1. Introduction
Human health benefits from diets being rich in fruits and vegetables have been verified in a range of studies, and is partly due to an association with a reduction in cardiovascular disease and cancer mortality [
1]. Both fruit and vegetables, as well as other plant based foods, are rich in compounds that are suggested to have health beneficial properties [
2]. Of these compounds, in particular dietary fiber and bioactive compounds such as phenolics are reported as beneficial when sufficiently consumed [
3,
4,
5].
Dietary fiber is a term used for naturally occurring carbohydrate polymers that are not digested nor absorbed in the small intestine, and that have health beneficial properties [
6]. Dietary fiber can be divided into two fractions, soluble (SDF) and insoluble (IDF) dietary fiber, due to the solubility in water. Most plant foods contain a combination of SDF and IDF [
7]. Dietary fiber has been shown to promote health benefits, such as lowering cholesterol in the blood [
8], have an impact on the rate of gastric emptying [
9], and promote peristaltic movement in the intestines [
10]. In addition, dietary fiber is important as energy source for the gut microbiota, which will use the dietary fiber to produce short chained fatty acids (SCFA) [
11]. These SCFA can be absorbed and can help in regulating the metabolism and immune system of the host [
11]. A diet that contains several types of dietary fiber has been suggested to lead to a gut microbiota with an increased diversity, which in turn could have health beneficial effects [
11,
12,
13]. Fruit and vegetables have been shown to be good sources of dietary fiber [
14]. The edible parts of vegetables in the Brassica family usually contain dietary fiber in moderate to high amounts [
15,
16,
17]. Given the recent interest in increasing the resource efficiency by using side streams of different produce, broccoli leaves could be an attractive new source of dietary fiber.
In earlier studies, dietary fiber and phenolic compounds have been analyzed separately due to substantial differences in their chemical structure and biological properties, even though the phenolic compounds that are associated with the dietary fiber might have a significant contribution to the overall health [
18,
19]. Dietary fiber is proposed to bind phenolics [
20,
21,
22], enabling these compounds to escape digestive enzymes in the upper gastrointestinal tract and instead reach the colon intact [
23,
24]. There, the gut microbiota can ferment both the dietary fiber and the phenolics to more easily absorbable compounds.
Phenolic compounds are defined as substances possessing an aromatic ring bearing one or more hydroxyl group including their functional derivatives [
25]. In plants, the phenolic compounds have various functions, such acting as anti-feedant, anti-pathogenic, and protective agents (e.g., for UV light) [
25]. They also provide pigmentation of plants, are attractants for pollinators, make the cell walls impermeable for gas and water, and contribute to physical stability of the plant [
25]. Phenolic compounds are often complex molecules, that are transformed into molecules of smaller size by the gut microbiota before absorption, which increases the bioavailability of these compounds [
26]. Most phenolic compounds have antioxidative properties, hence protecting the cells from, e.g., free radicals [
27]. Furthermore, the phenolic compounds have been implicated as involved in improving the vascular health [
28], lower the risk for developing certain types of cancer [
29] and lower the risk of chronic inflammations [
3,
30]. Phenolic compound may also have an impact on the diversity of the gut microbiota, if they can reach the colon intact [
26]. Leafy green vegetables usually contain high levels of phenolic compounds [
31]. In Brassica vegetables, including broccoli, a large number of phenolic compounds have been identified [
32,
33,
34], mainly from the parts already used as food, such as the broccoli florets and kale leaves. This indicates that broccoli leaves should contain phenolic compounds in comparable amounts.
The florets in broccoli (
Brassica oleracea Italica group) have been shown to contain health beneficial compounds, such as vitamin K and C, minerals, dietary fiber, phenolic compounds, glucosinolates and folic acid [
35,
36,
37]. The broccoli leaves, on the other hand, are not as well studied as the florets, but have been shown to have higher levels of phenolic compounds as compared to the florets [
38,
39]. The stem in broccoli contains large amount of insoluble fiber and low amounts of soluble fiber [
40].
From the currently applied greenhouse production systems of broccoli, it has been estimated that only 10% of the above ground biomass ends up as broccoli florets for consumption. The rest (90%) of the above ground broccoli plants (which includes stems, leaves, and inflorescences of insufficient size) becomes waste [
41]. Previous experiments have shown that 70% of the total weight of the broccoli plants is wasted in the field, while 45–50% of the harvested edible broccoli florets are wasted during processing and transportation [
42]. Such parts of the broccoli plant, today cultivated and edible but not used as food, are interesting sources for use as novel food products. These side streams have a potential to be used as functional ingredients to improve the nutritional values of different food products.
The aim of this study was to evaluate the content and composition of dietary fiber and phenolic compounds in broccoli leaves, and to investigate potential relationships between the content and composition of these groups of compounds. A second aim was to discuss possible impact on health from consumption of broccoli leaves, based on the evaluated content and composition of these compounds. Furthermore, the study aimed to describe possible food applications of broccoli leaves as a side stream from commercial broccoli (florets) production.
4. Discussion
The present study clearly showed that broccoli leaves, today commonly not used as food, have high content of both dietary fiber and phenolic compounds and also that the content of some of the dietary fiber constituents and phenolic compounds co-varied. Broccoli leaves turned out as having high content of compounds regarded as healthy, which make them of interest as potential component for the food industry. Environmental and climate change concern has increased the interest in using edible side-streams of food production for new food products, which also would increase the amount of food available globally. Furthermore, a high content of dietary fiber and phenolic compounds combined is of interest from a health perspective. The co-variation of these compounds might be of specific relevance as a major factor affecting the uptake mechanism in the human intestine.
Here, we have for the first time, to our knowledge, shown a co-variation in broccoli leaves among content of certain phenolic and dietary fiber, i.e., some of the phenolic acids showed a positive correlation with some of the SDF (Sol fuc, Sol xyl, Sol man and Sol glc; (
Table 4)). Three of the mentioned dietary fiber constituents (Sol fuc, Sol xyl and Sol glc) are known as being the main parts of the complex soluble dietary fiber xyloglucan [
57]. Previous studies have suggested a possibility that phenolic compounds are bound to the complex dietary fiber xyloglucan [
58].
Previous results have indicated that phenolic compounds can be strongly bound to dietary fiber, thereby they should be considered as one collective group, denominated as antioxidant dietary fiber [
19,
23,
59]. However, previous studies have also pointed out that phenolics are a large and diverse group of compounds localized in several parts of the plant cell; in the vacuole, in the chloroplast, in the nuclei, and also in the cell wall [
60]. In a study of chicory leaves, the fractions of foliar parenchyma cells were found to have higher concentration of phenolics as compared to vein fractions [
61], indicating that cells in the veins with thicker cell walls, constituting of dietary fiber, had lower concentrations of phenolics. The results from the present study showed corresponding results, i.e., in this investigation the dietary fiber constituents of the broccoli leaves present in highest concentration in this investigation (Insol UA, Insol xyl, and Insol glc) showed in general no significant correlation with the analyzed phenolics, and some of both IDF and SDF constituents showed negative correlation with different phenolic groups. Hence, the major part of the phenolics found in this investigation should not be bound to cell walls, i.e., the dietary fiber, but rather be present in other parts of the cells or in cells with thinner cell wall. However, the phenolic compounds are possibly not easily extracted from the fiber matrix with only organic solvent. As described in the materials and method section, we have used methanol extraction following similar procedure as recommended and used in other publications and also by us on other brassica species [
48,
49,
50]. However, the results from the present study indicate that additional phenolic compounds might be present in broccoli leaves not able to be extracted with methods generally adopted and commonly used for phenolics extraction in plants. To be able to evaluate content of all phenolic compounds, and including all cell wall bound phenolic compounds, alternative extraction procedures with a more efficient disruption of the cell wall can be considered, including enzymatic [
62], ultrasonic [
63] and ultrasonic assisted enzymatic extraction [
64,
65].
Broccoli leaves, with their mean content of TDF at 26%–32% of the DW, have an intermediate content of TDF, as compared to other types of food and vegetables (
Table 6). Thus, the content of dietary fiber in broccoli leaves is higher than that in oat brans, carrots and apples, but lower content as compared to onions, cabbage outer leaves, kale leaves and the broccoli florets. This makes broccoli leaves an interesting raw material for food from a health perspective.
Despite, as discussed above, that content of phenolics might possibly be higher in broccoli leaves than possible to measure with the applied methodology, the levels were found similar as previously reported for kale, and higher as compared to the broccoli florets (
Table 7). Thus, from perspective of phenolic content, the broccoli leaves are an interesting component for the food industry. The content of conjugated phenolics in the present study varied between the years, with 10.8–15.2 mg/g DW for 2017 as compared to 6.3–7.5 mg/g DW for 2018. At the same time, the content of phenolic acids did not vary significantly between years, but were approximately 3.6–5.7 mg/g DW.
Both the content of dietary fiber and phenolics varied between the two years of this study, though the former to somewhat lower degree. This might be due to the different weather conditions during these years, with an exceptionally warm and dry summer in Sweden 2018 (maximum and mean temperature in 2018 were 28.6 °C and 16.4 °C respectively, compared to 20.8 °C and 14.3 °C respectively in 2017, according to Swedish Meteorological and Hydrological Institute (SMHI)). The levels of phenolic compounds in kale, another member of the Brassica family, have been shown to increase when the temperature decreases due to an accumulation of secondary metabolites [
67,
68]. The amount of phenolic compounds in Brassica also depend on genetic variation (both within and among species) and on environmental factors as well as biotic and abiotic stresses (e.g., insect attacks, light, temperature, nutrients, water, growing conditions, and UV radiation) [
51]. Furthermore, in this investigation the broccoli leaves were collected at commercial farms applying crop rotation, i.e., the same fields were not used for broccoli production during the two years. Instead plant materials were collected from fields in the same area both years, resulting in that variation between the two years might also be due to differences between fields. Lastly, water content in the broccoli leaves differed significantly between the years, which also indicate differences in environmental factors which might impact variation in phenolic and dietary fiber between the years. Similar water content have been reported earlier [
37,
42].
In this investigation we have used the common categorization of the dietary fiber in soluble and insoluble fiber. However, recently it has been questioned if these two categories are sufficient when describing the functionality of the specific type of fiber, and the perceived health effects [
4,
69]. At present, there is insufficient knowledge of how the individual components of both the dietary fiber and the phenolics influence the various health effects, and also possible interactions between these groups. In addition, the structural diversity of the different fiber, both within a plant, but also depending of the plant species, is likely to influence the digestion of the fiber, and thereby possibly the health effects.
Health beneficial effects from phenolic compound have been suggested to be a result of some phenolics having the opportunity to travel along the intestines to reach the colon, and the gut microbiota, intact [
3,
26]. Phenolics are suggested to be strongly bound to dietary fiber, and to not be released from the food matrix by mastication, acid pH or human digestive enzymes [
70]. The phenolic compounds that travels inside the gastrointestinal tract for a long time together with the dietary fiber might also have the effect that they lower the amounts of reactive oxygen species (e.g., free radicals) in the gastrointestinal tract, which would also be beneficial [
19]. Dietary fiber from kale has been shown to bind bile acid and simultaneously release phenolic compounds from the matrix, thus bile acids can increase the bioaccessibility of the phenolic compounds [
71], and has also been shown to have a beneficial impact on the cholesterol levels in the blood [
72]. In connection to this, the gut microbiota has been shown to be altered by consumption of dietary fiber rich cruciferous vegetable, such as broccoli, cauliflower and cabbage, which could ultimately influence gut metabolism of bioactive food components and host exposure to these beneficial compounds [
73]. Phenolic compounds in themselves have been shown to be beneficial for health, e.g., by increasing weight loss in obese mice and humans [
74], and also to lower the mortality of some chronic diseases, mainly cardiovascular diseases and cancer [
75].
The average daily intake of dietary fiber in most Western countries (15–25 g dietary fiber/day) is low compared with the recommended daily intake of dietary fiber in Europe (20–38 g/day for adults) [
4]. The content of dietary fiber found in this investigation in broccoli leaves, 26%–32% of the DW, are in line with earlier studies which showed that the levels of TDF in a mixture of broccoli leaves and stems were approximately 36% [
76] with lower amount of fiber in the leaves compared to the stems [
42]. Hence, if broccoli side streams are used in every day food products, this will contribute to an increase in total dietary intake of fiber towards the recommended levels, while at the same time lessen the amount of the broccoli plant not used as food. Dietary fiber ingredients can also be used to improve functional properties in, e.g., meat, dairy, and wheat flour-based products [
77].
Production of food requires resources such as water, fertilizers, farmland, and energy. Currently, the generated amount of food waste correspond to production on 0.9 million hectares of farmland, release of 3.49 GT carbon dioxide equivalents (CO
2e), and use of 306 km
3 of drinkable water [
78]. In these calculations, the biomass not harvested but that could be eaten was not included. A more complete use of the agriculturally produced biomass would contribute to an increased productivity with less field waste, which would have a beneficial impact on the global climate. Furthermore, the different side steams from fruit and vegetable production are a readily available resource, and can be used as new food products, but also as a raw material for extraction of valuable compounds [
79].
In the case of broccoli, the florets only make up 15% of the total biomass of the broccoli plant, while the leaves make up a total of 47%, and stems and roots make up the remaining 38% [
37]. Broccoli powder from dried florets, leaves or stalks can be used as a natural food supplement since these powders contain high levels of amino acids and fatty acids and also good physicochemical properties [
37,
42]. An addition of 10% vegetable powder (carrot, tomato, broccoli florets, and beetroot), has been shown to increase the nutritional and functional attributes in oil-free bread [
80]. Broccoli florets increased levels of protein, fat, vitamin E and also antioxidant capacity in these breads. Broccoli leaves and stems have been shown to increase the phenolic content and antioxidant capacity in bread when added in a concentration of 2% (w/w), while still have an overall acceptability [
81]. In addition, broccoli leaf powder has been proposed for use in gluten free sponge cake to increase the content of minerals, antioxidant capacity and protein [
82], and also to increase the technological and sensory quality of gluten free sponge cake [
83]. Hence, with the levels of dietary fiber and phenolic compounds found in broccoli leaves in this study, future food uses of this side stream would be of interest as a food supplement to increase nutritional values. Furthermore, added-value use of the side streams of broccoli leaves contributes to socio-economic and environmental sustainability to the bioeconomy of our modern society.