Development of novel fortified beef patties with added functional protein

ingredients for the elderly

Sephora Baugreet, Joseph P. Kerry, C. Botineştean, Paul Allen, Ruth M.

Hamill

PII: S0309-1740(16)30214-5
DOI: doi: 10.1016/j.meatsci.2016.07.004
Reference: MESC 7052

To appear in: Meat Science

Received date: 26 February 2016

Revised date: 7 July 2016
Accepted date: 8 July 2016

Please cite this article as: Baugreet, S., Kerry, J.P., Botineştean, C., Allen, P. & Hamill,
R.M., Development of novel fortified beef patties with added functional protein ingredi-
ents for the elderly, Meat Science (2016), doi: 10.1016/j.meatsci.2016.07.004

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 ACCEPTED MANUSCRIPT

Development of novel fortified beef patties with added functional protein

ingredients for the elderly

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Sephora Baugreet1, Joseph P. Kerry2, C. Botineştean1, Paul Allen1 and Ruth M. Hamill1

1
Teagasc Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland

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2
Food Packaging Group, School of Food and Nutritional Sciences, Food Science Building, University College Cork, Ireland

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Email: Ruth.Hamill@teagasc.ie

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Abstract

The effects of clean label functional protein ingredients; pea protein isolate (PPI), rice protein
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(RP) and lentil flour (LF), at 3% and 7% inclusion levels on technological and shelf life
parameters of beef patties were evaluated over 12 days. Protein content in the RP7 treatment was
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higher (P<0.05) than controls, but did not differ significantly from PPI7 and RP3. No effects on
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moisture, fat or ash content were detected. LF addition reduced product hardness, cohesiveness,
gumminess and chewiness compared with controls. RP-enriched and control patties were
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associated with lower lipid oxidation over storage than PPI- and LF-enriched patties. RP had a
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differential effect on (L*), (a*) and (b*) when compared with controls. Microbiological
characteristics for all treatments were acceptable after 12 days. Protein fortified beef patties with
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a softer texture and acceptable technological properties targeted for ease of consumption by older
adults were produced in an attempt to reach dietary targeted protein requirements for this
segment.

Keywords: Elderly, sarcopenia, clean label, functional ingredients, protein-enriched, beef patties

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1. Introduction

Between 1990 and 2013, the elderly cohort of the world population increased from 9.2% to
11.7% (United Nations, 2013). Moreover, it is forecast that, by 2025, the proportion of older

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adults aged over 65 in Europe will rise by a further 20%, coupled with an even more rapid
increase in the numbers of people over 80 years of age (European Commission, 2015). The

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prevalence of many age-related conditions faced by older adults, such as; sarcopenia, dementia
and dysphagia, is therefore highly likely to increase in the coming years. Sarcopenia is a

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condition pertaining to the gradual loss of skeletal muscle mass that is associated with an
increased susceptibility to falls and fractures, which leads to reduced mobility and independence,

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and can greatly affect quality of life. The prevalence of sarcopenia among older adults aged 60-
70 years of age is between 5-13%, while the prevalence in those aged 80+ ranges from 11-50%
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(Santilli, Bernetti, Mangone, & Paoloni, 2014), indicating that this condition is especially
prevalent among the frail elderly.
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Higher protein intakes are recommended for older adults than for younger adults in order to
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reduce the prevalence/severity/onset of sarcopenia and other age-related diseases (Volpi et al.,
2013; Wolfe et al., 2008). While current protein reference nutrient intake (RNI) is set at 0.8g
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protein/kg/bodyweight/day for healthy adults of all ages, a protein intake of at least 1.0 to 1.2g
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protein/kg/bodyweight/day is recommended for healthy older adults aged 65+ and 1.2 to 1.5g
protein/kg/bodyweight/day for older adults with chronic illnesses. Achievement of recommended
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dietary protein intakes by more elderly people would result in improvements in/greater
maintenance of muscle mass, physical function and bone strength in this cohort. However,
despite these recommendations, as an adult advances in age, dietary protein intake will normally
decrease for various reasons such as reduced appetite, loss of chemosensory perception,
consuming a less varied diet, dental issues and the consumption of smaller portions (Nicklett &
Kadell, 2013), which makes attainment of dietary recommendations more difficult to achieve.

Because food preferences by older adults are well established (Bernstein & Munoz, 2014), they
are more likely to maintain traditional food habits and choices, for example, regular consumption
of meat and dairy. Red meat is a high biological-value protein source (Symons et al., 2007) and
is nutritionally balanced with micronutrients, such as; iron, zinc, selenium, potassium and a

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range of B-vitamins. Because it forms a traditional part of the diet of many elderly people, red
meat is a suitable vehicle for further fortification with nutrients targeted at elderly people, such
as protein. Addition of dietary proteins from sources such as pulses or legumes in fortified meat
products could fulfil this requirement in compliance with clean label requirements. Non-meat

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proteins from legumes such as bean flour (Dzudie, Scher, & Hardy, 2002) and corn flour
(Serdaroğlu & Değırmencioğlu, 2004) have previously been used as binders and extenders in

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comminuted meat products, but have not been tested as ingredients to enhance protein content

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and intakes for vulnerable population cohorts. Legumes are composed of a balanced amino acid
profile and display good protein digestibility as well as containing fibre, vitamins (folate) and

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minerals that are ideal to enhance the nutritional characteristics of meat products (de la Hera,
Ruiz-París, Oliete, & Gómez, 2012). They have furthermore been shown to have beneficial
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effects on bone health (Alekel et al., 2000). They also display good technological and sensorial
properties, as well as being relatively cheap and stable (Silva-Cristobal, Osorio-Díaz, Tovar, &
Bello-Pérez, 2010).
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The objective of this study was to investigate the inclusion of clean label ingredients rich in
protein: pea protein isolate (PPI), rice protein (RP) and lentil flour (LF) in a meat model product
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(beef patty). The technological and shelf-life parameters of the applied protein ingredients were
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determined with a view to developing protein-enriched meat products aimed at enhancing protein
intake in the elderly segment of the population.
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2. Materials and Methods

2.1 Preparations of samples, packaging and storage

Approximately 20 kg of beef pieces [95% visual lean (VL)] were obtained from Kepak (Dublin,
Ireland) and chilled overnight (1 to 4°C). Excess fat was trimmed from the muscle which was
then roughly diced. Approximately 2-3 kg of the meat was used to prepare each of seven
experimental treatments. This was added to a mixer mincer (La Minerva Model No. CIE701,
Bologna, Italy) and minced once through a mincing attachment of 5mm in diameter and mixed
for 8 min. Seven experimental groups were prepared; non-treated control sample (control),
samples with part of the weight replaced with protein ingredients at 3% and 7% (PPI3% and

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PPI7%), (RP3% and RP7%) and (LF3% and LF7%). The whole experiment was repeated three
times using separate batches of beef which were treated equally under the same conditions. Once
the protein ingredient was added, each batch was manually mixed (3min) then minced through a
3.5mm sieve plate to obtain a homogeneous mixture. Beef patties were formed using a

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conventional patty maker (~75g/patty), to average dimensions of 10cm diameter and 1cm
thickness. Patties were packaged in black amorphous polyethylene terephalate (APET/PE)

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padded retail trays, then gas flushed (Ilpra Foodpack VG 400 Packaging Machine, Italy) using a

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modified atmosphere packaging (MAP) of 80% O2: 20% CO2 (Air Products and Chemical Inc.,
Dublin, Ireland), sealed with a low oxygen permeable barrier film (3cm3/m2/24h at STP)

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polyvinyl-chloride film (Versatile Packaging, Ireland) and stored for 12 days at 4°C. Samples
were analysed during storage for the following parameters: pH (day 1, 6, 12), colour (day 0, 3, 6,
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9, 12) composition analysis (day 1), TPA/cook loss (day 1, 6, 12), TBARS measurements (day 0,
3, 6, 9, 12) and microbiological analysis (day 1, 12).
All reagents used were purchased from Sigma-Aldrich, Ireland and Fannin, Ireland. Pea protein
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isolate (83% protein) was supplied by Redbrook Ingredients (Dublin, Ireland) and rice protein
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(79% protein) and lentil flour (Lens Esculenta, 23.8% protein) from Healy Group (Dublin,
Ireland).
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2.2 Proximate composition analysis for raw beef patties

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Compositional analysis was carried out on raw samples thawed at 4°C overnight. Protein was
determined using a LECO FP-628 (LECO® Corp., MI, USA) according to AOAC official
method 992.15, 1990. Fat and moisture were determined using the Smart System 5 microwave
moisture drying oven and NMR Smartrac rapid analyser (CEM Corporation, USA) as described
by Keenan, Resconi, Kerry, and Hamill (2014). Ash content was determined by a standard
method (ISO 936:, 1998).

2.3 Measurement of pH of beef patties

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The pH values of raw beef patties were measured using a glass probe pH electrode (Thermo
Scientific pH meter 420A, Orion Research Inc) and readings were obtained in triplicate from
each sample. pH of the beef patties was recorded on days 1, 6 and 12 of MAP chilled storage.

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2.4 Texture measurements and cook loss

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Texture Profile Analysis was performed on cooked beef patties based on a method described by

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Keenan et al. (2014) on days 1, 6 and 12 of chilled storage. Beef patties were cooked on a frying
pan on an electric hob at low heat, turning every 2 min until it reached an internal core

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temperature of 72°C (Thermometer PT1000, Eurolec Instrumentation Ltd, Dublin).
Measurements were carried out on an Instron Universal Testing Machine (4464) (Instron Ltd.,
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High Wycombe, UK). Nine cores (diameter 25mm) were taken from random locations in three
cooked beef patties per treatment. Cylindrical samples were compressed twice axially to 90% of
their original height at a crosshead speed of 100mm/min using a 500 N load cell. Textural
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parameters were expressed as described by Keenan et al. (2014).

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Cooking loss values were determined by calculating the difference in weight of three patties both
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before and immediately after cooking. Before recording weights, samples were blotted with a
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paper towel to remove excess surface moisture.

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2.5 Measurement of colour stability

Surface colour was measured at room temperature using the CIE L*a*b* system with a dual
beam xenon flash spectrophotometer (UltraScan XE, Hunterlab) on days 1, 3, 6, 9 and 12 of
storage. The Ultrascan XE was standardised using a black light trap and white tile. The
illumination (D65, approximates to daylight, 10°) with 8° viewing angle and a 10 mm port size
was used. Chroma (C*) and hue-angle (h) were also calculated by the following formulae: C* =
(a*2+b*2)1/2, h = tan−1(b*/a*). Each package was opened and left to bloom for 30 min before
measurements were recorded. The average of six readings per patty per treatment was recorded.

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2.6 Measurement of lipid oxidation

Thiobarbituric acid reactive substance (TBARS) values were measured as an indicator of lipid
oxidation, according to the filtration method described by Siu and Draper (1978). TBARS values

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were measured in raw beef patties on days 0, 3, 6, 9 and 12 of storage. Three individual beef
patties from each treatment were analysed in duplicate each time. Approximately 2.5g of sample

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was homogenized in 10ml of 5% trichloroacetic acid and 10mg butythydroxytoluene for 60 sec
at high speed using an Ultraturrax homogeniser (Labortechnik, Staufen, Germany).

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Homogenised samples were centrifuged at 2823 G for 40 minutes at 4°C. They were then filtered
through filter paper (Whatman number 4) and 3ml of the filtrate was mixed with 3ml 2-

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thiobarbituric acid (TBA) solution (0.289% in distilled water). Tubes were covered with tin foil
and placed on a heating block for 1 hr. at 100°C. Absorbance was subsequently measured
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spectrophometrically on a UV-Vis Spectrophotometer Shimadzu UV-1700 (Columbia, USA) at
wavelength 532nm. Results were expressed as mg malonaldialdehyde (MDA)/kg beef.
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2.7 Microbial Analysis

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After aerobic storage, all minced samples (10g) were aseptically transferred into sterile filter
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stomacher bags and stomached for 30 sec using a model BA6024 Colworth stomacher (Seward,
UK) in 90ml of Oxoid maximum recovery diluent (MRD) to form a meat homogenate. Five
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dilutions of meat homogenates were prepared in 9ml MRD tubes. To analyse mesophilic and
psychrotrophic bacteria, meat homogenates were examined by duplicate plating 0.1ml volumes
onto Oxoid plate count agar (PCA) and incubated at 30°C and 6°C for 3 and 10 days respectively,
to estimate total viable count (TVC) (ISO 4833-1:, 2013; ISO 17401:, 2001). Pseudomonas
numbers were determined in Oxoid Pseudomonas selective agar, with CFC supplement
incubated at 30°C for 3 days (ISO 13720:, 2010) and lactic acid bacteria (LAB) numbers were
estimated in Oxoid de Man-Rogosa-Sharpe agar (MRS), over poured with the same medium
incubated at 30°C for 3 days (ISO 15214:, 1998). Enterobacteriaceae (ENT) numbers were
determined in Oxoid violet red bile glucose agar (VRBGA) medium, over poured with a further
10ml of VRBGA and incubation at 37°C for 22 hr. (ISO 25128-1:, 2004).

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2.8 Statistical Analysis

Three replicates of the experiment were carried out. Each measurement was performed in
duplicate and mean values ± standard errors were reported. The factors were fixed effects and the

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experimental design was a split-plot in time analysis of variance (ANOVA) with treatment or a
factorial arrangement of treatments on the main plot and storage days on the sub plot analysed

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using the GenStat Statistical Package (Release 14.1, Hertfordshire, UK). Differences between
replicates were not significant (P<0.05) so this term was removed from the final model.

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Significant differences among treatments were assessed using Fisher’s LSD test, the level of
significance was set as P<0.05.

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3. Results and Discussion
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3.1 The effect of inclusion of pea protein isolate, rice protein and lentil flour in beef patties on
proximate analysis
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Composition analysis of controls and enriched beef patties formulated with PPI, RP and LF at
two inclusions levels are presented in Table 2. The goal of the study was to enrich beef patties
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with additional protein over and above the intrinsic protein content of 95% VL beef. Protein
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content in 100% beef patty controls was 22.17%. Protein content was significantly (P<0.05)
higher in RP7-enriched beef patties in comparison to controls. However, the other treatments did
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not significantly increase protein relative to the control. While RP7 was significantly higher in
protein than LF patties, it did not differ significantly in protein content compared to PPI7 and
RP3. Plant-based protein ingredients have previously been used in meat products for
technological purposes, as extenders for cost reduction and for their nutritional composition.
However, they can provide nutritional benefits by increasing the protein content and balancing
the amino acid profile (Jimenez Colmenero, 2004). For example, Owusu‐Ansah & McCurdy
(1991) reported pea protein used as a filler for sausages improved protein content compared to
controls. With a protein content of 79g/100g and 83g/100g dry solids, rice protein and pea
protein isolate are considered a rich source of protein and have been proposed as a potential
replacer for commonly used, but allergenic, proteins such as soy and whey in selected food
products (Souza, Sbardelotto, Ziegler, Marczak, & Tessaro, 2016). For this reason, the actual

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protein content of the patty was analysed and it should have significantly increased in the case of
RP and PPI. The actual protein ingredient was analysed and found to be (77% in PPI), (79% in
RP) and (26% in LF). According to the ingredients percentages mentioned above, PPI7 enriched
patties should have resulted in higher protein content (26%). However, this increase was not

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evident and could possibly be due to ingredient loss during processing. This could be
investigated further in future work. Under the current Regulation (EU) No. 1047/2012 of the

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European Parliament and the Council on nutrition claims, a ‘high protein’ claim could be made

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for the RP7-enriched patties because at least 20% of the energy value of these patties is provided
by protein. Lentil flour is not enriched in protein, and has a protein content of 23.8g/100g dry

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solids. Hence, it is not surprising that addition of lentil flour did not enhance the protein content.
Serdaroǧlu, Yildiz-Turp, and Abrodímov, (2005) observed a decrease in protein level with the
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addition of LF to low-fat meatballs which may be attributed to the contribution of carbohydrates
from LF since the protein and fat content of lentil is lower than that of meat. No significant
differences (P>0.05) in the moisture, fat and ash contents between any of the experimental beef
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patties was recorded; therefore, the addition of PPI, RP or LF into the patties had no influence on
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these compositional parameters of beef patties.

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3.2 pH measurements of enriched beef patties

There was no treatment effect or interaction between treatment and storage day among pH values
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(Table 3). This is consistent with the findings of Elif Bilek and Turhan (2009) who reported no
significant difference among beef patties enhanced with flaxseed flour at different levels.
Nevertheless, statistical analysis indicated that the storage period (between day 6 and day 12)
significantly affected all patty formulations (P<0.01). There was a significant increase in pH
values from day 1 to day 6 and a decrease from day 6 to the end of storage (day 12), which could
be at least partially due to breakdown of protein in the patties by microorganisms (Soltanizadeh
& Ghiasi-Esfahani, 2015).

3.3 Texture profile analysis and cook loss of beef patties

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Meat intake is often reduced in elderly people since meat is a complex matrix that can present a
challenging substrate from a texture perspective. Hardness, cohesiveness, chewiness and
gumminess value of beef patties were significantly affected by treatment (Table 4). Hardness
was influenced by the addition of PPI, RP and LF, with the lowest value found in LF7-enriched

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patties. This was significantly lower than controls (82.5). In contrast, hardness was higher
(P<0.05) in PPI7 and RP7 in comparison to controls. As both of these protein ingredients (PPI

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and RP) contain a low moisture content (<10%) (as per product specification), the increase in dry

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matter in treatments may have caused the observed increase in hardness. These results are in line
with Youssef and Barbut (2011) who reported that adding whey protein to meat batters increased

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hardness compared to controls. Also, an increase in hardness values has been previously
described in food emulsions which can be explained as a result of an imbalance in the emulsion
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process, causing water and fat from the protein molecules to separate (Fernández-Ginés,
Fernández-López, Sayas-Barberá, & Pérez-Alvarez, 2005). Similarly to LF-enriched beef patties
in this study, lower hardness values were reported when glutinous rice flour was added to beef
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patties, in comparison to controls (Yi et al. 2012). Any increase in hardness in a product
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targeting elderly people, who suffer from dentition issues and impaired ability to chew hard
textures must be carefully considered. The softening effect obtained by the addition of the LF
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may be explained due to the low density of lentil flour particles which hydrate rapidly, thus
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contributing a softer texture to a product and this was evident in noodle preparation with fine
powder flour (Hatcher, 2001). Springiness can be described as the rate at which the deformed
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beef patty springs back after the first compression. No significant difference was found between
any treatment and the control. Cohesiveness is described as the degree to which the beef patty
can be deformed before it breaks. Addition of LF reduced (P<0.05) cohesiveness in comparison
to controls, and also PPI and RP which did not differ significantly from each other. In fact,
cohesiveness values increased comparably with hardness values observed in PPI and RP. This
trend was also observed for gumminess (i.e., the energy required to break down a semi-solid
food before swallowing) and chewiness (i.e., the energy required to chew a solid food before
swallowing). Both parameters increased (P<0.05) with the addition of PPI7 and RP7 and were
reduced (P>0.05) in LF7-enriched beef patties compared to controls. The textural characteristics
of LF-enriched patties could be considered especially appropriate for the elderly because of
easier mastication during eating. Based on the composition and texture analysis results, it could

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be speculated that a combination of rice protein and lentil flour as additives to beef patties could
serve to enhance protein content while offsetting some of the hardening effects of the protein
enrichment.

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Cook loss in meat products is influenced by numerous variables for example, composition,
cooking methods, temperature and sample dimensions etc. as indicated by Yi et al. (2012). There

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were significant effects of added functional ingredients on cook loss (Table 4). Addition of LF7-,
PPI7- and RP-enriched beef patties improved (P<0.05) cooking yield in beef patties in

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comparison to control samples. According to the product specification, the carbohydrate content
of the utilised lentil flour was approximately 53.2g/100g. The polysaccharides in lentil flour,

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either alone or in interaction with proteins may form a network that traps water and prevents its
release (Soltanizadeh & Ghiasi-Esfahani, 2015). The high water holding capacity of lentil flour
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(Zayas, 1997) could therefore serve to retain the water released from the meat component of the
patty during thermal processing. Pea protein isolate has been reported to absorb 1- to 3-times its
weight in water; hence it could be similarly useful in comminuted products, as it imparts
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structure (swelling) to the meat product (Owusu-Ansah & McCurdy, 1991). All the ingredients
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used in this study therefore provided increased capability of retaining moisture and fat during the
cooking process, and lower cook loss compared with controls, exerting a beneficial effect on the
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beef patties’ textural properties.

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3.4 Colour stability of raw beef patties containing added pea protein isolate, rice protein and
lentil flour

Colour, appearance and presentation of the product are very important for the consumer, and
greatly influence purchasing decisions (Nassu et al, 2012). Ideally, novel functional meat
products should have similar colour characteristics to their conventional alternative. Instrumental
colour parameters are presented in Table 5. Treatments and storage days affected (P<0.05)
colour parameters in beef patties. Addition of RP7 increased (P<0.001) lightness (L*) values in
comparison to controls from 49.11 to 52.69 on day 12. LF7 also increased lightness to a lesser
extent compared to controls (51.80). Similarly, Youssef and Barbut (2011) reported that meat
batters prepared with 1.5% soy protein and whey protein showed a significant increase in (L*)

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values. This increase may be attributed to natural colour of the ingredients RP and LF, which are
paler than meat. The increased in lightness in RP could also be associated to the fat content (5%
as per product specification), which may drive an increase in the overall light scattering
properties. Similarly, increases in L* have been reported with flaxseed flour with 20% fat

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inclusion in beef patties (Elif Bilek and Turhan 2009). Sensory evaluation would need to be
carried out to confirm if these differences are visually detectable. Significant (P<0.001)

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variations were detected between day 1, 6 and 9 across all treatments. No interaction between

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treatment and day of storage was observed (Table 5).

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For beef, consumers prefer bright red beef rather than purple or brown. Redness (a*) values were
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increased (P<0.001) following the addition of RP at both levels (3 and 7%). No significant
changes were found in redness (a*) with the addition of LF compared to controls. While it is
generally desired to closely match the physical appearance of novel products to conventional
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alternatives, an increase in a parameter such as redness in a fresh meat product such as a beef
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patty could be viewed as a desirable improvement. Additionally, a high O2 atmosphere

contributes to the bright red colour of meat. Acceptability would need to be assessed using
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consumer panels. Regarding storage time, a decline (P<0.001) in a* values was observed from
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day 6 to day 12 of patties enriched with PPI, RP and LF at both levels, but this trend did not
differ from controls. The reduction in the intensity of red colour in meats at latter time periods of
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storage could be explained due to the correlation between lipid oxidation and colour oxidation
(Lynch & Faustman, 2000). This change can occur due to the production of free radicals during
oxidation, which denatures the myoglobin molecules in meat products. On the other hand, the
observed pH change between day 6 and 12 could also have affected redness.

Rice protein enriched patties at 7% had the highest (b*) values (12.85) in comparison to controls
(10.70) on day 12. Yellowness (b*) decreased to a small extent and in a similar fashion across all
treatments after day 6. Chroma (C*) values were higher (P<0.001) in RP7 in comparison to
controls indicating increased saturation index of the red colour of beef patties. Hue (h*) values
(i.e. indication of meat browning (shifting from red to yellow)), were inversely related to (C*)
values. No differences were detected for (h*) values between beef patties formulated with PPI,
RP3 and LF while control samples showed the lowest (h*) values.

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It can be concluded that PPI had no differential effect on colour parameters (L* and b*) when
compared with controls. On the other hand, RP (at 3 and 7%) enhanced redness values (a*) for

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beef patties.

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3.5 Lipid stability (TBARs) of raw beef patties containing added pea protein isolate, rice protein

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and lentil flour

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Lipid oxidation of beef patties were affected (P<0.001) by treatment and storage time, with
significant (P<0.001) interactions between the two factors (Table 6). Addition of PPI7 and LF (at
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3 and 7%) increased TBARS values significantly in comparison to other treatments and controls
between day 9 and 12 of storage. Upon patty storage, RP-enriched patties had similar TBARS
values to controls. At the end of storage, RP7-enriched patties had the numerically lowest degree
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of oxidation (0.264 mg/MDA/kg meat) but the difference in comparison to controls (0.692
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mg/MDA/kg meat), was not significant. On day 9, TBARS values for PPI7 and LF (at 3 and 7%)
increased (P<0.001) in comparison to other treatments and controls. The high water retention in
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LF-enriched patties could have increased lipid oxidation due to increased water availability. As
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the small molecular weight of lipid peroxides and free-radicals from oxidation products in meat
are more water soluble, they move towards the more hydrophilic part of the meat (Weiss, Gibis,
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Schuh, & Salminen, 2010), causing the water content in the meat to indirectly affect/or increase
lipid oxidation. Lipid oxidation is perceived as one of the limiting factors for the quality and
acceptability of meat products (Serdaroǧlu, Yildiz-Turp, & Abrodímov, 2005). Among the three
different ingredients added to beef patties, RP was the most effective at reducing the formation
of MDA while PPI and LF contributed to lipid oxidation. A sensory threshold value of 1mg
malonaldehyde/kg meat is suggestive for perceiving sensorial rancidity (Alakali et al., 2010). A
similar increase observed over time in other studies has been attributed to deteriorative reactions
(microbiological and enzymatic) (Brannan, 2008) or high O2 levels in packs (Sun & Holley,
2012). After day 12 of storage period, only RP among other treatments indicated a reducing-
effect of TBARS values. This may be due to the low fat content that decreases the substrate for

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oxidation. The presence of RP at both levels reduced (P<0.001) lipid oxidation relative to control
throughout storage.

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3.6 Microbial analysis of raw beef patties containing pea protein isolate, rice protein and lentil
flour

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None of the ingredients added (at 3% or 7%) had an effect on the microbiological parameters

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analysed, with all products behaving similarly to the controls over time. Table 7 shows the
effects of storage on psychrotrophic, mesophilic, pseudomonas, LAB and ENT in raw beef

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patties. Storage time had a significant effect (P<0.001) on psychrotrophic and LAB with an
increase in bacterial counts while pseudomonas and ENT had a significant increase between day
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6 and day 12. However, even by the end of the storage period, on day 12, all values were within
the acceptable thresholds for TVC (6log10 CFU/g) (O'Sullivan, Le Floch, & Kerry, 2015). The
fact that these clean label ingredients did not have a negative effect on microbial parameters is
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important from the point of view of industry and in particular, elderly vulnerable consumers.
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4. Conclusions
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Fortification of muscle-based foods could help older adults achieve targeted protein requirements
in order to decrease the effects of age-related diseases such as sarcopenia. Addition of plant
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proteins had significant and variable effects on texture characteristics of beef patties. Patties
fortified with each ingredient displayed both beneficial and slightly negative effects on different
parameters. Addition of rice protein at 7% enhanced the protein content of beef patties compared
to controls. Rice protein-enriched patties were also more effective in inhibiting oxidative rancidity
throughout storage in comparison to PPI- and LF-enriched patties. Meat texture is considered a
challenging matrix and often difficulties such as dysphagia or diminished muscular strength can
hinder food intake among the elderly. While products made using LF were generally softer than
controls and demonstrated good textural properties, this ingredient did not increase protein content
as targeted, probably due to its significantly lower intrinsic protein content. Addition of pea protein
did not significantly increase protein content, and displayed a hardening effect on beef patties,
compared to controls. They also produced a strong rancid aroma which was observed by

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experimenters throughout storage. Rice protein and lentil flour are thus the most promising
ingredients for enhancing protein content and textural parameters respectively and should be
considered to have complementary features, which would justify further exploration in a mixture
design experiment. Meat products enriched with proteins from plant sources would not only

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display improved nutritional and textural properties but also promote the use of traditional staple
food products as vehicles to help attain targeted protein intakes in older adults.

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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
MA
value of meat products for older adults and the Walsh Fellowship Scheme. The authors also wish
to acknowledge the technical assistance of Mr. Des Walsh, Ms. Karen Hussey, Mr. Eugene Vesey,
Ms. Sarah Lynch, Ms. Lauren Van-Rooyen and Ms. P. Reid, Teagasc for advice on statistical
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analysis.
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Table 1 Proximate composition (%) of raw beef patties formulated with different levels of
protein ingredients (means ± standard error)
Attribute Control PPI3 PPI7 RP3 RP7 LF3 LF7
Moisture 71.46 ± 70.31 ± 67.23 ± 70.54 ± 67.23 ± 69.00 ± 69.34 ± 1.31
2.08 1.84 1.51 1.79 2.47 0.57

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1
Protein 22.17 ± 22.65 ± 24.51 ± 24.19 ± 25.28 ± 21.69 ± 21.79 ±
ab ab bc abc c a a
0.28 1.15 0.99 1.03 3.31 0.38 0.32

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Fat 6.90 ± 5.64 ± 7.78 ± 5.52 ± 5.49 ± 6.02 ± 6.03 ± 1.53
2.12 1.37 1.94 1.86 2.07 2.39

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Ash 2.46 ± 4.61 ± 4.03 ± 1.15 ± 1.04 ± 2.22 ± 3.15 ± 3.74
2.54 4.45 2.40 0.24 0.14 3.15
1
N x 6.25

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abc
Means in the same row that do not share a common superscript are significantly different
(P<0.05) MA
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Table 2 pH values of raw beef patties formulated with different levels of protein ingredients over
12 days shelf-life at 4°C (means ± standard error)
Treatments Storage days
Day 0 Day 6 Day 12 Source of variation P Value
Control 5.58 ± 0.26WX 5.56 ± 0.18X 5.33 ± 0.22W Treat 0.330
WX X W

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PPI3 5.39 ± 0.14 5.62 ± 0.24 5.22 ± 0.03 Day <0.01
PPI7 5.46 ± 0.18WX 5.69 ± 0.21X 5.42 ± 0.20W Day x Treat 0.977
WX X W
RP3 5.50 ± 0.16 5.61 ± 0.14 5.46 ± 0.30

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RP7 5.53 ± 0.12WX 5.58 ± 0.19X 5.47 ± 0.06W
WX X
LF3 5.46 ± 0.11 5.57 ± 0.20 5.29 ± 0.02W
5.47 ± 0.09WX 5.56 ± 0.26X 5.43 ± 0.03W

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LF7
WX
Means in the same row that do not share a common superscript are significantly different
(P<0.05).
Treat: Treatment effect

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Day: Storage time effect
Day x Treat: Interaction between treatment and storage day
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Table 3 Mean values and standard deviations of the texture profile analysis and cooking loss
over 12 days shelf-life (means ± standard error)
Parameters Treatments Storage
days
Day 1 Day 6 Day 12 Source of P Value
variation
Hardness (N) Control 79.0 ± 87.5 ± 80.9 ± Treat <0.05
13.95aW 20.60aX 8.32aX
PPI3 84.2 ± 114.6 ± 104.8 ± Day <0.001
5.43abW 21.34abX 18.85abX
PPI7 119.4 ± 126.6 ± 138.6 ± Day x 0.092
bW bX bX
23.24 41.93 40.99 Treat
RP3 89.5 ± 99.9 ± 118.5 ±

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13.38abW 21.54abX 33.76abX

RP7 120.2 ± 119.7 ± 129.8 ±
29.97bW 30.88bX 20.05bX
LF3 70.2 ± 74.7 ± 76.9 ±
19.37cW 22.31cX 20.69cX

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LF7 69.1 ± 65.3 ± 70.3 ±
9.81cW 11.85cX 15.90cX

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Springiness Control 5.84 ± 5.69 ± 5.73 ± Treat 0.336
(mm) 0.23 0.62 0.29

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PPI3 4.45 ± 6.25 ± 6.10 ± Day 0.089
3.13 0.26 0.74
PPI7 6.81 ± 6.84 ± 6.45 ± Day x 0.840
0.90 0.84 0.81 Treat

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RP3 6.37 ± 6.60 ± 6.39 ±
0.34 0.23 0.52
RP7 6.51 ± 6.06 ± 6.16 ±
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0.32 0.74 0.42
LF3 6.28 ± 6.21 ± 6.15 ±
0.60 0.14 0.32
LF7 6.35 ± 6.08 ± 6.14 ±
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0.16 0.23 0.27

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Cohesiveness Control 0.89 ± 0.89 ± 0.89 ± Treat <0.01

0.02cW 0.02cWX 0.03cX
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PPI3 0.87 ± 0.88 ± 0.90 ± Day <0.05

0.03cW 0.02cWX 0.02cX
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PPI7 0.87 ± 0.86 ± 0.88 ± Day x 0.899

0.03bcW 0.05bcWX 0.02bcX Treat
RP3 0.88 ± 0.89 ± 0.89 ±
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0.02cW 0.01cWX 0.02cX

RP7 0.88 ± 0.89 ± 0.89 ±
0.03cW 0.03cWX 0.03cX
LF3 0.83 ± 0.84 ± 0.85 ±
0.01abW 0.04abWX 0.01abX
LF7 0.81 ± 0.83 ± 0.83 ±
0.03aW 0.00aWX 0.01aX

Chewiness (N Control 254 ± 282 ± 255 ± Treat <0.01

x mm) 54.26abW 75.39abX 17.55abX
PPI3 283 ± 403 ± 374 ± Day <0.01
20.17bcW 92.29bcX 87.87bcX
PPI7 444 ± 458 ± 514 ± Day x 0.410
104.46cW 212.37cX 193.80cX Treat
RP3 309 ± 365 ± 424 ±
40.38bcW 86.68bcX 138.52bcX

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RP7 423 ± 422 ± 447 ±

113.88cW 118.47cX 69.88cX
LF3 189 ± 211 ± 228 ±
61.39abW 91.57abX 68.28abX
LF7 171 ± 173 ± 197 ±
8.95aW 31.29aX 43.73aX

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Gumminess Control 43.5 ± 48.8 ± 44.9 ± Treat <0.01

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7.82abW 8.17abX 4.62abX
PPI3 43.9 ± 64.6 ± 58.6 ± Day <0.001
4.85bcW 12.49bcX 6.24bcX

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PPI7 53.7 ± 67.5 ± 78.0 ± Day x 0.259
22.34cW 22.04cX 19.19cX Treat
RP3 42.2 ± 55.2 ± 65.4 ±

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13.85bcW 12.21bcX 16.61bcX
RP7 62.8 ± 68.3 ± 72.1 ±
11.39cW 11.46cX 8.96cX
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LF3 29.9 ± 33.7 ± 36.7 ±
7.14aW 14.39aX 9.86aX
LF7 26.0 ± 28.6 ± 31.0 ±
1.06aW 6.19aX 7.70aX
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Cooking loss Control 26.24 ± 30.92 ± 29.75 ± Treat <0.001

dW dWX dX
(%) 3.83 3.61 2.70
PPI3 22.45 ± 24.24 ± 24.53 ± Day <0.01
P

cW cWX cX
0.63 0.99 1.40
PPI7 17.14 ± 16.56 ± 17.48 ± Day x 0.148
CE

bW bWX bX
2.75 4.37 0.75 Treat
RP3 22.75 ± 23.28 ± 26.23 ±
1.42cW 0.63cWX 1.00cX
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RP7 14.83 ± 20.70 ± 20.88 ±

6.24bW 1.72bWX 0.87bX
LF3 17.84 ± 19.17 ± 18.65 ±
1.66bW 1.38bWX 2.70bX
LF7 10.09 ± 8.06 ± 16.39 ±
2.30aW 1.10aWX 5.62aX
abc
Means in the same column for each parameter (different treatments) that do not share a
common superscript are significantly different (P<0.05)
WX
Means in the same row (different storage days) that do not share a common superscript are
significantly different (P<0.05)
Treat: Treatment effect
Day: Storage time effect
Day x Treat: Interaction between treatment and storage day

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Table 4 Effect of pea protein isolate, rice protein and lentil flour (at 3 and 7%) on colour
evaluation in raw beef patties over 12 days shelf-life at 4°C (means ± standard error)

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Parameter Treatment Storag Source P
s s e Days of Value
Variatio

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n
1 3 6 9 12
L* Control 47.66 ± 47.83 ± 48.44 47.76 ± 49.11 ± Treat <0.00

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1.94aW 0.70aXY ± 1.03aX 1.26aY 1
1.52aY
PPI3 48.42 ± 47.53 48.05 46.63 ± 50.19 ± Day <0.00
aW
MA
1.31 ± ± 1.40aX 0.58aY 1
1.26aXY 1.32aY
PPI7 48.73 ± 48.88 48.48 48.41 ± 49.75 ± Day x 0.449
0.71abW ± ± 1.40abX 0.79abY Treat
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abX abY
1.53 0.57
Y
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RP3 50.43 ± 50.10 50.83 49.59 ± 50.83 ±

1.65cW ± ± 1.26cX 0.43cY
0.89cXY 0.43cY
P

RP7 51.96 ± 52.08 53.62 51.83 ± 52.69 ±

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0.87dW ± ± 1.81dX 0.44dY

0.97dXY 0.80dY
LF3 47.80 ± 49.48 49.45 49.60 ± 51.19 ±
AC

0.17bcW ± ± 0.73bcX 0.75bcY

1.09bcX 0.85bcY
Y

LF7 48.65 ± 50.40 49.87 49.85 ± 51.80 ±

0.79cW ± ± 1.92cX 0.87cY
1.59cXY 1.36cY

a* Control 11.84 ± 11.40 10.87 10.63 ± 8.60 ± Treat <0.00

0.77abZ ± ± 1.33abX 1.48abW 1
0.12abZ 0.70abY
PPI3 14.82 ± 14.82 12.12 8.64 ± 6.81 ± Day <0.00
0.21bcZ ± ± 0.88bcX 0.65bcW 1
1.02bcZ 0.65bcY
PPI7 15.24 ± 16.95 13.16 8.50 ± 6.74 ± Day x <0.00
1.34cdZ ± ± 0.43cdX 0.51cdW Treat 1
0.85cdZ 0.47cdY

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RP3 13.35 ± 13.57 13.05 11.76 ± 10.45 ±

1.02cdZ ± ± 0.50cdX 0.53cdW
0.83cdZ 0.79cdY
RP7 15.35 ± 13.58 12.25 11.78 ± 11.13 ±
1.06dZ ± ± 0.98dX 0.24dW
0.79dZ 0.71dY

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LF3 11.90 ± 10.25 9.67 ± 8.22 ± 7.82 ±
1.84aZ ± 1.81aY 0.74aX 0.64aW

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0.81aZ
LF7 11.87 ± 9.88 ± 9.61 ± 8.55 ± 7.58 ±
1.68aZ 1.11aZ 1.53aY 0.93aX 0.77aW

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b* Control 10.75 ± 11.05 10.62 11.10 ± 10.70 ± Treat <0.00
0.45aX ± ± 0.73aW 0.88aW 1

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0.59aX 0.79aW
PPI3 11.90 ± 12.37 10.68 10.13 ± 9.97 ± Day <0.00
0.58aX ± ± 0.86aW 0.94aW 1
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0.37aX 0.48aW
PPI7 11.96 ± 12.88 11.23 9.69 ± 10.04 ± Day x <0.00
0.21abX ± ± 0.36abW 0.27abW Treat 1
0.48abX 0.87ab
D

W
TE

RP3 12.43 ± 12.91 12.54 12.21 ± 11.75 ±

1.41cX ± ± 0.41cW 0.31cW
0.08cX 0.63cW
P

RP7 14.60 ± 13.48 12.63 13.08 ± 12.85 ±

0.68dX ± ± 1.10dW 0.40dW
CE

0.46dX 1.02dW
LF3 11.39 ± 11.39 11.56 10.82 ± 11.77 ±
0.87abX ± ± 0.08abW 0.62abW
AC

0.25abX 1.33ab
W

LF7 12.54 ± 11.13 12.10 11.95 ± 11.97 ±

1.23bcX ± ± 0.56bcW 0.65bcW
0.89bcX 1.34bc
W

C* Control 16.02 ± 15.89 15.21 15.40 ± 13.77 ± Treat <0.00

0.49aZ ± ± 1.41aX 1.45aW 1
0.49aZ 1.04aY
PPI3 19.08 ± 19.32 16.19 13.34 ± 12.11 ± Day <0.00
2.70abZ ± ± 1.22abX 1.10abW 1
1.02abZ 0.80abY
PPI7 19.49 ± 21.31 17.32 12.92 ± 12.11 ± Day x <0.00
0.95bcZ ± ± 0.50bcX 0.32bcW Treat 1
0.95bcZ 0.92bcY

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RP3 18.29 ± 18.76 18.12 16.97 ± 15.77 ±

1.51cdZ ± ± 0.64cdX 0.40cdW
0.59cdZ 1.01cdY
RP7 21.21 ± 19.15 17.61 17.61 ± 17.01 ±
1.00dZ ± ± 1.44dX 0.45dW
0.79dZ 1.23dY

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LF3 16.50 ± 15.35 15.11 13.62 ± 14.21 ±
1.92aZ ± ± 0.52aX 0.68aW

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0.61aZ 2.01aY
LF7 17.31 ± 14.91 15.48 14.73 ± 14.18 ±
1.89abZ ± ± 0.94abX 0.95abW

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1.37abZ 2.00abY

h* Control 12.85 ± 13.10 12.48 13.20 ± 12.35 ± Treat <0.05

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0.88aY ± ± 1.11aWX 1.20aW
0.84aY 1.30aX
PPI3 13.92 ± 14.72 12.51 12.36 ± 11.34 ± Day <0.00
MA
0.55abY ± ± 1.32abWX 1.13abW 1
0.07abY 0.58abX
PPI7 13.18 ± 14.87 13.18 11.41 ± 11.50 ± Day x <0.05
1.20aY ± ± 0.11aWX 0.29aW Treat
D

0.88aY 1.15aX
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RP3 13.96 ± 14.52 14.00 13.93 ± 13.10 ±

1.86bcY ± ± 0.73bcWX 0.29bcW
0.17bcY 0.88bcX
P

RP7 15.78 ± 14.60 13.30 14.24 ± 13.78 ±

0.82cY ± ± 1.58cWX 0.31cW
CE

0.60cY 1.21cX
LF3 13.51 ± 13.08 13.26 12.46 ± 13.02 ±
1.03abY ± ± 0.27abWX 0.53abW
AC

0.37abY 1.61abX
LF7 14.56 ± 12.59 13.73 13.56 ± 13.08 ±
1.20abc ± ± 1.08abcW 0.76abc
Y
1.38abcY 1.79abc X W
X
abcd
Means in the same column for each parameter (different treatments) that do not share a
common superscript are significantly different (P<0.05)
WXYZ
Means in the same row (different storage days) that do not share a common superscript are
significantly different (P<0.05)
Treat: Treatment effect
Day: Storage time effect
Day x Treat: Interaction between treatment and storage day

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PT
RI
SC
Table 5 Effect of pea protein isolate, rice protein and lentil flour on lipid stability (TBARS,
mgMDA/kg muscle) in raw beef patties (means ± standard error)

NU
Treatments Storage days TBARS (mg/MDA/kg Meat)
Day 0 Day 3 Day 6 Day 9 Day 12 Source of P Value
variation
MA
Control 0.53 ± 0.60 ± 0.76 ± 0.60 ± 0.69 ± Treat <0.001
abW abX abXY abY abZ
0.33 0.16 0.05 0.23 0.42
PPI3 0.55 ± 0.69 ± 0.80 ± 0.85 ± 1.15 ± Day <0.001
bW bX bXY bY bZ
0.33 0.20 0.38 0.04 0.36
D

PPI7 0.60 ± 0.94 ± 0.96 ± 1.54 ± 1.98 ± Day x <0.01

cW cX cXY cY cZ
0.33 0.21 0.65 0.06 0.20 Treat
TE

RP3 0.32 ± 0.38 ± 0.37 ± 0.38 ± 0.52 ±

0.12aW 0.26aX 0.22aXY 0.16aY 0.45aZ
P

RP7 0.26 ± 0.29 ± 0.52 ± 0.25 ± 0.26 ±

0.05aW 0.08aX 0.44aXY 0.04aY 0.24aZ
CE

LF3 0.63 ± 1.05 ± 1.38 ± 1.40 ± 1.72 ±

0.21cW 0.40cX 0.21cXY 0.20cY 0.18cZ
LF7 1.05 ± 1.27 ± 1.39 ± 1.60 ± 1.87 ±
AC

0.38cW 0.29cX 0.66cXY 0.08cY 0.14cZ

abc
Means in the same column (different treatments) that do not share a common superscript are
significantly different (P<0.05) across each individual day
WXYZ
Means in the same row (different storage days) that do not share a common superscript are
significantly different (P<0.05) over 12 days storage period
Treat: Treatment effect
Day: Storage time effect
Day x Treat: Interaction between treatments and storage day

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Table 6 Effect of MAP storage on microbiological characteristics (Log10 CFU/g sample) of beef

PT
patties (means ± standard error)
Parameters Treatments Storage days (Log CFU/g)

RI
Day 0 Day 6 Day 12 Source of P
variation Value
TVC Control 3.39 ± 4.69 ± 5.16 ± Treat 0.957

SC
Psychrotrophic 0.25W 0.78Y 0.48Z
PPI3 2.95 ± 4.52 ± 5.40 ± Day <0.001
0.30W 0.65Y 0.07Z

NU
PPI7 2.98 ± 4.34 ± 5.64 ± Day x Treat 0.641
W Y Z
0.05 0.48 0.14
RP3 3.09 ± 4.52 ± 5.28 ±
MAW Y
0.18 0.86 0.22Z
RP7 3.03 ± 4.11 ± 5.35 ±
W Y
0.06 0.33 0.01Z
LF3 3.34 ± 4.53 ± 5.29 ±
D

W Y
0.24 0.53 0.38Z
TE

LF7 3.57 ± 4.44 ± 4.98 ±

0.71W 0.80Y 0.41Z
P

TVC Mesophilic Control 4.31 ± 5.15 ± 5.85 ± Treat 0.637

1.25W 0.85X 0.71X
CE

PPI3 4.18 ± 5.35 ± 5.53 ± Day <0.001

0.99W 0.60X 0.70X
PPI7 4.62 ± 5.35 ± 5.62 ± Day x Treat 1.000
AC

0.90W 0.76X 0.71X

RP3 4.75 ± 5.25 ± 5.56 ±
0.47W 0.69X 0.73X
RP7 4.21 ± 5.31 ± 5.23 ±
0.85W 0.52X 1.62X
LF3 4.03 ± 5.10 ± 5.76 ±
0.29W 0.80X 0.97X
LF7 4.29 ± 4.92 ± 5.69 ±
0.91W 0.95X 0.92X

Pseudomonas Control 3.13 ± 3.52 ± 4.42 ± Treat 0.078

0.53W 0.42W 0.04X
PPI3 2.61 ± 3.28 ± 3.93 ± Day <0.001
0.53W 0.70W 0.19X
PPI7 2.89 ± 2.69 ± 3.23 ± Day x Treat 0.779
0.83W 0.64W 0.95X

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RP3 2.85 ± 3.53 ± 4.42 ±

0.30W 0.29W 0.08X
RP7 2.79 ± 2.54 ± 4.09 ±
0.47W 0.94W 0.31X
LF3 3.03 ± 3.66 ± 4.33 ±
0.33W 0.42W 0.21X

PT
LF7 3.06 ± 3.48 ± 4.44 ±
0.10W 0.17W 0.12X

RI
Lactobacilli Control 2.81 ± 4.63 ± 5.04 ± Treat 0.940
0.47W 0.41X 0.19Y

SC
PPI3 2.87 ± 4.72 ± 5.09 ± Day <0.001
0.53W 0.31X 0.48Y
PPI7 2.98 ± 4.28 ± 5.20 ± Day x Treat 0.999

NU
0.63W 0.42X 0.18Y
RP3 2.85 ± 4.61 ± 5.19 ±
0.54W 0.45X 0.48Y
MA
RP7 2.88 ± 4.36 ± 4.95 ±
0.54W 0.19X 0.68Y
LF3 2.78 ± 4.52 ± 4.91 ±
0.58W 0.51X 0.57Y
D

LF7 2.82 ± 4.55 ± 5.02 ±

0.54W 0.77X 0.10Y
TE

Enterobacteriaceae Control 2.23 ± 2.32 ± 2.94 ± Treat 0.148

P

W W X
0.05 0.23 0.45
PPI3 2.12 ± 2.05 ± 2.31 ± Day <0.001
CE

0.08W 0.27W 0.74X

PPI7 2.17 ± 2.21 ± 2.26 ± Day x Treat 0.866
0.03W 0.09W 0.31X
AC

RP3 2.23 ± 2.21 ± 2.77 ±

0.05W 0.34W 0.15X
RP7 2.16 ± 2.01 ± 2.28 ±
0.10W 0.18W 0.46X
LF3 2.12 ± 2.32 ± 2.60 ±
0.23W 0.15W 0.54X
LF7 2.19 ± 2.23 ± 2.48 ±
0.13W 0.09W 0.30X
WXY
Means in the same row that do not share a common superscript are significantly different
(P<0.05). Since microbiological analysis was not influenced (P>0.05) by treatment, data
reported are the mean values across all treatments over 12 storage days
Treat: Treatment effect
Day: Storage time effect
Day x Treat: Interaction between treatment and storage day

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Highlights

 The potential of PPI, RP and LF as clean label ingredients for enhancing protein content in beef
patties were evaluated
 Beef patties enriched with rice protein at 7% increased protein

PT
 Redness values remained stable in rice protein-enriched patties
 Addition of plant proteins had significant and variable effects on texture characteristics

RI
SC
NU
MA
D
P TE
CE
AC

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