pH of raw beef patties were measured on days 1, 6 and 12 using a glass probe pH electrode

(Thermo Scientific pH meter 420A, Orion Research Inc). Beef patties were cooked under a grill
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unit (Salamander, Stephens, Falcon Food Service Equipment, Scotland) for 3 min until they

reached an internal core temperature of 70°C (Thermometer No. PT1000, Eurolec

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Instrumentation Ltd, Dublin) was achieved. Cooking loss values were recorded as described by

Baugreet, Kerry, Botineştean, Allen, & Hamill (2016). Thiobarbituric acid-reactive substances

(TBARs) assay was performed as described by Siu and Draper (1978), as modified by Baugreet,

Kerry, Botineştean, Allen, & Hamill (2016). WHC was determined on raw beef patties after two

days of MAP storage as described by Baugreet, Kerry, Botineştean, Allen, & Hamill (2016).
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2.4 Measurement of colour stability

Colour parameters of raw beef patties were measured as described by Baugreet, Kerry,

Botineştean, Allen, & Hamill (2016). All values were the mean of six independent measurements

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obtained at random from triplicate beef patties taken on days 1, 6 and 12 of storage.

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2.5 Texture measurements

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Texture profile analysis (TPA) was carried out on cooked beef patties based on a method
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described by Baugreet, Kerry, Botineştean, Allen, & Hamill (2016).
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2.6 Microbial Analysis

Psychrotrophic, mesophilic and Pseudomonas of raw beef patties were analysed as per methods
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described by Baugreet, Kerry, Botineştean, Allen, & Hamill (2016).

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2.7 Sensory Analysis

Sensory evaluation was assessed by trained panellists who were experienced in sensory

evaluation and consumed beef patties regularly. Instructions were provided before they evaluated

the beef patties. The sensory analysis was conducted on patties from day 6 of their storage life.

Patties were grill-cooked as specified in section 2.3. Cross-sectional slices of each beef patty

were cut into eight portions, wrapped in aluminium foil and labelled with a 3-digit code. During
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tasting sessions, samples were presented randomly to panellists in order to prevent the order of

presentation and first-order carryover effects. Panellists were asked to indicate their score on a

10 cm line scale ranging from 0 to 10. Each point was converted to a numerical value. Eight

sensory descriptors were used to evaluate the patties; aroma, off-odour, tenderness, chewiness,

juiciness, greasiness, after taste and off-flavour. Six coded samples were presented to the

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panellist in each session and they were instructed to rinse their palate with water between

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samples. All samples were presented four times.

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2.8 Experimental design and statistical Analysis
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A mixture design was carried out and analysed by using Design Expert (v. 7.6.1, Stat-Ease Inc.,

Minneopolis, MN, USA). The effect of ingredient proportions on beef patty characteristics were
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studied with a D-optimal mixture system (Table 1) with constraints which comprised [A1 90-
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100% (M), B2 0-10% (RP), and C3 0-10% (LF)]. The constraints (minimal/maximal) for each
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ingredient were established previously in preliminary trials. 17 experimental formulations with

three independent variables (M: 90-100%, RP: 0-10% and LF: 0-10%) were employed within
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this study. In order to design a robust model, experimental formulations were replicated e.g. run

5, 12 (controls), run 16 was the centre point and run 1, 8 represented equal amounts of both LF
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and RP. The mixture design was created to enable the study of the effects of meat, lentil flour

and rice protein as well as supporting the importance of ingredient interaction (Arteaga, Li-Chan,

Nakai, Cofrades, & Jimenez-Colmenero, 1993); all of which added to the total of 100% of total

weight of beef pattie. Technological parameters: composition (fat, moisture, protein and ash),

cook loss, WHC, texture, colour, lipid oxidation and sensory characteristics were evaluated.
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After data analysis linear [Eq. (2(a)), quadratic Eq. (2(b)) or Scheffe’s special cubic models (Eq.

(2(c))] (as per the degree of fit, predictive power and robustness of the model) for three

components was used to model the responses:

[2(a)]

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[2(b)]

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Where:
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Y = predicted dependent variable such as moisture, protein etc,

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β = equation coefficient,

A1, B2 and C3 = proportion of the pseudo-components [M, LF, RP], respectively.

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The dependent variables (e.g. colour, TBARS, cook loss) were analysed and the models were

subject to variance analysis (ANOVA) to determine the significance (P < 0.05), determination
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coefficient (R2) and lack of fit. Significant dependent variables were consequently analysed

(maximised or minimised) using the software’s optimisation tool and used to predict optimal

beef patty formulations. An optimal beef patty formulation was predicted using the Design

Expert optimisation tool with the goal of enhancing protein content in beef patty with acceptable

technological and sensorial properties. The experimental data for optimised beef patties and
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control for both sensory analysis and model validation physical parameters were determined by

analysis of variance (ANOVA) as described by Baugreet, Kerry, Botineştean Allen, & Hamill,

(2016). Differences were considered significant where P < 0.05. The standard error of the

difference between the means (SED) was also calculated. Each formulation was performed in

triplicate.

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2.8.1 Model validation

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The predictive performance of the developed models was validated using the accuracy factor

(AF) [Equation 3(a)] and bias factor BF [Equation 3(b)] described by Rößle, Ktenioudaki, &

Gallagher (2011).
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(3(a))
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(3(b))

AF and BF are important in describing the effects of the independent variables (M, LF and RP)

on the dependent variables, hence resulting in a validated final optimised beef patty. Accuracy

factor values close to 1.00 suggest a perfect model fit where the predicted and actual response
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values are similar (Hossain, Brunton, Martin-Diana, & Barry-Ryan, 2010). Bias factor is

generated based on the agreement between the model and experimental data. The average mean

deviation (AMD; Equation 4) was used to determine how efficiently the data fit the model

(O’Shea, Arendt, & Gallagher, 2014).

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(4)

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Where:
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∑ = average mean deviation,

= number of experimental data,

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= experimental value and is the calculated value.

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3. Results and Discussion

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Table 2 presents the model characteristics of the responses with significant effect (P < 0.05) for
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beef patties and the significance, determination coefficient (R2) and lack of fit values of the

individual, interaction and quadratic effects. Fig. 1-5 show the representation of the predicted
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values of the response surface models as contour plots. Mean values for all recorded

technological responses are presented in supplementary Tables 2A, 3A and 4A. Results

suggested that partial substitution of RP and LF for meat increased protein content, enhanced

tenderness and aroma of beef patties and demonstrate that it is possible to develop innovative,

nutrient-enriched and healthy meat products for older adults.

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3.1 Compositional analyses

Protein, fat, moisture and ash were determined for raw patties. Compositional data for ash and fat

for raw beef patties could not be fitted and are not presented here.

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Protein content of raw beef patties was fitted with a quadratic model (P < 0.001; R2 = 0.97)

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(Table 2). Significant interaction terms for M*LF and M*RP were observed. Maximal predicted

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protein content was 28.7% with protein positively correlated with RP, but not LF (Fig. 1a). This

compares favourably with the predicted protein content for 100% beef patties which was 22%.

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Current EU Regulation No. 1047/2012, on nutrition claims states that, a ‘high protein’ claim can
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be made where at least 20% of the energy value of a consumed foodstuff is provided by protein.

All patties in this experiment would meet this criterion; however the elderly require higher
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intakes of protein compared with younger consumers and they consume smaller portions
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(Baugreet, Kerry, Botineştean Allen, & Hamill, 2016). Therefore this enrichment of protein in

the fortified patty by approximately 30% would help older adults consume adequate protein from
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a smaller portion size. Here, RP contrasts with LF because RP is a protein extract (79 g/100g dry
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solids), while LF is a leguminous seed (pulse) (23.8 g/100g dry solids) (Baugreet, Kerry,

Botineştean Allen, & Hamill, 2016). The high protein content of rice protein (79 g/100g dry
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solids) is similar to soy and whey protein. The elderly population would benefit from consuming

a protein rich diet to help counter age-associated muscle loss and fortification with RP could

facilitate this.

Moisture data was fitted with a linear model (P < 0.001; R2= 0.87) (Table 2), and was positively

correlated with meat content. The moisture content decreased as the (%) of LF and RP increased,
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perhaps partly due to an increase in dry matter content in the formulations (Fig. 1b). Valenzuela,

Camou, Torrentera, Álvarez, González, Avendaňo & González (2014) also observed reduced

moisture levels in beef patties formulated with 10% flaxseed flour compared to 100% meat.

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3.2 Physicochemical parameters

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No correlation was observed with RP and LF for pH values. Examination of the response surface

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shows that cook loss was positively correlated with M on both analysis days (Table 2; Fig.

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2a/2b). Cook loss was negatively linearly correlated with LF inclusion. The high carbohydrate

content (53.2g/100g according to product specification) of LF could enhance the ability of

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products with LF inclusion to retain moisture in the matrix. The presence of carbohydrates in

lentil flour, either alone or by interacting with proteins may form a structure that traps water and
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prevents its release (Soltanizadeh & Ghiasi-Esfahani, 2015, Baugreet, Kerry, Botineştean, Allen,
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& Hamill, 2016). For WHC, a linear model was fitted (P < 0.001; R2 = 0.76). Contour plots (Fig.
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2c) showed that water retention in beef patties was positively correlated with increasing LF, in

combination with decreasing RP. RP did not correlate with enhanced WHC which contrasts with
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Youssef and Barbut (2011), who reported that the addition of soy and whey protein improved

WHC.
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TBARS values were fitted with a quadratic model (P = 0.0397; R2 = 0.64) (Table 2). LF content

in the mixture was associated with an increase in TBARS, whereas RP-enrichment of beef

patties was lipid stable (Fig. 2d). No interaction effects were observed. This supports the findings

of our previous study, wherein beef patties enriched with RP were less susceptible to lipid

oxidation than LF-enriched beef patties. The high water retention in LF-enriched patties may
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promote lipid oxidation. As the small water-soluble molecular weight lipid peroxides and free

radicals in meat move towards the more hydrophilic part of the product, this occurrence has been

postulated to promote lipid oxidation (Weiss, Gibis, Schuh, & Salminen, 2010, Baugreet, Kerry,

Botineştean, Allen, & Hamill, 2016). It has been reported that rice protein exhibits antioxidant

activity (Zhang, Zhang, Wang, Guo, Wang & Yao 2009), and here appears to have been effective

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against TBARS formation.

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3.3 Instrumental colour

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As shown in Fig. 3a; LF and RP inclusion was associated with increased lightness (L*) (maximal
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lightness was 51.32 compared to 47.77 in 100% beef formulations). A special cubic model was

fitted for a* (redness) (Fig. 3b). It can be seen in Fig. 3b that maximal values for redness were
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observed at approximately 4-5% of both LF and RP, while minimum values (7.65) were
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observed at highest LF inclusion levels (above 9%) (Fig. 3b). A linear relationship for
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yellowness (b*) was observed for LF and RP inclusion (Fig. 3c).

All colour parameters were significantly affected by novel ingredient inclusion. With regard to
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potential visual acceptance, novel fortification with LF (5-3.33%) and RP (1-3.33%) increased
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redness (a*), compared to 100% meat formulations, from 9.45 to 10.24. The bright red colour in

meat is perceived as a positive attribute for consumer acceptance, LF and RP fortification at

these levels may enhance visual acceptance of the product by consumers. However, the increased

lightness and yellowness with LF and RP may be a negatively perceived change.

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3.3 Texture profile analysis

The identification of meat product formulations that would fulfil nutritional requirements, while

maintaining palatability and ease of eating was an important goal of this study. Experimental

data obtained for textural parameters are shown in Table 2. According to Table 2, special cubic

was the best model for hardness, chewiness and gumminess, while for cohesiveness and

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springiness linear models were fitted. M, LF and RP all had a significant (P < 0.01) impact on

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hardness, cohesiveness and springiness, whereas only M and RP were significant (P < 0.01) for

chewiness and gumminess. Response surfaces for hardness, chewiness and gumminess showed

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similar trends in relation to the ingredient inclusion levels (Fig. 4a, 4b, 4c). The contour plots

demonstrate that these parameters were maximal with inclusion levels between 3.33-4% for both
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LF and RP. As well as main effects, interaction effects (M*LF*RP) on hardness, chewiness and
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gumminess were observed (Table 2). Springiness was positively correlated with RP inclusion

and negatively correlated with LF (Fig. 4d). Cohesiveness declined as LF levels increased (Fig.
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4e). The observed increase in textural parameters with RP could be attributed to enhanced gel
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formation in the product matrix due the increased protein content (Youssef & Barbut, 2011).

These observations are similar to protein-enriched savoury products prepared using defatted
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groundnut cake flour (Purohit & Rajyalakshmi, 2011). The observed texture modification and
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softening caused by lentil flour could be related to hydration of the low density particles which

comprise this ingredient and is supported by our observations on water-holding capacity (cook

loss). This effect was also evident in cakes prepared with flour with low particle size to obtain

better texture (Gómez, Ruiz-París, & Oliete, 2010).

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While the RP was advantageous in terms of boosting protein content of enriched patties,

inclusion of LF seems to provide a beneficial effect on all textural parameters, helping to offset

any negative effects of RP inclusion on hardness, chewiness and gumminess. This would be an

important consideration in the development of new product for the elderly i.e. a softer texture is

beneficial for consumers with reduced or impaired mastication.

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3.4 Microbiological Analysis

Significant models were obtained for aerobic mesophillic bacteria (P < 0.01; R2 = 0.71),

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psychrotrophic bacteria (P < 0.001; R2 = 0.79) and pseudomonas (P < 0.01; R2 = 0.85) (Fig. 5a-c;
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Table 2). However all treatments were found acceptable after 12 days at refrigerated storage

(levels >106 = spoilage in mesophillic, psychrotrophic and pseudomonas bacteria) (O'Sullivan,

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Le Floch, & Kerry, 2015, European Commision, 2007). Plant-based protein ingredients, PPI, RP
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and LF did not affect microbial parameters hence they can be commercially viable for the
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development of functional meat products for elderly consumers.

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3.5 Optimisation and validation of enriched beef patty formulation

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Product optimization establishes conditions that simultaneously satisfy the requirements placed

on each of the responses and conditions. To obtain simultaneous optimisation, individual

parameters can be maximised, minimised or set within limits. In this study, inclusion levels of

RP and LF were both maximised, while significant dependent factors such as protein, moisture

lipid oxidation, hardness (Table 3) were either minimised/maximised depending on their

beneficial effect on the desired final product. This tool predicted two optimised formulations
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(OF) which maximised protein and generated predicted values for each dependent variable

(Table 3) which had acceptable overall desirability levels (0.50) for rice protein and lentil flour

addition. These consisted of: OF1) RP 3.63% and LF 6.22% and OF2) RP 3.95% and LF 6.05%.

Validation is an important step in RSM in order to assess that the model and limits are accurate

and precise. Table 3 illustrates the predicted and experimental results for the optimised

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formulations and Table 4 shows the performance of the model indices. The models showed a

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good fit for both formulations as shown by the accuracy and bias factors which are close to 1.00

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for the key responses. Our results demonstrate that a mixture design approach can be applied to

develop an optimised enhanced protein, softer beef patty formulation with acceptable

technological properties.
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3.6 Sensory analysis of optimised formulations

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OF1, OF2 and control (100% beef) formulations were assessed by panel members (Table 5).
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Significant differences were observed between formulations for beef aroma and tenderness.

Texture attribute scores improved from 4.19 (control) to 6.62 and 7.27 (OF1 and OF2)
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respectively. Gao, Zhang, and Zhou (2014) also observed an increase in tenderness in pork

patties with the addition of glutinous rice flour. In an unexpected finding, RP and LF inclusion
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also intensified the aroma quality of beef patties (P < 0.01). Other attributes of off-odour,

chewiness, juiciness, greasiness and off-flavour of beef patties were comparable to controls (P >

0.05). While there was a significant effect of RP and LF inclusion on instrumental colour, the

sensory analysis indicated that any differences in colour were not perceptible to panel members

because OF1 and OF2 did not differ from controls in subjective colour (P > 0.05).
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4. Conclusion

A mixture design approach was found to be an effective technique to optimise inclusion of plant-

based protein (RP and LF) in fortified beef patties. Overall, a substitution of approximately 4-7%

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lentil flour and 1-4% rice protein can elevate protein content of beef patties in comparison to

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controls and do not compromise, but may enhance sensory attributes and technological

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parameters such as cooking loss, hardness, lipid oxidation and microbiological growth. The

validation of the models, OF1 and OF2, has successfully showed that LF (at 6.22 and 6.05%) and

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RP (at 3.63 and 3.95%) produced a fortified-protein beef pattie with acceptable textural
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parameters. The development of fortified beef patties with the inclusion of plant-based protein

ingredients could help older adults achieve targeted protein requirement in smaller portion size,
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thus reducing risk of sarcopenia.

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5. Acknowledgements
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This research was funded by the FIRM programme administered under the Irish Department of

Agriculture, Food and the Marine (11/F/045): enhancement of texture, flavour and nutritional
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value of meat products for older adults and the Walsh Fellowship Scheme.

The authors also wish to acknowledge Dr. Derek F. Keenan and Dr. Norah O’Shea for their useful

comments in the preparation of this manuscript and statistical advice and the technical support of

Dr. Clare Corish, Dr. Eimear Gallagher, Mr. Eugene Vesey, Mr. Des Walsh and Ms. Sarah Lynch.
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a)

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Fig. 1 Contour plots for protein and moisture content (%) analysis on day 0
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d)

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Fig. 2 Contour plots for cook loss on storage day 1 and 12 respectively, water holding capacity on day 2
and lipid oxidation on day 12 in enriched protein beef patties
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Fig. 3 Contour plots for instrumental colour (L*, a* and b*) on day 12
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Fig. 4 Contour plots for texture profile analysis (TPA) on day 12

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Fig. 5 Contour plots for microbiological analysis (log CFU/g) on day 12

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Table 1 Experimental design of three components in beef patty formulation

Run Order A1: % Meat (M) B2: % Lentil Flour C3:% Rice protein
(LF) (RP)
1 90.00 5.00 5.00
2 95.00 0.00 5.00
3 90.00 0.00 10.00
4 95.00 0.00 5.00
5* 100.00 0.00 0.00
6 90.00 10.00 0.00

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7 95.00 5.00 0.00

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8 90.00 5.00 5.00
9 95.00 5.00 0.00

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10 90.00 10.00 0.00
11 90.00 10.00 0.00
12* 100.00 0.00 0.00
13 95.00 0.00 5.00

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14 95.00 5.00 0.00
15 90.00 0.00 10.00
16 93.33 3.33 3.33
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17 90.00 0.00 10.00
Where: (low) 90 <A1: Meat < 100 (high); (low) 0 <B2: Lentil flour < 10 (high); (low) 0 <C3: Rice protein
< 10 (high): and A1 + B2 + C3 = 100% of total weight beef patty, * Controls
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Table 2 Analysis of variance of the regression models and regression coefficients for significant quality
and textural parameters of beef patties
Dependent Model Independent Variables
variables fitted
M LF RP A1 * A1 * B2 * A1 * B2 * R2 p P
A1 B2 C3 B2 C3 C3 C3 (Mod (Lac
el) k of
fit)
Protein Quadra 22.6 22.1 28.70 - - - 0.9 0.001 0.71
tic 6 0 4.34* 6.28 0.05 7
** 0 ns
Moisture Linear 72.5 65.7 66.35 0.8 0.001 0.08
1 3 7
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TBARS Quadra 1.52 2.43 1.67 -1.05 1.62 -1.83 0.6 0.040 0.75
tic ns ns ns 4
Cook loss Linear 28.5 13.0 22.92 0.8 0.001 0.31
7 0 5
WHC Linear 94.0 96.4 94.68 0.7 0.001 0.65
5 6 6
L* Linear 48.3 50.9 50.99 0.4 0.038 0.52
9 1 0
a* Special 8.93 7.64 8.90 - - -4.72 92.45** 0.7 0.026 0.76
cubic 6.00* 4.74 ns 4

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b* Linear 9.61 11.9 11.74 0.4 0.017 0.68

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2 7
Hardness Special 200. 124. 328.4 - 50.7 - 7090.14 0.8 0.005 0.96

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cubic 58 38 9 166.2 6 ns 128. ** 3
1 ns 17 ns
Cohesivene Linear 0.84 0.75 0.83 0.6 0.002 0.00
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Chewiness Special 558. 172. 1013. - 250. - 28091.1 0.8 0.005 0.85
cubic 53 89 42 705.2 17 ns 909. 0** 2
2 ns 08 ns
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Gumminess Special 96.4 30.2 144.3 - 50.7 - 3432.91 0.8 0.002 0.65
cubic 6 8 0 120.4 0 ns 111. ** 6
7 ns 06 ns
Springiness Linear 5.85 5.55 7.07 0.4 0.015 0.50
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Mesophillic Quadra 7.51 7.38 7.41 - -0.09 -0.32 0.7 0.018 0.47
tic 1.07* ns ns 1
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Psychrotro Linear 7.47 7.08 7.51 0.7 0.001 0.72
phic 9
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Pseudomon Special 7.34 7.19 7.36 - 0.04 -0.33 6.66** 0.8 0.002 1.00
as cubic 0.46* ns ns 5
Where A1*B2 = interaction between meat and lentil; A1*C3 = interaction between meat and rice, B2*C3 =
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interaction between lentil and rice, A1* B2*C3 = interaction between meat, lentil and rice; *, ** and ***
are significance levels at P < 0.05; P < 0.01; and P < 0.001; ns = not significant
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Table 3 Optimised formulations and predicted responses based on constraints applied to significant
variables compared to experimental data
Variables Optimisation Formulation 1 Experimental Formulation 2 Experimental
criteria Formulation Formulation
1 2

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Independent
A: M Maximised 90.15 90.15 90 90

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B: LF Maximised 6.22 6.22 6.05 6.05
C: RP Maximised 3.63 3.63 3.95 3.95

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Total (A + B + 100 100 100 100
C)

Dependent

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Moisture Maximised 66.06 68.74 65.98 69.06
Protein Maximised 24.42 24.29 24.69 24.21
Lipid oxidation Minimised 1.73 0.71 1.70 0.55
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Cook loss Minimised 16.83 16.87 16.92 16.08
WHC Maximised 95.77 96.78 95.75 97.18
L* Range: 46.67 - 50.89 49.21 50.94 49.49
52.18
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a* Maximised 7.27 8.06 7.01 7.92

b* Range: 8.95 - 11.82 12.98 11.85 13.43
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Hardness Minimised 192.84 107.61 174.37 120.20

Cohesiveness Range: 0.73 – 0.78 0.83 0.78 0.83
0.87
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Chewiness Minimised 366.05 244.64 287.63 297.25

Gumminess Minimised 58.07 47.54 48.77 52.56
Springiness Range: 4.98 - 6.11 5.14 6.15 5.68
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7.74
Mesophilic Minimised 7.31 4.16 7.31 4.09
bacteria
Psychrotrophic Minimised 7.24 4.08 7.25 4.13
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bacteria
Pseudomonas Minimised 7.20 2.35 7.18 2.70
Desirability Minimised 0.52 0.52
Where: Moisture, protein, cook loss = %; WHC = water holding capacity = %; Lipid oxidation =
mg/MDA/kg of meat; Hardness, gumminess = N; Chewiness = J; Springiness = mm; L*, a*, b* and
cohesiveness = unitless, mesophilic, psychrotrophic, pseudomonas = (log10 CFU/g)
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Table 4 Calculated model performance indices accuracy factor (AF), bias factor (BF) and average mean
deviation (∑%) values for optimised formulation 1 and 2
Formulation 1 Formulation 2
Accuracy Bias ∑ Accuracy Bias ∑
Variables Factor Factor (%) Factor Factor (%)
Moisture 1.04 0.96 3.90 1.05 0.96 4.46
Protein 0.99 1.01 0.54 0.98 1.02 1.98
143.6 209.0
Lipid oxidation 0.41 2.44 6 0.32 3.09 9

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Cook loss 1.00 1.00 0.24 0.95 1.05 5.22
WHC 1.01 0.99 1.04 1.01 0.99 1.47

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L* 0.97 1.03 3.41 0.97 1.03 2.93
a* 1.11 0.90 9.80 1.13 0.89 11.49

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b* 1.10 0.91 8.94 1.13 0.88 11.76
Hardness 0.56 1.79 79.20 0.69 1.45 45.07
Cohesiveness 1.06 0.94 6.02 1.06 0.94 6.02

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Chewiness 0.67 1.50 49.63 1.03 0.97 3.24
Gumminess 0.82 1.22 22.15 1.08 0.93 7.21
Springiness 0.84 1.19 18.87 0.92 1.08 8.27
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Mesophilic bacteria 0.57 1.76 75.72 0.56 1.79 78.73
Psychrotrophic
bacteria 0.56 1.77 77.45 0.57 1.76 75.54
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206.3 165.9
Pseudomonas 0.33 3.06 8 0.38 2.66 3
AF and BF = values close to 1.00 suggests a good model fit; ∑ (%) = variation between predicted and
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Table 5 Sensory attributes of optimised formulations compared to control beef patties

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Attributes Control OF1 OF2 LSD
a b ab
Aroma 3.6 ± 1.9 6.6 ± 1.7 5.5 ± 2.3 2.1

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Off-odour 2.5 ± 1.0 3.8 ± 2.7 3.6 ± 2.7 2.4
a b b
Tenderness 4.2 ± 1.5 6.6 ± 1.3 7.3 ± 2.5 1.9
Chewiness 5.3 ± 2.4 3.6 ± 2.6 3.3 ± 2.5 2.6

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Juiciness 3.1 ± 1.8 5.1 ± 2.3 6.0 ± 2.8 2.4
Greasiness 2.3 ± 1.4 3.7 ± 1.5 4.7 ± 2.4 1.9
After taste 2.6 ± 1.1 5.1 ± 2.8 4.5 ± 2.4 2.3
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Off-flavour 2.5 ± 1.1 4.6 ± 2.6 3.4 ± 2.1 2.1
ab
Means in the same row that do not share the same superscript are significantly different according to
Fisher’s protected test (P < 0.01): attributes were evaluated by means of 10cm line scale where aroma,
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off-odour, tenderness, chewiness, juiciness, greasiness, after taste and off-flavour were ranked (0 = not
intense/ very hard/not chewy/dry/not greasy/not intense/dislike; 10 = intense/very tender/very chewy/very
fatty/very intense/like); where each sample was consumed randomly 4 times with 8 panellists and 8
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sessions in total
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