Food Chemistry 179 (2015) 159–169

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Phytochemical profile of commercially available food plant powders: their potential role in healthier food reformulations M. Neacsu a,⇑, N. Vaughan a, V. Raikos a, S. Multari a, G.J. Duncan b, G.G. Duthie a, W.R. Russell a a b

Natural Products Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen AB21 9SB, UK Mass Spectrometry Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen AB21 9SB, UK

a r t i c l e

i n f o

Article history: Received 28 September 2014 Received in revised form 18 December 2014 Accepted 29 January 2015 Available online 4 February 2015 Keywords: Food plant powders Food reformulation Phytophenols Flavonoids Healthier foods

a b s t r a c t Reformulation of existing processed food or formulation of new foods using natural products (plantbased) will inherently confer to new products with less calories, fat, salt, phosphates and other synthetic components, and higher amounts of fibre, antioxidants, vitamins and other beneficial components. Plant ingredients, such as food plant powders, are currently being used in food manufacturing, predominantly for flavouring and colouring purposes. To expand their use as a food ingredient, freeze-dried powders representing major vegetable groups were characterised by targeted LC–MS/MS analysis of their phytochemicals. All the plant powders were found to be rich in flavonoids, phenolic acids and derivatives; total content in these compounds varied from around 130 mg kg 1 (green pea) to around 930 mg kg 1 (spinach). The food plant powders’ phytochemical content represents valuable information for the food industry in the development of healthier novel foods and for the reformulation of existing food products in relation to antioxidants, food preservatives and alternatives to nitrite use. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Food reformulation is defined as ‘‘changing the nutrient content of a processed food product to either reduce the content of negative nutrients, such as sodium, saturated fat, trans fat or energy (kilojoules), or to increase the content of beneficial nutrients, such as dietary fibre, wholegrain, fruit, vegetables and unsaturated fats’’ (NHFA, 2012). Reformulation of existing processed food or formulation of new foods using natural products (i.e. plant based ingredients) has the potential to confer several benefits; products lower in calories, fat, salt, phosphates and other synthetic components, and rich in fibre, antioxidants, and other bioactives. At the same time, the reformulation of processed foods provides a realistic opportunity to improve the health of a population by modifying the nutritional characteristics of commonly consumed processed foods (NHFA, 2012). The use of plant ingredients, such as food plant powders, by the food and drink industry is limited; therefore knowledge of their phytochemical content could be useful to expand their use as food ingredients. Macronutrients, such as protein, fibre, carbohydrates (sugars) and fats, are essential for human health and are present in food ⇑ Corresponding author at: Rowett Research Institute, University of Aberdeen, Greenburn Road, Aberdeen AB21 9SB, Scotland, UK. Tel.: +44 1224 438700; fax: +44 1224 438760. E-mail address: [email protected] (M. Neacsu). http://dx.doi.org/10.1016/j.foodchem.2015.01.128 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

products for reasons of nutrition and functionality. For example, vegetable proteins have been shown to play an important role in weight control and satiety similar to animal proteins (Neacsu, Fyfe, Horgan, & Johnstone, 2014). Functional properties of proteins include viscosity enhancement, water binding, gelation, aeration and foaming; and emulsification with consequent contributions to a food’s flavour, texture and colour (Protein Trends & Technologies, 2013). Dietary fibre, the indigestible cell wall component of plant material, plays an important role in human diet and health (Smith & Tucker, 2011) for example ameliorating negative changes in gut fermentation seen with high protein diets (Russell et al., 2011). In the UK most people do not eat enough fibre (the average intake is 12.8 g/day for women and 14.8 g/day for men). The recommended average intake for adults is 18 g (non starch polysaccharides) per day (BNF, 2012). Thus, it is incumbent on the food industry to develop products with appropriate nutritional content particularly by lowering the fat and salt and by optimising minerals, vitamins and fibres (van Raaij, Hendriksen, & Verhagen, 2008). Phytochemicals are compounds present in plants which are currently classified as non-nutrient constituents of the human diet. Among the most abundant are the phenolic acids and flavonoids. Phenolic acids are broadly distributed throughout the plant kingdom and they are attracting much scientific attention due to their potential bioactivity. For example, hydroxycinnamic acid was

Red pepper Free

Spinach Bound

Free

Carrot Bound

Free

Yellow pea Bound

Free

Broccoli Bound

Free

Green pea Bound

Free

Swede Bound

Free

Beet root Bound

Free

Tomato Bound

Free

Celery Bound

Free

160

Table 1 Phytophenol content from food plant powders, concentration of benzoic acids (A); concentration of benzaldehydes, benzenes and acetophenones (B); concentration of cinnamic acids (C); concentration of phenylpropionic, phenylacetic, phenylpyruvic and phenyllactic acids (D); quinadilic acid, coniferyl alcohol, 4-hydroxy 3-methoxy ciannamyl alcohol, and 4-methylcatechol (E); catechins (F); lignans (G) and flavonoid, isoflavonoid and catechin content in the food plant powders (H). Onion Bound

Free

Bound

A Benzoic acid

5.22 ± 2.1

4.83 ± 0.59

3.48 ± 0.49

4.87 ± 0.55

1.47 ± 0.2

5.31 ± 0.52

1.57 ± 0.14

4.14 ± 1.13

1.44 ± 0.16

3.9 ± 0.92

1.60 ± 0.37 4.43 ± 0.85

1.02 ± 0.47

4.86 ± 0.49

1.28 ± 0.25

4.38 ± 0.65

1.94 ± 0.35

4.23 ± 0.6

1.22 ± 0.28 5.83 ± 0.5

Salicylic acid

0.24 ± 0.03

4.02 ± 0.67

1.27 ± 0.11

2.61 ± 0.32

0.01 ± 0.02

1.79 ± 0.34

n/d

1.02 ± 0.15

0.18 ± 0.3

3.02 ± 0.31

n/d

1.12 ± 0.16

n/d

2.39 ± 0.58

0.21 ± 0.37

1.19 ± 0.08

0.85 ± 0.05

3.48 ± 0.74

5.16 ± 0.8

1.21 ± 0.28 5.32 ± 0.57

13.31 ± 1.66 n/d

0.14 ± 0.14

m-Hydroxybenzoic acid

n/d

128.17 ± 42.15 n/d

n/d

n/d

109.68 ± 2.15 n/d

n/d

n/d

n/d

n/d

43.50 ± 38.84 n/d

97.60 ± 11.11 n/d

97.95 ± 22.44

n/d

99.99 ± 16.19 n/d

n/d

n/d

p-Hydroxybenzoic acid

n/d

6.39 ± 0.6

1.87 ± 0.14

4.49 ± 0.46

2.82 ± 0.58

25.06 ± 2.78

2.41 ± 0.64

2.89 ± 0.47

n/d

3.36 ± 0.3

n/d

1.97 ± 0.22

n/d

1.7 ± 0.18

n/d

0.53 ± 0.05

n/d

6.87 ± 0.62

n/d

6.19 ± 1.52

n/d

1.35 ± 0.13

2,3-Dihydroxybenzoic acid

0.1 ± 0.01

0.64 ± 0.06

0.18 ± 0.01

0.1 ± 0.01

n/d

0.32 ± 0.07

n/d

n/d

n/d

0.36 ± 0.04

n/d

0.02 ± 0.04

0.09 ± 0.02

0.53 ± 0.05

n/d

0.08 ± 0.01

n/d

0.41 ± 0.05

0.03 ± 0

0.21 ± 0.02

n/d

0.2 ± 0.05

2,5-Dihydroxybenzoic acid

n/d

1 ± 0.16

2.66 ± 0.09

0.17 ± 0.3

n/d

0.55 ± 0.05

n/d

0.52 ± 0.06

n/d

3.08 ± 0.33

n/d

0.75 ± 0.03

n/d

2.03 ± 0.15

0.02 ± 0.04

0.41 ± 0.07

0.11 ± 0.01

8.82 ± 0.4

0.27 ± 0.06 5.79 ± 1.19

n/d

0.42 ± 0.08

Protocatechuic acid

4.95 ± 0.75

2.29 ± 0.19

1.3 ± 0.07

3.47 ± 2.07

1.45 ± 0.18

0.79 ± 0.12

1.49 ± 0.1

4.67 ± 0.29

0.26 ± 0.07

1.08 ± 0.1

0.33 ± 0.06 1.69 ± 0.12

0.39 ± 0.07

0.76 ± 0.05

0.36 ± 0.44

0.44 ± 0.11

1.59 ± 0.1

1.39 ± 0.03

0.2 ± 0.05

0.29 ± 0.02

2.84 ± 0.3

4.76 ± 0.57

0.09 ± 0.08

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.32 ± 0.04

2,4-Dihydroxybenzoic acid

n/d

n/d

222.02 ± 15.81

n/d

n/d

n/d

n/d

n/d

0.03 ± 0.03

n/d

n/d

n/d

2,6-Dihydroxybenzoic acid

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.08 ± 0.01 n/d

n/d

n/d

o-Anisic acid

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

12.43 ± 0.47 n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Gallic acid

0.25 ± 0.08

0.17 ± 0.05

0.15 ± 0.02

n/d

n/d

n/d

n/d

0.12 ± 0.02

n/d

n/d

n/d

n/d

n/d

n/d

0.06 ± 0.02

0.1 ± 0.03

0.02 ± 0.04

n/d

n/d

n/d

n/d

n/d

Vanillic acid

1.62 ± 0.03

6.55 ± 0.74

2.98 ± 0.07

5.46 ± 1

0.53 ± 0.03

6.76 ± 0.74

0.52 ± 0.01

1.05 ± 0.09

0.22 ± 0.06

2.04 ± 0.14

0.09 ± 0.01 1.28 ± 0.06

0.09 ± 0.02

2.07 ± 0.17

0.35 ± 0.07

4.61 ± 0.54

n/d

1.21 ± 0.13

0.54 ± 0.15 14.52 ± 2.01 0.19 ± 0.03 1.1 ± 0.16

Syringic acid

0.19 ± 0.02

0.52 ± 0.05

0.32 ± 0.03

0.18 ± 0.12

n/d

0.09 ± 0.03

n/d

0.13 ± 0.01

0.13 ± 0.04

0.37 ± 0.07

n/d

0.19 ± 0.02

0.03 ± 0.03

0.81 ± 0.12

0.31 ± 0.12

5.98 ± 0.67

0.01 ± 0.02

0.2 ± 0.03

n/d

0.07 ± 0

n/d

0.07 ± 0.07

3,4-Dimethoxybenzoic acid

n/d

n/d

22.3 ± 0.99

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

p-Hydroxybenzaldehyde

0.74 ± 0.15

1.37 ± 0.24

n/d

4.69 ± 0.32

0.19 ± 0.02

0.39 ± 0.15

0.29 ± 0.01

1.08 ± 0.24

0.37 ± 0.06

1.69 ± 0.22

0.55 ± 0.19 1.01 ± 0.1

n/d

0.80 ± 0.13

n/d

0.43 ± 0.08

n/d

0.93 ± 0.05

0.36 ± 0.04 2.81 ± 0.28

n/d

0.14 ± 0.05

Protocatachaldehyde

0.55 ± 0.08

1.47 ± 0.31

n/d

0.41 ± 0.08

0.3 ± 0.05

0.64 ± 0.09

0.25 ± 0.08

0.3 ± 0.08

0.11 ± 0.03

0.42 ± 0.04

0.04 ± 0.04 0.09 ± 0.02

0.53 ± 0.08

0.55 ± 0.11

0.49 ± 0.15

1.04 ± 0.23

0.04 ± 0.07

1.22 ± 0.08

0.08 ± 0.01 0.27 ± 0

n/d

0.16 ± 0.01

Vanillin

0.56 ± 0.54

0.85 ± 0.12

0.63 ± 0.38

1.88 ± 0.12

0.77 ± 0.43

0.79 ± 0.06

0.74 ± 0.06

0.41 ± 0.1

0.4 ± 0.28

0.74 ± 0.09

0.09 ± 0.02 0.47 ± 0.05

0.12 ± 0.02

1.06 ± 0.09

0.42 ± 0.13

1.28 ± 0.06

0.05 ± 0.01

0.71 ± 0.14

0.76 ± 0.12 3.47 ± 0.17

0.07 ± 0.01 2.11 ± 0.45

Syringin

0.07 ± 0.01

0.26 ± 0.05

0.01 ± 0.01

0.31 ± 0.06

0.01 ± 0.01

0.03 ± 0.02

0.02 ± 0

0.22 ± 0.06

0.28 ± 0.11

0.47 ± 0.05

0.01 ± 0.01 0.08 ± 0.01

0.04 ± 0

0.49 ± 0.08

0.02 ± 0.01

0.24 ± 0.06

n/d

0.04 ± 0.01

n/d.01

0.12 ± 0.01

n/d

0.03 ± 0.03

3-Methoxybenzaldehyde

n/d

0.71 ± 1.23

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

4.23 ± 0.17

1.76 ± 3.05

5.29 ± 1.51

n/d

4.32 ± 0.11

n/d

n/d

n/d

10.38 ± 0.1

B

Phenol

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

3.58 ± 0.81

n/d

10.34 ± 0.68

n/d

n/d

n/d

n/d

n/d

1.69 ± 1.65

n/d

8.36 ± 0.31

4-Ethylphenol

n/d

n/d

n/d

0.10 ± 0.03

n/d

n/d

n/d

0.02 ± 0.01

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

1,2-Hydroxybenzene

n/d

1.08 ± 0.13

n/d

n/d

0.18 ± 0.16

0.49 ± 0.14

0.1 ± 0.21

n/d

n/d

n/d

n/d

n/d

0.64 ± 0.09

0.35 ± 0.13

n/d

n/d

n/d

0.25 ± 0.04

n/d

n/d

n/d

n/d

1,2,3-Trihydroxybenzene

0.22 ± 0.38

0.43 ± 0.1

n/d

n/d

0.28 ± 0.07

n/d

0.35 ± 0.31

0.12 ± 0.2

0.13 ± 0.23

1.01 ± 0.03

0.13 ± 0.22

n/d

0.11 ± 0.19

n/d

n/d

Ellagic acid

n/d

n/d

n/d

n/d

n/d

0.13 ± 0.23

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

4-Hydroxyacetophenone

0.11 ± 0

0.26 ± 0.03

0.05 ± 0.01

0.55 ± 0.06

n/d

0.02 ± 0

0.25 ± 0.24

0.08 ± 0.02

n/d

0.02 ± 0

0.25 ± 0.15

0.06 ± 0.01

n/d

0.02 ± 0

n/d

0.08 ± 0.01

n/d

0.01 ± 0.01 0.08 ± 0.01

n/d.01

0.09 ± 0.02

0.03 ± 0.01

0.08 ± 0.01

0.05 ± 0.01

0.31 ± 0.01

0.21 ± 0.06 0.26 ± 0.03

0.16 ± 0.03 0.73 ± 0.09

4-Hydroxy-3-methoxyacetophenone

0.46 ± 0.22

1.31 ± 0.28

0.14 ± 0.06

0.6 ± 0.16

0.06 ± 0.02

0.07 ± 0.01

0.06 ± 0.03

0.16 ± 0.05

0.03 ± 0.03

0.35 ± 0.04

n/d

0.06 ± 0.01

n/d

0.11 ± 0.03

0.05 ± 0.04

0.46 ± 0.06

0.13 ± 0.01

0.77 ± 0.1

0.02 ± 0.03 0.3 ± 0.04

0.72 ± 0.12 3.41 ± 0.37

4-Hydroxy-3,5-dimethoxyacetophenone

0.12 ± 0.01

0.35 ± 0.03

0.28 ± 0.03

2.19 ± 0.15

n/d

n/d

n/d

0.14 ± 0.03

n/d

0.78 ± 0.08

n/d

0.05 ± 0.05

n/d

n/d

n/d

3.74 ± 0.45

n/d

0.13 ± 0.06

0.09 ± 0.01 0.10 ± 0.05

n/d

3,4-Dimethoxyacetophenone

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.13 ± 0.02 n/d

Cinnamic acid

0.80 ± 0.11

1.82 ± 0.32

2.94 ± 0.25

1.68 ± 0.08

0.84 ± 0.2

1.54 ± 0.19

0.89 ± 0.12

1.41 ± 0.14

0.79 ± 0.1

1.48 ± 0.22

0.73 ± 0.11 1.48 ± 0.17

0.71 ± 0.13

0.90 ± 0.07

0.78 ± 0.2

1.46 ± 0.13

0.6 ± 0.19

1.18 ± 0.16

0.77 ± 0.04 1.01 ± 0.05

1.00 ± 0.09 0.96 ± 0.12

4-Methoxycinnamic acid

n/d

n/d

1.30 ± 0.12

0.55 ± 0.07

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

3,4,5-Trimethoxycinnamic acid

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.03 ± 0

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

o-Coumaric acid

n/d

5.86 ± 0.14

n/d

n/d

n/d

6.84 ± 0.29

n/d

n/d

n/d

n/d

n/d

5.48 ± 0.75

n/d

5.84 ± 0.43

n/d

8.17 ± 0.37

n/d

7.7 ± 0.7

n/d

6.67 ± 0.89

n/d

8.35 ± 1.17

p-Coumaric acid

n/d

9.65 ± 1.68

15.49 ± 0.76

53.69 ± 3.65

n/d

0.82 ± 0.21

0.43 ± 0

1.26 ± 0.35

n/d

7.48 ± 0.38

n/d

1.32 ± 0.28

n/d

n/d

1.79 ± 0.84

2.95 ± 0.73

4.28 ± 0.15

4.37 ± 0.35

n/d

0.84 ± 0.09

1.24 ± 0.04 0.27 ± 0.23

Caffeic acid

1.32 ± 0.07

8.51 ± 0.84

0.53 ± 0.04

1.49 ± 0.21

3.7 ± 0.36

14.01 ± 2.03

2.4 ± 0.23

0.63 ± 0.09

0.47 ± 0.09

27.93 ± 1.32

0.06 ± 0.05 0.21 ± 0.05

n/d

0.25 ± 0.03

1.12 ± 1.73

0.49 ± 0.12

5.61 ± 0.38

14.15 ± 0.68

0.65 ± 0.15 3.28 ± 0.36

0.12 ± 0.01 0.16 ± 0.17

Chlorogenic acid

17.90 ± 1.21 n/d

Ferulic acid

6.33 ± 0.3

26.1 ± 2.81

17.02 ± 0.13

135.27 ± 4.11 1.38 ± 0.15

0.78 ± 0.09

1.25 ± 0.11

5.77 ± 1.27

3.53 ± 0.87

50.83 ± 2.65

0.44 ± 0.05 1.34 ± 0.16

0.12 ± 0.03

0.55 ± 0.05

1.64 ± 0.46

208.18 ± 19.99 0.61 ± 0.06

2.18 ± 0.07

6.22 ± 1.64 6.56 ± 1.06

0.96 ± 0.09 2.28 ± 2.41

Ferulic dimer (5-5 linked)

n/d

n/d

0.04 ± 0

4.35 ± 0.35

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.06 ± 0.01

9.21 ± 2.34

n/d

n/d

n/d

n/d

n/d

0.03 ± 0.04

C

2.67 ± 0.59

n/d

148.11 ± 8.73 n/d

n/d

97.22 ± 10.69 n/d

4.93 ± 0.24

0.10 ± 0.04

0.15 ± 0.03 n/d

n/d

0.08 ± 0.01

n/d

6.18 ± 10.59 n/d

35.09 ± 1.87 n/d

n/d

9.60 ± 1.38 n/d

n/d

n/d

n/d

n/d

0.03 ± 0.06

Ferulic dimer (8-8 linked)

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.06 ± 0.03

n/d

n/d

n/d

n/d

n/d

n/d

Hydrogenated ferulic dimer

0.15 ± 0.14

n/d

n/d

n/d

n/d

0.07 ± 0.01

n/d

n/d

n/d

n/d

n/d

0.06 ± 0.01

n/d

0.1 ± 0.01

n/d

0.1 ± 0.09

n/d

0.13 ± 0

n/d

n/d

n/d

0.43 ± 0.06

Sinapic acid

9.04 ± 0.23

36.64 ± 3.22

0.78 ± 0.03

1.24 ± 0.18

0.11 ± 0.02

0.34 ± 0.06

0.25 ± 0.03

14.91 ± 3.27 14.52 ± 1.69 228.15 ± 11.93 0.21 ± 0.02 1.81 ± 0.26

0.87 ± 0.19

3.60 ± 0.31

0.20 ± 0.11

1.78 ± 0.34

0.53 ± 0.02

1.76 ± 0.17

n/d

0.23 ± 0.04

0.67 ± 0.11 0.87 ± 0.05

3,4-Dimethoxycinnamic acid

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.95 ± 0.03

n/d

n/d

n/d

n/d

n/d

0.04 ± 0

n/d

n/d

n/d

n/d

n/d

Phenylpropionic acid

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

2.20 ± 0.17

n/d

n/d

n/d

n/d

n/d

n/d

n/d

4-Hydroxyphenylpropionic acid

n/d

2.32 ± 0.35

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

6.60 ± 0.2

n/d

n/d

n/d

2.01 ± 0.65

4-Hydroxy 3-methoxyphenylpropionic

0.25 ± 0.02

0.23 ± 0.03

0.11 ± 0.02

0.11 ± 0.09

0.08 ± 0.01

0.23 ± 0.02

0.07 ± 0

0.22 ± 0.03

0.12 ± 0.02

0.11 ± 0.01

n/d

0.19 ± 0.03

n/d

n/d

0.05 ± 0.09

0.22 ± 0.04

0.32 ± 0.03

3.95 ± 0.25

0.08 ± 0.01 0.36 ± 0.09

0.02 ± 0.03 0.13 ± 0.01

n/d

D n/d

acid 3-Hydroxyphenylpropionic acid

n/d

n/d

n/d

0.23 ± 0.01

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

3,4-Dihydroxyphenylacetic acid

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.58 ± 0.14

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Phenylacetic acid

2.93 ± 0.42

1.03 ± 0.13

6.35 ± 0.04

0.9 ± 0.15

0.81 ± 0.16

0.88 ± 0.11

0.77 ± 0.22

0.79 ± 0.1

1.13 ± 0.04

0.92 ± 0.16

0.89 ± 0.06 0.83 ± 0.06

0.73 ± 0.03

0.74 ± 0.09

0.73 ± 0.04

0.63 ± 0.11

0.76 ± 0.04

0.97 ± 0.13

0.84 ± 0.24 0.83 ± 0.14

0.29 ± 0.07 0.46 ± 0.05

3-Hydroxyphenylacetic acid

n/d

2.05 ± 0.39

2.33 ± 0.12

n/d

n/d

1.43 ± 0.29

n/d

n/d

n/d

n/d

n/d

0.72 ± 0.04

n/d

0.94 ± 0.24

n/d

0.83 ± 0.24

n/d

2.09 ± 0.16

n/d

0.72 ± 0.13

n/d

0.81 ± 0.06

3,4-Dihydroxymandelic acid

n/d

0.67 ± 0.09

0.13 ± 0.12

0.16 ± 0.02

0.28 ± 0.02

0.7 ± 0.09

0.19 ± 0.03

n/d

0.1 ± 0.09

0.35 ± 0.08

n/d

0.1 ± 0.09

n/d

0.43 ± 0.06

0.1 ± 0.09

n/d

0.1 ± 0.09

0.17 ± 0.03

n/d

n/d

n/d

n/d

Phenylpyruvic acid

0.42 ± 0.72

4.03 ± 0.21

1.87 ± 0.23

17.79 ± 3.99

2.35 ± 0.12

3.95 ± 0.42

2.38 ± 0.15

3.5 ± 0.33

1.04 ± 0.11

4.19 ± 0.33

2.31 ± 0.59 4.49 ± 0.12

1.56 ± 0.23

4.08 ± 0.43

4-Hydroxyphenylpyruvic acid

34.99 ± 3.64 163.44 ± 29.03 442.07 ± 77.6 37.29 ± 11.91 43.65 ± 8.63

61.98 ± 21.59 27.59 ± 3.67

10.18 ± 2.42 7.90 ± 1.8

22.05 ± 2.87

6.06 ± 1.32 24.95 ± 7.56

50.31 ± 4.84 68.78 ± 7.28

21.16 ± 2.61 8.57 ± 2.51

74.55 ± 5.69 89.19 ± 38.13 8.51 ± 0.56 26.77 ± 8.71 7.89 ± 1.5

Phenyllactic acid

2.26 ± 0.49

0.44 ± 0.06

0.41 ± 0.04

0.54 ± 0.07

1.65 ± 0.11

0.22 ± 0.04

1.67 ± 0.15

0.25 ± 0.05

0.19 ± 0.03

0.17 ± 0.03

0.69 ± 0.25 0.28 ± 0.05

0.12 ± 0.03

0.24 ± 0.05

0.16 ± 0.01

0.23 ± 0.06

0.67 ± 0.08

0.29 ± 0.06

0.58 ± 0.14 1.06 ± 0.19

0.15 ± 0.04 0.4 ± 0.08

4-Hydroxyphenyllactic cid

n/d

1.55 ± 0.17

n/d

0.07 ± 0.12

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.62 ± 0.09

2.76 ± 0.15

0.63 ± 0.14 4.68 ± 1.47

n/d

n/d

1.45 ± 0.06

4.54 ± 0.46

2.93 ± 0.24

6.19 ± 0.57

2.16 ± 0.35 4.51 ± 0.26

1.84 ± 0.03 5.06 ± 0.38 100.36 ± 83.6

n/d

M. Neacsu et al. / Food Chemistry 179 (2015) 159–169

n/d

E n/d

0.09 ± 0.02

0.13 ± 0.01

0.3 ± 0.06

n/d

0.04 ± 0.01

0.04 ± 0

0.12 ± 0.05

0.02 ± 0

0.26 ± 0.08

0.08 ± 0.02 0.25 ± 0.07

n/d

0.08 ± 0.01

n/d

0.08 ± 0.01

n/d

0.02 ± 0.01 0.07 ± 0.01

n/d

0.02 ± 0.01

Quinadilic acid

0.01 ± 0

0.16 ± 0.02

0.13 ± 0.03

0.11 ± 0.02

n/d

0.43 ± 0.06

n/d

0.01 ± 0

0.03 ± 0.01

0.07 ± 0.01

0.02 ± 0

0.03 ± 0

0.02 ± 0

0.04 ± 0

0.01 ± 0

0.11 ± 0.02

n/d

0.36 ± 0.12

0.01 ± 0

n/d

0.03 ± 0

Coniferyl alcohol

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

1.65 ± 0.78

0.22 ± 0.02

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.03 ± 0.02

1.44 ± 0.67 2.78 ± 1.57

0.35 ± 0.22 n/d

4-Hydroxy 3-methoxy ciannamoyl

n/d

0.43 ± 0.74

n/d

0.14 ± 0.25

n/d

0.31 ± 0.54

0.19 ± 0

n/d

n/d

1.02 ± 0.37

0.29 ± 0.06 n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.55 ± 0.96 n/d

0.03 ± 0.01

n/d

n/d

n/d

0.05 ± 0.01

0.04 ± 0

0.04 ± 0

0.05 ± 0.03

n/d

0.01 ± 0

n/d

0.01 ± 0

0.12 ± 0.03

0.11 ± 0.05

n/d

0.01 ± 0

n/d

0.01 ± 0.02

n/d

0.02 ± 0

n/d

0.06 ± 0.01

Catechin

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Epicatechin

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Gallocatechin

n/d

2.05 ± 0.24

n/d

0.78 ± 0.05

n/d

1.87 ± 0.33

n/d

0.26 ± 0.02

n/d

0.89 ± 0.1

n/d

1.57 ± 0.07

n/d

3.91 ± 0.17

n/d

2.08 ± 0.24

n/d

2.83 ± 0.49

n/d

1.75 ± 0.49

n/d

3.1 ± 0.33

Epigallocatechin

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Epigallocatechin gallate

0.12 ± 0.04

n/d

0.11 ± 0.04

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Secoisolariciresinol

n/d

n/d

n/d

0.37 ± 0.07

0.20 ± 0.02

0.38 ± 0.03

0.21 ± 0.02

0.17 ± 0.04

n/d

0.16 ± 0.05

n/d

0.2 ± 0.03

n/d

n/d

n/d

n/d

n/d

n/d

0.02 ± 0.04 0.39 ± 0.14

0.04 ± 0.01 0.05 ± 0.05

Matairesinol

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.04 ± 0.01

n/d

Enterodiol

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.01 ± 0

Enterolactone

n/d

0.01 ± 0

n/d

0.01 ± 0

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.01 ± 0.01

n/d

0.02 ± 0

Syringaresinol

n/d

n/d

0.08 ± 0.02

alcohol 4-Methylcatechol F

G

0.03 ± 0.02

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

1.60 ± 0.13

0.32 ± 0.55

n/d

n/d

n/d

n/d

n/d

Pinoresinol

n/d

n/d

n/d

n/d

0.13 ± 0.01

0.22 ± 0.04

0.14 ± 0.01

n/d

n/d

n/d

n/d

n/d

0.09 ± 0.01

n/d

n/d

n/d

n/d

n/d

0.35 ± 0.06 0.17 ± 0.02

n/d

n/d

Lariciresinol

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

1.18 ± 0.23 n/d

n/d

n/d

Hydroxymatairesinol

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

H 0.40 ± 0.01

0.47 ± 0.1

0.09 ± 0.04

0.04 ± 0.01

0.02 ± 0.03

0.1 ± 0.01

0.09 ± 0.01

0.04 ± 0.01

n/d

n/d

0.13 ± 0.01

Psoralen

0.02 ± 0.01

2.39 ± 0.37

n/d

0.08 ± 0

n/d

0.01 ± 0.02

0.04 ± 0

n/d

n/d

1.80 ± 0.42

n/d

8-Methylpsoralen

0.04 ± 0

3.82 ± 1.06

n/d

0.17 ± 0.01

n/d

n/d

0.03 ± 0

n/d

n/d

8.08 ± 3.44

n/d

Tangeretin

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Coumesterol

n/d

n/d

n/d

0.02 ± 0

0.01 ± 0

0.03 ± 0

n/d

n/d

n/d

n/d

0.01 ± 0

Isoliquiritigenin

n/d

n/d

n/d

0.03 ± 0

n/d

0.02 ± 0

n/d

n/d

n/d

n/d

n/d

Phloretin

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Eriocitrin

n/d

n/d

n/d

0.01 ± 0

n/d

n/d

n/d

n/d

0.02 ± 0

n/d

n/d

Naringenin

3.58 ± 0.23

0.34 ± 0.19

n/d

0.35 ± 0.18

0.13 ± 0.13

0.11 ± 0.01

n/d

n/d

7.94 ± 2.69

0.3 ± 0.18

0.04 ± 0

Hesperitin

0.25 ± 0.01

0.33 ± 0.33

n/d

n/d

0.05 ± 0.02

n/d

n/d

n/d

0.79 ± 0.26

0.15 ± 0.07

0.11 ± 0.01

Kaempferol

0.39 ± 0.03

2.45 ± 2.25

n/d

11.74 ± 1.18

13.44 ± 5.51

3.86 ± 0.22

0.02 ± 0.03

n/d

1.4 ± 0.31

0.11 ± 0.02

12.56 ± 0.17

Morin

n/d

n/d

0.02 ± 0.04

0.08 ± 0.07

n/d

n/d

n/d

n/d

0.02 ± 0.03

0.04 ± 0.03

n/d

Quercetin

23.74 ± 1.05

1.7 ± 1.58

0.01 ± 0.01

0.91 ± 0.15

13.1 ± 4.52

2.66 ± 0.14

0.24 ± 0.02

0.01 ± 0

19.1 ± 2.18

0.39 ± 0.12

49.38 ± 2.32

Myricetin

n/d

0.01 ± 0.01

n/d

0.29 ± 0.07

n/d

0.11 ± 0.01

n/d

n/d

n/d

n/d

n/d

Quercetin-3-Glucoside

2.99 ± 0.2

0.45 ± 0.42

n/d

0.04 ± 0.01

6.05 ± 0.54

0.04 ± 0.01

0.07 ± 0.01

n/d

2.4 ± 0.18

0.03 ± 0.01

1.64 ± 0.18

Taxifolin

0.49 ± 0.05

0.06 ± 0.06

n/d

0.29 ± 0.07

0.03 ± 0.02

0.62 ± 0.02

n/d

0.01 ± 0.01

0.65 ± 0.09

0.03 ± 0.01

0.44 ± 0.02

Genistein

n/d

0.43 ± 0.12

n/d

0.03 ± 0.01

n/d

0.01 ± 0

n/d

n/d

0.02 ± 0

0.08 ± 0

n/d

Scopoletin

0.03 ± 0.01

0.19 ± 0.16

4.19 ± 0.27

0.1 ± 0.01

1.35 ± 0.24

0.01 ± 0.02

n/d

n/d

0.03 ± 0

9.02 ± 0.46

n/d

Umbelliferone

0.02 ± 0

0.23 ± 0.11

n/d

0.01 ± 0

n/d

n/d

n/d

n/d

0.02 ± 0.03

2.37 ± 0.06

n/d

7,8-Dihydroxy-6-methyl coumarin

0.01 ± 0.01

0.02 ± 0.01

0.28 ± 0.01

0.06 ± 0.01

n/d

n/d

n/d

n/d

0.02 ± 0

5.5 ± 0.22

n/d

Biochanin A

n/d

0.22 ± 0.17

0.01 ± 0

0.02 ± 0

n/d

0.03 ± 0

n/d

0.02 ± 0.02

n/d

n/d

n/d

Poncirin

0.04 ± 0.01

n/d

n/d

0.04 ± 0.01

0.04 ± 0

n/d

n/d

n/d

n/d

n/d

Didymin

0.03 ± 0

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Phloridzin

0.01 ± 0.01

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.01 ± 0.01

n/d

n/d

Daidzein

n/d

n/d

n/d

0.01 ± 0

n/d

0.01 ± 0

n/d

n/d

n/d

n/d

n/d

Galangin

n/d

0.02 ± 0.04

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d.01

Luteolin

23.7 ± 1.72

6.89 ± 5.48

0.12 ± 0.01

0.01 ± 0

0.04 ± 0.01

0.33 ± 0.06

0.02 ± 0.01

0.01 ± 0.01

0.03 ± 0

4.34 ± 0.44

0.04 ± 0

Equol

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Fisetin

n/d

n/d

n/d

0.06 ± 0.01

n/d

0.05 ± 0

n/d

n/d

n/d

n/d

n/d

Luteolinidin

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Neoeriocitrin

n/d

n/d

n/d

0.01 ± 0.01

n/d

n/d

n/d

n/d

0.01 ± 0

n/d

n/d

Isorhamnetin

3.27 ± 0.35

11.45 ± 10.15

n/d

0.08 ± 0.02

3.35 ± 1.69

0.05 ± 0.02

0.03 ± 0

0.02 ± 0.02

0.26 ± 0.03

0.86 ± 0.19

7.84 ± 0.48

Formononetin

n/d

n/d

n/d

0.02 ± 0

n/d

0.05 ± 0

n/d

n/d

n/d

n/d

n/d

Apigenin

0.37 ± 0.02

68.45 ± 16.54

0.01 ± 0

0.07 ± 0.02

0.01 ± 0

0.02 ± 0.02

0.07 ± 0.02

0.01 ± 0

0.02 ± 0

12.67 ± 0.93

0.02 ± 0.02

Gossypin

n/d

n/d

n/d

n/d

0.23 ± 0.04

n/d

n/d

n/d

n/d

n/d

n/d

Tyrosol

0.81 ± 0.04

0.39 ± 0.27

0.16 ± 0.03

0.11 ± 0.02

0.67 ± 0.15

0.71 ± 0.04

0.01 ± 0.02

0.11 ± 0.1

1.89 ± 0.15

2.8 ± 0.23

Hydroxityrosol

0.03 ± 0.01

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.01 ± 0.01

n/d

n/d

Catechin

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

0.01 ± 0.01

0.06 ± 0.01

n/d

Epicatechin

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Gallocatechin

0.15 ± 0.03

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Epigallocatechin

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Epigallocatechin Gallate

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

n/d

Data is given as mean ± standard deviation and is expressed mg kg

1

n/d

M. Neacsu et al. / Food Chemistry 179 (2015) 159–169

Coumarin

n/d

0.59 ± 0.04

dry weight. n/d = not detected (i.e. below the detection level).

161

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M. Neacsu et al. / Food Chemistry 179 (2015) 159–169

reported to inhibit peroxidation of low-density lipoproteins (Cheng, Dai, Zhou, Yang, & Liu, 2007). Several studies provide correlations between consumption of foods rich in phenolic acids like cereal fibres and whole-grain (Neacsu et al., 2013) and human health benefits (Smith & Tucker, 2011). Flavonoids are plant secondary metabolites with well-known beneficial effects on human health. High intake of flavonoids is associated with low mortality from coronary heart disease and low incidence of myocardial infarction (Hertog et al., 1993), cancer protective effects (Le Marchand, 2002) and type 2 diabetes mellitus (T2DM) (Knekt et al., 2002) Isoflavones are a subclass of flavonoids, also known as phytoestrogen compounds because they exhibit oestrogenic activity, found in high quantities in Leguminosae family (Boniglia et al., 2009). Genistein and daidzein are the most extensively investigated to date. They may exhibit beneficial effects in ameliorating postmenopausal symptoms (Howes, Howes, & Knight, 2006), reducing the risk of cardiovascular diseases and breast and prostate cancer (Espin, Garcia-Conesa, & Tomas-Barberan, 2007). Catechins are another type of flavonoids; most of the beneficial health properties associated with catechins is attributed to their antioxidant activity. By virtue of their capacity to inhibit LDL oxidation, cathechins exert cardioprotective effects (Heim, Tagliaferro, & Bobilya, 2002), moreover, they may act as anticarcinogens and anti-inflammatory agents (Katiyar & Mukhtar, 1996). To benefit the rapidly growing food industry and the health of the consumer, there is now a crucial need to identify the phytochemical profiles of food ingredients. The objective of this research is to quantitatively determine the composition of phytochemicals in food plant powders commonly used by the food industry. These include: onion, celery, broccoli, beetroot, red pepper, green pea, yellow pea, tomato, spinach, carrot, swede. The potential role of these powders for use as food ingredients in healthier food reformulation, based on their phytochemicals composition is discussed. 2. Materials and methods 2.1. General reagents Standard phytophenols and general laboratory reagents were purchased from Sigma/Aldrich [Gillingham, England] or synthesised as described previously (Russell, Burkitt, Forrester, & Chesson, 1996; Russell, Burkitt, Scobbie, & Chesson, 2003). 2.2. Food plant powders The food plant powders used in this work were obtained from J.L. Priestley & Co. They were chosen to represent commonly consumed food crops, such as red pepper (Capsicum annuum) and tomato (Solanum lycopersicum) from the Solanaceae family; spinach (Spinacia oleracea) and beetroot (Beta vulgaris) from the Amaranthaceae family; carrot (Daucus carota) and celery (Apium graveolens) from the Apiaceae family; yellow pea (Lathyrus aphaca) and green pea (Pisum sativum) from the Fabaceae family; broccoli (Brassica oleracea) and swede (Brassica napobrassica) from the Brassicaceae family and onion (Allium cepa) from the Amaryllidaceae family. All of these powders are used as ingredients in food industry predominantly for flavorings and colour purposes. 2.3. Extraction and analysis of phytochemicals from food plant powders According with the manufacturer’s specifications, the plant foods powders were obtained as follows: beetroot was washed, sorted, trimmed, blanched, dried, milled; carrot was washed, peeled, diced, blanched, dried; mature broccoli heads were

washed, sorted, trimmed, diced, blanched, dried, milled; mature Celeriac roots were washed, sorted, trimmed, diced, dried milled; white onions, free from rot and other defects, were washed, sliced, dried, milled; garden peas were mechanically harvested, washed, scarified, blanched, air dried, sieved, milled; peas were dried, graded, sorted, cooked, re-dried and milled; ripe fruit of bell peppers, were washed, cored, trimmed, diced, dried, granulated; first quality tomato paste was concentrated, spray dried; swedes were washed, trimmed, diced, blanched, dried and spinach leaves were washed, air dried, milled. The powders’ particle sizes were in the range of 400 to 800 lm. All the powders were metal detected and produced in accordance with good manufacturing practice and complying with all UK & EU Food Laws. The extraction procedures used for the analysis of phytochemicals from the plant material were divided into three methods: one method for the isolation of phytophenols, one for catechins, and another for flavonoids and isoflavonoids. 2.3.1. Phytophenols extraction method The method used for phytophenol extraction was as described elsewhere (Neacsu et al., 2013). Samples (approx. 0.1 g dry weight; n = 3) were suspended in HCl (0.2 mol dm 3; 3 cm3), extracted into ethyl acetate (EtOAc; 5 cm3) and the layers were separated by centrifugation (5 min; 1800g; 4 °C). The extraction was repeated three times and the EtOAc extracts combined. The organic layer was left to stand over sodium sulphate (anhydrous) for 1 h and filtered. The combined organic layers were then evaporated to dryness under reduced pressure at a temperature not exceeding 40 °C and stored in a desiccator prior to analysis by HPLC. The extract produced in this step represents the ‘‘free fraction’’ as reported in Table 1. The pH of the aqueous fraction was increased to pH 7 with NaOH (4 mol dm 3). NaOH (4 mol dm 3) was added to give a final concentration of 1 mol dm 3 and the sample stirred at room temperature for 4 h under nitrogen. The pH was reduced to pH 2 with HCl (6 mol dm 3) and the samples extracted into EtOAc (5 cm3  3) and processed as described above. The pH of the aqueous fraction was then increased to pH 7 with NaOH (4 mol dm 3). HCl (10 mol dm 3) was added to give a final concentration of 2 mol dm 3 and the sample incubated with stirring at 95 °C for 30 min. This was cooled and extracted with EtOAc (5 cm3  3) and processed again as described above. The extracts obtained after alkaline and acid hydrolysis represents the ‘‘bound fractions’’ as presented in Table 1. The extracts were then re-dissolved in methanol (0.5 cm3) and an aliquot (20 ll) transferred to an eppendorf for HPLC–MS/MS analysis as described before (Neacsu et al., 2013). Internal standard 1 for negative mode mass spectrometry (IS1; 13C benzoic acid; 2 ng ll 1 in 0.02% acetic acid in 75% methanol; 20 ll), internal standard 2 for positive mode mass spectrometry (IS2; 2-amino-3,4,7,8-tetramethylimidazo[4,5f] quinoxaline; 0.5 ng ll 1 in 0.02% acetic acid in 75% methanol; 20 ll) and acidified (0.4 mol dm 3 HCl) methanol (40 ll) were added, vortexed and centrifuged (12,500g; 5 min; 4 °C). 2.3.2. Catechins extraction method Samples (approx. 0.25 g dry weight; n = 3) were suspended in a solvent extraction mixture (acetone: water: acetic acid; 70: 28: 2; v: v: v; 5 cm3), placed in an ultrasound bath for 10 min and then the supernatant was separated by centrifugation (5 min; 3220g; 4 °C). The extraction was repeated three times and the supernatants were combined. The organic solvent from the combined extracts was then evaporated under reduced pressure at a temperature not exceeding 40 °C. The water phase extract was freezedried and then re-dissolved in methanol (1.5 cm3, 50%) for HPLC– MS/MS analysis and mixed with IS1 for negative ion mass spectrometry. The ‘bound’ catechins were also isolated using the phy-

M. Neacsu et al. / Food Chemistry 179 (2015) 159–169

tophenols method (see Section 2.3.1) and are reported as well in the Table 1. 2.3.3. Flavonoids and isoflavonoids extraction method Samples (approx. 1 g dry weight; n = 3) were suspended in methanol (60%; containing acetic acid 0.1%; 8 cm3), placed in an ultrasound bath for 60 min and then the supernatant was separated by centrifugation (5 min; 3220g). The extraction was repeated twice and the supernatants were combined. These were then evaporated to dryness under reduced pressure at a temperature not exceeding 40 °C. The dried extracts were re-suspended in hydrochloric acid (1 mol dm 3; 4 cm3) and the samples incubated at 90 °C for one hour. Following acid hydrolysis the samples were extracted into ethyl acetate (10 cm3) and the layers separated by centrifugation (5 min; 3220g; 4 °C). The extraction was repeated three times and the ethyl acetate extracts combined. The combined organic layers were then evaporated to dryness under reduced pressure at temperature not exceeding 40 °C and re-dissolved in methanol (1.0 cm3) for HPLC–MS/MS analysis and mixed with IS1 and IS2 for negative ion and positive ion mass spectrometry respectively as described at Section 2.3.1 (see Table 2). 2.3.4. LC–MS/MS analysis The LC–MS/MS analysis method used for all phytophenols presented in the results Table 1A–F is described elsewhere (Russell et al., 2011; Neacsu et al., 2013) with the exception of the metabolites (flavonoids, isoflavonoids and catechins) presented in the results Table 1H. Liquid chromatography separation of these metabolites was performed on an Agilent 1100 HPLC system (Agilent Technologies, Wokingham, UK) using a Zorbax Eclipse™ 5 lm, 150 mm  4 mm column (Agilent Technologies). Two different gradients were used to separate the different categories of metabolites and the mobile phase solvents in each case were water containing 0.1% acetic acid and acetonitrile containing 0.1% acetic acid, respectively. In all cases the flow rate was 300 ll min 1 with an injection volume of 5 ll. The LC eluent was directed into, without splitting, an API 3200 triple quadrupole mass spectrometer (Applied Biosystems, Warrington, UK) fitted with a Turbo Ion Spray™ (TIS) source. For LC method 1, the mass spectrometer was run in negative ion mode using a solvent gradient and the mass spectrometer was set with the following source parameters: ion spray voltage 4500 V, source temperature 400 °C, gases 1 and 2 set at 15 (units) and 40 (units) respectively and the curtain gas set to 10 (units). LC method 2 used a solvent gradient program, and the mass spectrometer was run in positive ion mode with the following source settings; ion spray voltage 5500, source temperature 400 °C, gases 1 and 2 set at 14 (units) and 40 (units) respectively and the curtain gas set at 10 (units). All the metabolites were quantified using multiple reaction monitoring (MRM). Standard solutions (10 ng ll 1) for all metabolites were prepared and pumped directly via a syringe pump. For all the phytochemical quantifications the standard calibrations were over a concentration interval of 2 ng ll 1 to 10 pg ll 1. The threshold used for quantification was a signal to noise ratio of 3 to 1. The ion transitions for each of the metabolites were determined based upon their molecular ion and a strong fragment ion; their voltage parameters; declustering potential, collision energy and cell entrance/exit potentials were optimised individually for each metabolite; see Table 1. For several categories of compounds, it was inevitable that their molecular ion and fragment ion would be the same, but this was overcome by their differing elution times. All the data were averaged from three repeats coming from three different batches of samples and are reported as means and standard deviation. The data was analysed by principal component

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analysis (PCA), unit variance (UV)-scaled using Simpca P+ 12 (Umetrics, Cambridge).

3. Results and discussion 3.1. Phytochemical qualitative and quantitative analysis Principal component analysis (UV-scaled) of the food plant powders indicates that the phytochemical profiles of the spinach and celery powders discriminated from the rest of the powders (Fig. 1). Onion, swede, beetroot, carrot, green pea, yellow pea, broccoli and tomato powders had similar total metabolite profiles with green pea, yellow pea and tomato clustering more closely. Red pepper powder was found to have a slightly different metabolic profile. The PCA also indicates that celery and carrot belonging to the Apiaceae family are both situated in the lower quadrants of the diagram; the spinach and beetroot from the Amaranthaceae family are situated both in the upper quadrants; and the red pepper and tomato from the Solanaceae family are situated both in the upper quadrants, but this time they are much closer. On the PCA there is a strong alignment for swede and broccoli (Brassicaceae family) and for the yellow pea and green pea (Fabaceae family). Green pea powder had the lowest total content of phytochemicals of all the food plant powders studied. The total amount of phytophenol molecules present in the powders analysed varied in a range of approximately 0.1 to 1 g kg 1 of powder, in the following order: spinach > red pepper > onion > carrot > tomato > broccoli > beetroot > swede > celery > yellow pea > green pea. The quantitative analysis of the phytochemicals in the food plant powders (Table 1) reveals that 4-hydroxyphenylpyruvic acid was one of the most abundant metabolites present in all the powders and the main metabolite in red pepper, spinach, swede and tomato. This metabolite could be also derived from protein. Another metabolite found in rich amounts in most of the powders is m-hydroxybenzoic acid, being the second most abundant in tomato, swede, beetroot, carrot and red pepper; the most abundant in onion and green pea, but totally absent in spinach, yellow pea, broccoli and celery. Chlorogenic acid was the main metabolite in carrot and yellow pea powders and was also detected in red pepper, tomato and celery. Ferulic acid was present in all the powders, being the main metabolite determined in beetroot and the second most abundant in broccoli and spinach. It was also present in red peppers, yellow pea and celery. The main metabolite in broccoli was sinapic acid, this also being one of the main metabolites in red pepper, yellow pea, swede and also found in all other food plant powders. The gallocatechin was the only catechin ubiquitous in all the food plant powders. This was in the bound fraction obtained (after acid hydrolysis) from the phytophenol method of extraction (see Table 1F and H). Any other catechins were below detection limits (i.e. below 1 mg kg 1). The isoflavonoid and lignin compounds were absent or only present in low concentrations (see Table 1G and H). The quantitative analysis of flavonoids from the vegetables powders (Table 1H) reveals that the dominant (above 2.5 mg/kg powder) flavonoid in red pepper was quercetin followed by luteolin, isorhamnetin and naringenin; in spinach, apigenin, isorhamnetin and luteolin were the main flavonoids; in yellow pea, kaempferol; in broccoli, kaempferol, quercetin and isorhamnetin; in green pea, kaempferol and quercetin; in tomato, quercetin and naringenin; in celery, apigenin and luteolin and in onion, quercetin and kaempferol. Carrot, swede and beetroot powders were the poorest sources of flavonoids and none of the individual flavonoids were present at a concentration of more than 2.5 mg kg 1 powder. The concentrations of the ten most predominant phytophenols determined in this study, quantified by LC–MS/MS in each powder

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Table 2 MRM transition values for metabolites analysed with LC–MS/MS method 1⁄ and LC–MS/MS method 2⁄⁄. Metabolite

Negative mass spectrometry method, MRM transition values M H+ (m/z)

Fragment (m/z)

289.2 289.2 305.1 305.1 457.1 255.1 273.1 595.4 271.1 579.3 301.1 285.1 301.1 301.1 317.1 463.1 303.1 269.1 191.1 161.1 207.1 609.5 609.5 447.1 283.1 593.3 593.3 435.1 253.1 269.1 285.1 241.1 285.1 595.3 267.1 269.1 479.1 227.1 137.1 153.1 315.1 122.1

109.1 109.1 125.1 125.1 169.1 135.1 167.1 151.1 151.1 271.1 164.1 93.1 125 179.1 151.1 300.1 125.1 133.1 176.1 133.1 192.1 301.1 301.1 300.1 268.1 285.1 285.1 167.1 91.1 169.1 133.1 121.1 135.1 151.1 252.1 117.1 317.1 143.1 106.1 123.1 300.1 77.1

Voltage parameters⁄⁄⁄⁄ DP

Catechin Epicatechin Gallocatechin Epigallocatechin Epigallocatechin gallate Isoliquiritigenin Phloretin Eriocitrin Naringenin Naringin Hesperetin Kaempferol Morin Quercetin Myricetin Quercetin-3-glucoside Taxifolin Genstein Scopoletin Umbelliferone 7,8-di-OH-6-methylcoumarin Neohesperidin Hesperidin Quercitrin Biochanin A Poncirin Didymin Phloridzin Daidzein Galangin Luteolin Equol Fisetin Neoeriocitrin Formononetin Apigenin Gossypin Reservatrol Tyrosol Hydroxytyrosol Isorhamnetin 13 C-Benzoic acid (IS1)⁄⁄⁄

54 54 49 49 36 40 43 62 52 91 56 85 56 51 75 93 52 69 26 51 42 91 91 71 59 99 99 46 72 69 86 44 60 99 57 61 59 57 29 38 68 24

CEP 19.1 19.1 22 22 16 11.8 17 28.1 15.5 28.1 14.3 11.9 11.3 12 12.7 18.5 12.2 11.6 11.9 9.5 11.2 25 25 18.8 11.9 22.5 22.5 22.7 14 17.2 13.7 13.6 13 21.5 9.5 12.2 14.9 9.2 12.8 10.4 10.7 7.7

CE 35 35 30 30 25.5 23 23 53 26.5 44.5 33 49 29.5 27 35 41 31.5 43.5 21.5 29 22 47 47 37 31 51 51 43 51 37 45.5 20 28.5 53 30.5 52.5 31.5 36 23 21 31.5 16.5

CXP 1.8 1.8 3 3 2.4 3.8 4.2 1.8 2.2 3.2 1.8 2.2 2.8 1.8 3.2 4.2 2.8 1.4 2 1.4 2.2 4.4 4.4 4.4 4 3.2 3.2 2.4 1.8 2.6 3.4 2.8 2.8 4 4.8 2.8 3.8 3.2 2.3 2.8 4.2 5.4

Positive mass spectrometry method, MRM transition values M+H+ (m/z)

Fragment (m/z)

Voltage parameters DP

Coumarin Psoralen 8-Methylpsoralen Bergapten Tangeretin Coumesterol Luteolinidin Glycitein 4,7,8-TriMeIQx (IS2)⁄⁄⁄

Time (min) ⁄

147.1 187.1 217.2 217.2 373.1 269.1 271.1 285.1 242.1

A (%)

91.1 131.1 202.1 202.1 343.3 213.1 115.1 270.2 227.1

49.5 54 54 54 60 73 82 70 58

CEP

CE

CXP

12.3 11.1 15 15 17.6 16.5 14.7 16 16

34 31.5 28 28 35 35 67 35 34

1.6 2.4 3.6 3.6 5.5 4 1.8 4.5 3.9

B (%)

LC–MS/MS method 1, gradient solvent condition 0.00

85.0

40.00

5.0

95.0

43.00

5.0

95.0

44.00

85.0

15.0

50.00

85.0

15.0

⁄⁄

15.0

LC–MS/MS method 2, gradient solvent condition

0.00

75.0

15.0

20.00

5.0

95.0

23.00

5.0

95.0

24.00

75.0

15.0

32.00

75.0

15.0

Where A: 0.1% acetic acid; solvent B: acetonitrile + 0.1% acetic acid. ⁄⁄⁄ IS1-internal standards 1, 13C benzoic acid; IS2-internal standard 2, 2-amino-3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline. ⁄⁄⁄⁄ DP, declustering potential; CE, collision energy and CXP, collision cell exit potential.

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Amaranthaceae Amaryllidaceae Apiaceae

12 10 8

Brassicaceae

6

Fabaceae Solanaceae

4

spinach

red pepper

t[2]

2

swede

0 carrot

-2 -4 -6 -8 -10 celery

-12

-15

-14

-13

-12

-11

-10

-9

-8

-7

-6

-5

-4

-3

-2

-1

0 t[1]

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Fig. 1. Principal component analysis (UV-scaled) analysis of the full metabolite profiles (analysed in present study) in all food plant powders.

129.77

green pea

Total phytochemical content

yellow pea

214.45

celery

217.06 297.24

swede

419.05

beetroot broccoli

447.08

tomato

449.07 465.88

carrot

480.92

onion

581.96

red pepper

928.43

spinach 0

100

200

300

400

500

600

700

800

900

1000

mg Kg-1 sample

Fig. 2. Total phytochemical content (mg kg 1) in the food plant powders obtained by summing the individual compounds concentration and the ten highest individual phytochemical concentrations (mg kg 1) in the food plant powders obtained by quantitative LC–MS/MS analysis.

are presented in Fig. 2. The total phytochemicals content was estimated by summing the individual values for the phytochemicals obtained from LC–MS/MS quantitative analysis for each vegetable powder (Fig. 2). Spinach was the richest in total phytochemicals content (928.43 mg kg 1), followed by red pepper (581.96 mg kg 1) and onion (480.92 mg kg 1). The total ion current (TIC) chromatogram for the main flavonoids found in the food powders is shown in Fig. 3A and the TIC chromatograms for the main phytophenols is shown in Fig. 3B.

3.2. Health benefits and possible food applications The food industry continuously seeks to adapt and develop new formulations designed to increase shelf life, quality and safety. Lipid oxidation generates undesirable products from the sensory point of view; it also causes the degradation of fat soluble vitamins and essential fatty acids, and interferes with the integrity and safety of foods through the formation of potentially toxic compounds, such as malonaldehyde (Selani et al., 2011). The food

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A

B

Fig. 3. The TIC (total ion current) chromatograms for the flavonoids standards and main representatives found in the food powders analysed with the method described in the LC–MS/MS analysis (A), and the TIC chromatograms for the main phytophenols standards and main representatives found in the food powders analysed with methods previously described (Russell et al., 2011; Neacsu et al., 2013) (B). (A) 1, Gallocatechin, 7.41 min; 2, hydroxytyrosol, 8.75 min; 3, epigallochatechin, 8.83 min; 4, catechin, 11.53 min; 5, tyrosol, 12.27 min; 6, epicatechin, 13.00 min; 7, epigallochatechin gallate, 13.62 min; 8, eriocitrin, 14.38 min; 9, 7,8-dihydroxy-6-methoxycoumarin, 14.55 min; 10, neoeriocitrin, 14.82 min; 11, qercetin-3-glucoside, 15.36 min; 12, naringin , 16.34 min; 13, hesperidin, 16.52 min; 14, quercitrin, 16.79 min; 15, neohesperidin, 16.94 min; 16, scopoletin (A), 17.70 min; 17, taxifolin, 17.78 min; 18, gossypin, 17.97 min; 19, phloridzin, 17.99 min; 20, umbelliferone, 18.24 min; 21, myricetin, 19.48 min; 22, fisetin, 19.56 min; 23, didymin, 19.57 min; 24, poncirin, 19.97 min; 25, 13C-benzoic acid (IS), 20.77 min; 26, resveratrol, 21.15 min; 27, daidzein, 21.26 min; 28, luteolin (B), 22.01 min; 29, quercetin (C), 22.51 min; 30, phloretin, 24.36 min; 31, apigenin (D), 24.66 min; 32, genstein, 25.10 min; 33, naringenin (E), 25.16 min; 34, equol, 25.34 min; 35, kaempferol (F), 25.45 min; 36, isorhamnetin (G), 25.71 min; 37, morin, 25.86 min; 38, hesperetin, 25.86 min; 39, isoliquiritigenin, 27.01 min; 40, formononetin, 27.94 min; 41, galangin, 32.15 min; 42, biochanin a, 32.39 min. (B) 1, 3,4-dihydroxymandelic acid, 6.34 min; 2, 4-hydroxymandelic acid, 7.64 min; 3, gallic acid, 8.44 min; 4, 4-hydroxy-3-methoxymandelic acid, 8.96 min; 5, 3-hydroxymandelic acid, 11.48 min; 6, 1,2,3-trihydroxybenzene, 11.87 min; 7, 3,4,5-trihydroxybenzaldehyde, 13.35 min; 8, 3,5-dihydroxybenzoic acid, 14.24 min; 9, protocatechuic acid (A), 15.28 min; 10, 4-hydroxyphenyllactic acid, 16.04 min; 11, 3,4-dihydroxyphenylacetic acid, 16.65 min; 12, 2,5-dihydroxybenzoic acid (B), 17.56 min; 13, 1,3-dihydroxybenzene, 17.63 min;14, chlorogenic acid (C), 17.68 min; 15, 4-hydroxyphenylacetic acid (D), 19.34 min; 16, protocatachaldehyde, 19.81 min; 17, 2,3-dihydroxybenzoic acid (E), 20.00 min; 18, p-hydroxybenzoic acid (F), 20.31 min; 19, 2-hydroxybenzylalcohol, 20.35 min; 20, 3,4dihydroxyphenylpropionic acid, 20.75 min; 21, 2,4-dihydroxybenzoic acid, 21.23 min; 22, 1,2-dihydroxybenzene, 21.23 min; 23, 3-hydroxyphenylacetic acid , 21.27 min; 24 caffeic acid (G), 21.90 min; 25, vanillic acid (H), 21.94 min; 26, syringic acid (I) 22.12 min; 27, 4-hydroxy-3-methoxyphenylacetic acid, 22.71 min; 28, m-hydroxybenzoic acid (J), 23.35 min; 29, mandelic acid, 23.42 min; 30, 2,6-dihydroxybenzoic acid, 24.06 min; 31, p-hydroxybenzaldehyde (K), 25.33; 32, 4-hydroxyphenylpropionic acid (L), 25.58; 33, o-hydroxyhippuric acid 26.45 min; 34, isovanillin 26.67 min; 35, p-coumaric acid (M), 26.76 min; 36, 4-hydroxy-3-methoxyphenylpropionic acid (N), 26.84 min; 37, 4hydroxyacetophenone, 27.03 min; 38, vanillin (O), 27.23 min; 39, syringin, 27.62; 40, sinapic acid (P), 27.67; 41, 3-hydroxyphenylpropionic acid, 27.98; 42, ferulic acid (Q), 28.22 min; 43, 4-hydroxy-3-methoxyacetophenone (R), 28.78 min; 44, 4-hydroxy-3,5-dimethoxyacetophenone, 28.88 min; 45, 3,4-dimethoxybenzoic acid (S), 29.26 min; 46, o-anisic acid (T), 29.54 min; 47, m-coumaric acid, 29.65 min; 48, 2-hydroxyphenylpropionic acid, 30.96 min; 49, salicylic acid (U), 31.10 min; 50, indole 3-carboxylic acid, 31.24 min; 51, o-coumaric acid (V), 32.53 min; 52, 13C-benzoic acid (IS), 32.96 min; 53, phenylacetic acid (W), 33.76 min; 54, indole 3-acetic acid 34.16 min; 55, 4methoxyphenylacetic acid, 34.47 min; 56, p-anisic acid, 34.59 min; 57, 3,4-dimethoxycinnamic acid, 35.14 min; 58, m-anisic acid, 35.63 min; 59, 3,4,5-trimethoxycinnamic acid, 37.52 min; 60, indole 3-propionic acid, 39.30 min; 6, phenylpropionic acid (X), 39.87 min; 62, 3-methoxyphenylpropionic acid, 40.03 min; 63, cinnamic acid (Y), 40.34 min; 64, 4-methoxycinnamic acid, 40.56 min; 65, 3-methoxycinnamic acid, 41.85 min; 66, ethylferulate, 47.64 min.

M. Neacsu et al. / Food Chemistry 179 (2015) 159–169 Table 3 Fibre, fat and protein content in the food plant powders. Vegetable powder

Red pepper Spinach Carrot Yellow pea Broccoli Green pea Swede Beetroot Tomato Celery Onion

Macronutrient (%) Fibre

Protein

Fat

8.8 39.7 16.7 4.7 15.5 17 29.8 14.4 16.5 47 11.7

16.1 32.2 7.4 22 29.8 23 12.1 14.7 12.9 15 12.7

2.7 3.2 0.5 2 1.8 2.1 Trace 0.5 0.4 Trace 0.7

Data is supplied by the manufacturer and represents % of dry weight of the food plant powders.

industry uses synthetic additives with antioxidant properties to control this process. Due to concerns of possible toxic effects from synthetic antioxidants and to increasingly demanding consumer preferences for natural products and health benefits, the interest for alternative methods to retard lipid oxidation in foods, such as the use of natural antioxidants, has increased (Selani et al., 2011). Alternative methods using natural antioxidants could include the use of extracts from herbs (El-Alim, Lugasi, Hóvári, & Dworchák, 1999), fruit juice and rind powder (Naveena, Sen, Vaithiyanathan, Babji, & Kondaiah, 2008), and grape seeds (Brannan & Mah, 2007). The additive and synergistic effects of the complex mixture of phytochemicals present in whole fruit and vegetables and in their extracts are responsible for their potent antioxidant activity (Sun, Chu, Wu, & Liu, 2002; Liu, 2003). There are several scientific papers reporting the use of natural antioxidants in meat and meat products, such as phenolic diterpene, hydroxycinnamic acid derivatives, flavonoids and triterpene found in different herb essential oils (Oberdieck, 2004; SanchezEscalante, Djenane, Torrescano, Beltran, & Roncales, 2003). The food plant powders from the current study are found to be rich in flavonoids (spinach and onion being the richest), and in hydroxycinnamic compounds, such as broccoli and spinach powders, offering further applicability in the food industry as food additives and preservatives. Previous work has shown that in an accelerated oxidation system, six of these eleven food plant powders significantly (p < 0.05) improved oxidative stability of turkey patties by 20–30%, in the order: spinach < yellow pea < onion < red pepper < green pea < tomato (Duthie, Campbell, Bestwick, Stephen, & Russell, 2013). It has also been shown that the oxidative stability of egg protein-stabilized oil-in-water emulsions was significantly improved in most cases by adding these plant food powders, as indicated by the Rancimat method with broccoli exhibiting the highest increase in induction time (98.2%) compared with the control (Raikos, Neacsu, Morrice, & Duthie, 2014). The same study showed that the physical stability of egg protein-stabilized oil-in water emulsions remained unaffected by the addition of these powders (Raikos et al., 2014). Due to the antimicrobial effect of phenolic acids and flavonoids, food plant powders have the potential to be successfully used as preservatives (Cushnie & Lamb, 2005). A decreasing number of hydroxyl groups and their substitution with methoxy groups increased the antibacterial activity of hydroxybenzoic, but not of hydroxycinnamic acid (Sanchez-Maldonado, Schieber, & Ganzle, 2011). In the present work, all the vegetables were rich in benzoic and hydroxybenzoic acid, onion being remarkably high in mhydroxybenzoic acid (222.01 mg kg 1), followed by red pepper

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with 128.16 mg kg 1 and carrot (109.68 mg kg 1). The spinach powder was the only one with 3,4-dimethoxybenzoic acid (22.29 mg kg 1). In general, all the food plant powders were richer in benzoic rather than cinnamic acids. Quercetin, apigenin, luteolin, kaempferol were found to have strong antibacterial activity (Basilea, Giordanoa, Lopez-Sanchez, & Cobianchia, 1999). Quercetin, apigenin, and luteolin were present in all the vegetable powders, onion being the richest in quercetin (49.37 mg kg 1), followed by red pepper with 23.73 mg kg 1 and tomato (19.09 mg kg 1). The highest concentration of apigenin was found in spinach (68.45 mg kg 1), followed by celery (12.67 mg kg 1); and luteolin was found to be in the highest amount in red pepper (23.70 mg kg 1). Broccoli, yellow pea and onion were found to be rich sources of kaempferol (13.43 mg kg 1, 11.74 mg kg 1 and respectively 12.56 mg kg 1). Due to the high content of naturally occurring antimicrobials (phenolic acids and flavonoids) in the food plant powders subject of this study, another possible application of them is as an alternative to nitrites in meat products (Weiss, Gibis, Schuh, & Salminen, 2010). Fruit and vegetable residual flour obtained from juice production has been successfully used to reformulate cereal bars and biscuits for increasing their microbiological stability, water holding capacity and mineral and fibre content (Ferreira et al., 2013). These improvements are due to the carbohydrate, fibre and phytochemical content of the raw material used for the reformulation. The incorporation of dietetic fibre as prebiotic is also a possibility, where its use jointly with thermo-tolerant probiotic lactic acid bacteria could be a viable alternative to symbiotic low fat-reduced salt healthy meat products (Totosaus & Pérez-Chabela, 2013). In the UK most people do not eat enough fibre (the average intake is 12.8 g/day for women and 14.8 g/day for men) (BNF, 2012). The recommended average intake for adults is 18 g (NSP) per day (FSA, 2006). According to the manufacturer specifications, the food plant powders analysed in the present study have, besides their rich phytochemical content, a substantial fibre content (Table 3), varying from as low as 9% in red pepper powder up to 47% in celery powder, thus representing a potential rich source of fibre in human diet. All the food plant powders are also low in fat (Table 3). For some powders (swede and celery) the fat was detected only as a ‘trace’, and consequently will not add substantially to the total fat content if used in food reformulations. Currently proteins of plant origin are gaining interest as an alternative to animal proteins, being favoured by consumers due to health, animal-welfare and/or environmental reasons (Abou-Samra, Keersmaekers, Brienza, Mukherjee, & Macé, 2011). Plant protein (soya) rich diets could successfully compete with animal protein diets for weight loss purposes, both proteins sources equally enhancing the satiety and inhibiting hunger sensation in humans (Neacsu et al., 2014). The food plant powders used in this study could represent an important source of proteins (Table 3). The richest protein sources were spinach with 32.2%, broccoli 29.8%, followed by green pea 23% and yellow pea 22%. Therefore, based on the present findings and the literature discussed above if we are to use/recommend each food powder for a specific food application, based on its major phytochemicals, we could conclude/advise the following: Spinach could be used as a food additive in meat products formulation due to the presence of strong antioxidants, such as apigenin, ferulic and p-coumaric acid. Moreover, spinach was the only powder to have 3,4-dimethoxybenzoic acid which is a strong antimicrobial; Red pepper could be used to replace the nitrite from meat products due to the presence of the strong antimicrobial compounds, such as m-hydroxybenzoic acid, quercetin and luteolin; Onion since it is the richest in quercetin and m-hydroxybenzoic acid could also be used an antimicrobial ingredient; Carrot was the richest source in chlorogenic acid, known as a strong antioxidant and therefore could be

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used as food additive to extend shelf-life; Tomato powder was rich a source of m-hydroxybenzoic acid, chlorogenic and caffeic acid and could be used as preservative. Broccoli could be also used as preservative not only due to the high quantities of ferulic and caffeic but also as it was the richest powder in sinapic acid. Beetroot was the richest source of ferulic acid which is used as food additive to prevent lipid peroxidation. Swede, Celery, Yellow Pea and Green Pea had the lowest amounts of phytochemicals analysed (Fig. 2). However yellow pea was one of the richest sources in chlorogenic acid from the powders and also contained sinapic acid and kaempferol which may have antioxidant relevance as a good food additive. Swede and celery powders even though they were low in phytochemicals were both remarkably high in fibre content (Table 3) which is also an important aspect for food formulations. On this note, spinach powder was remarkably high in both fibre and protein and broccoli was high in protein, crucial for healthier food reformulations. Excess nourishment and a sedentary lifestyle lead to glucose and fatty acid overload, resulting in production of reactive oxygen species (ROS) and thus may underlay the development of insulin resistance, b-cell dysfunction, impaired glucose tolerance and T2DM. Excessive generation of reactive oxygen species (ROS) can arise from a wide range of environmental factors including ultraviolet stress, pathogen invasion, herbicide action and pollution (Gupta & Gupta, 2013). ROS have also been implicated in the progression of long-term diabetes complications, including micro- and macrovascular dysfunction (Wright, Scism-Bacon, & Glass, 2006) as well as in promoting rancidity of fats and damage to DNA. The molecules measured in this study in the food plant powders are found exclusively in fruits, vegetables and other food crops and have been ascribed bioactivities with potential health-protecting properties. For example their antioxidant and anti-inflammatory properties may reduce the risk of developing several diseases including cancer, heart disease and diabetes (Hertog et al., 1993; Le Marchand, 2002) where oxidative stress is implicated. The total intake of flavonoids (quercetin, myricetin, kaempferol, luteolin, and fisetin) was inversely correlated with the plasma total cholesterol and low-density lipoprotein (LDL) cholesterol concentrations (Arai et al., 2000). A prospective study involving 9959 men and women (age 15–99 years old) in Finland showed an inverse association between the intake of flavonoids and the incidence of all types of cancer combined (Knekt et al., 1997). High amounts of flavonoids were found in onion, red pepper, tomato, spinach, celery, broccoli and yellow pea powders, therefore their consumption maybe beneficial for human health. Ferulic acid, due to its potent antioxidant capacity in vitro, and inhibition of the expression and/or activity of cytotoxic enzymes including inducible nitric oxide synthase, caspases and cyclooxygenase-2, has been proposed for the treatment of several age-related diseases, such as cardiovascular diseases and cancer (Barone, Calabrese, & Mancuso, 2009). Ferulic acid was one of the main phytochemicals found in celery, red pepper, spinach, yellow pea, broccoli and in particular beetroot powders. Hydroxycinnamic acids have been consistently associated with a reduced risk of cardiovascular disease, cancer and other chronic diseases (Spencer, El Mohsen, Minihane, & Mathers, 2008). Hydroxycinnamic acids representatives like chlorogenic acid, coumaric acid, caffeic acid and sinapic acid are amongst the main phytochemicals found in the plant food powders studied, with marked quantities of sinapic acid in broccoli powder, caffeic acid in tomato powder and chlorogenic acid in carrot and yellow pea powders. Therefore adding these food plant powders rich in bioactive phytochemicals could confer benefits to food reformulations, improving both the food products’ quality and health promoting properties. Such an approach would lead to increased consumption of plant-based foods which is generally accepted as being beneficial for health.

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