*Article* **Assessing the Camelina (***Camelina sativa* **(L.) Crantz) Seed Harvesting Using a Combine Harvester: A Case-Study on the Assessment of Work Performance and Seed Loss**

**Walter Stefanoni 1, Francesco Latterini 1, Javier Prieto Ruiz 2, Simone Bergonzoli 3,\*, Nadia Palmieri 1 and Luigi Pari 1**

**Abstract:** The growing demand in food and non-food industries for camelina oil is driving the interest of farmers and contractors in investing in such feedstock. Nonetheless, the cost, performance and critical aspects related to the harvesting stage are still not properly investigated. In the present study, an ad-hoc test was performed in Spain in order to fulfill this gap. The results support the hypothesis to harvest camelina seeds with the same combine harvester used for cereal harvesting without further investment. Theoretical field capacity (TFC), effective field capacity (EFC), material capacity (MC), and field efficiency (FE) were 4.34 ha h−1, 4.22 ha h−1, 4.66 Mg h−1 FM, and 97.24%, respectively. The harvesting cost was estimated in 48.51 € ha−1. Approximately, the seed loss of 0.057 ± 0.028 Mg ha−1 FM was due to the impact of the combine harvester header and dehiscence of pods, whilst 0.036 ± 0.006 Mg ha−1 FM of seeds were lost due to inefficiency of the threshing system of the combine harvester. Adjustment of the working speed of the combine and the rotation speed of the reel may help to reduce such loss.

**Keywords:** work productivity; harvesting costs; harvesting efficiency; wheat header; seed loss; header impact

#### **1. Introduction**

The European Union is currently fostering the replacement of fossil-based products with bio-based surrogates [1,2]. Oil crops play a key role concerning this issue, thanks to their suitability to synthesize molecular structures which could be used to displace substantial amount of petroleum oil derived compounds [3,4]. Worldwide production of vegetable oil is given for 75% by few crops, such as soybean, oil palm, cottonseed, rapeseed and sunflower; while the remaining 25% is obtained from other minor oilseeds [1]. On the other hand, some of these minor oilseeds show particular features, which make them particularly suitable in the concept of bio-economy. In particular, camelina (*Camelina sativa* (L.) Crantz) belonging to *Brassicaceae* family [5] and originating from South-East Europe and South-West Asia [6], is a very promising oil crop for multiple reasons [7]. Camelina oil can indeed be used as edible oil rich in omega-3 fatty acids [8], and its oil and meal are also suitable sources of protein for both fish and ruminant diets [9–12]. Camelina oil has also multiple industrial applications, such as biodiesel and jet-fuel production, even if with some drawbacks related to cetane number, iodine value, oxidation stability and linolenic acid methyl ester content [13,14]. Furthermore, camelina oil can be used in the production of

**Citation:** Stefanoni, W.; Latterini, F.; Ruiz, J.P.; Bergonzoli, S.; Palmieri, N.; Pari, L. Assessing the Camelina (*Camelina sativa* (L.) Crantz) Seed Harvesting Using a Combine Harvester: A Case-Study on the Assessment of Work Performance and Seed Loss. *Sustainability* **2021**, *13*, 195. https://doi.org/10.3390/su13010195

Received: 30 October 2020 Accepted: 23 December 2020 Published: 28 December 2020

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/).

plasticizers, lubricants, polyols, resins, composites, coatings, elastomers, and adhesives [15]. Other interesting features of camelina are related to its cultivation. This species is indeed resistant to both drought and frost stress [16]. It has low nutritional requirements [17–19], with subsequent positive effects on the environment highlighted by life cycle assessment (LCA) studies [20,21], and can be grown on poor soils, also in a Mediterranean context [22], even if both seed yield and oil yield show substantial variability, i.e., 1.0–3.0 Mg ha−1 and 30–49% *w/w* respectively [23]. Finally, camelina, considering the presence of both winter and spring cultivar and the relatively brief life cycle, is suitable for double cropping with small grain cereals, soybean, and sunflower [24–29].

On the other hand, one of the main issues in camelina cultivation is the high costs of the supply chain [30]. Indeed, the higher percentage of costs for biodiesel production are related to the feedstock [31] and optimizing harvesting operation can lead to a substantial decrease of such costs [32].

According to this, costs of harvesting and logistic have to be evaluated, in order to make camelina cultivation fully sustainable and give support in the decision-making process to farmers and other stakeholders. Currently, mechanical harvesting of camelina is mainly carried out by using a combine harvester equipped with wheat header [33], only few experiences on cutting and swathing are reported [34]. However, seed loss can be very high, as a consequence of the tiny dimension of the seeds which are very small and light in weight [35,36] moreover, presence of weeds can further increase seed loss amount. Indeed, the entrance within the combine harvester of the green material of weeds, which generally shows higher moisture content than camelina, can reduce the efficiency of the threshing and cleaning system of the combine harvester, leading to higher seed loss [37]. Considering this, appropriate setting of the combine harvester and adjustment of working speed are fundamental to reduce seed loss [38]. However, combine harvester settings are not the only important aspects to take into account. In fact, camelina suffers seed loss for shattering as the ripeness is completed. Pods can easily open as consequence of external mechanical input, as the cutting bar of the combine harvester can provide. Hence, it is also important to finely regulate the rotary speed of the reel as well as the working speed of the machine in order to reduce such a phenomenon as much as possible. Some authors also suggest to consider the swathing method for harvesting in case of uneven ripeness [38].

Notwithstanding the centrality of this topic in the optic of a sustainable cultivation of camelina, few studies have focused on the evaluation of work performance, harvesting costs, and seeds loss.

The only comprehensive study reported in literature is Stefanoni et al. (2020), who reported a work productivity of 3.17 ha h−1, with harvesting costs of 65.97 € ha−1 and seed loss of 7.82% *w/w* for a John Deere combine harvester (John Deere, Moline, IL, USA) [39]. In a previous work related to harvesting loss evaluation using a plot combine Sintim et al. (2016) found seed loss of 11.60% *w/w* [40], while Stolarski et al. (2019) reported harvesting cost per surface unit of 46.70 € ha−1 with a New Holland (New Holland, PA, USA) combine harvester [41].

Considering what is written above, there is still a need to investigate such a topic with specific field tests, in order to fill the knowledge gap that still exists. The aim of the present work is properly to provide the literature with significant information for both farmers and contractors; about work performance, costs and seed loss when collecting camelina seeds by combine harvester.

#### **2. Materials and Methods**

#### *2.1. Experimental Field*

Harvesting test was performed in the town of Astudillo, Palencia (Castilla y Leon, Spain) during the 27th week of 2020 (Figure 1). The experimental field (WGS84-UTM30T coordinates 390,896 E; 4,661,826 N) was flat and it measured 24.00 ha in surface and 893 m a.s.l in altitude.

**Figure 1.** Experimental field location.

Camelina cultivation in the experimental field was carried out in conventional farming regime. Cultivar *Alba* (commercial variety provided by Camelina Company España) was sown in the first half of December 2019 with a seeding rate of 8 kg ha−1. The previous crop was Barley. Fertilization was provided two times, with a rate of 250 kg ha−1 of NPK 8-15-15 in winter using a trailed fertilizer spreader and 250 kg ha−1 of liquid Nitrogen fertilizer (32%) in April by means of a mounted liquid fertilizer spreader. Chemical control of weeds was carried out before the nitrosulphate ammonium distribution by using a graminicide (Pilot, Quizalofop-p-ethyl 10%) to control the narrow-leaf weeds.

#### *2.2. Pre-Harvest Test*

Prior to the harvesting operation, 10 squared sample plots of 1 m2 each were randomly established in order to assess the amount of the whole epigeous biomass (straw, siliques, and seeds). Camelina plants were cut at ground level with a shear, counted and then measured in both weight and height. Siliques and seeds were pulled and weighed separately. Consequently, siliques, seeds and a sample of straw from each plot were closed in sealed bags and transferred to the laboratory of Research Centre for Engineering and Agro-Food Processing (CREA-IT, Monterotondo, Rome, Italy) in order to perform further analysis. In particular, potential seed yield (PSY), dry weight (DW), 1000 seed-weight, bulk density and moisture content were evaluated. Dry weight and moisture content were estimated according to EN ISO 18134-2:2017 standard [42]. Seeds bulk density (kg m−3) was calculated according to ISO 17828:201 [43] in 15 randomly selected samples.

#### *2.3. Combine Harvester Model and Setting*

The harvesting machinery was provided by the contractor. In particular the operation was carried out with a Claas Lexion 570 (Westfalia, Harsewinkel, Germany) combine harvester equipped with a conventional cleaning shoe and a 6.6 m wide cereal header. The machine had 273 kW diesel engine and the applied setting was as follow: rotor speed 800 rpm, cleaning fan speed 700 rpm, opening of the upper sieve 5/22 mm while lower sieve was closed. The combine harvester was moreover equipped with a straw chopper system, to thresh the straw and spread it on the ground.

#### *2.4. Work Productivity*

Harvesting productivity was tested in 6 sample plots randomly established in the study area. The area of each plot ranged between 420 to 950 m2, and the evaluation of the working times was performed according to the methodology developed by Reith et al. (2017) [44]. The investigated parameters were: working speed (km h−1), Theoretical Field Capacity (TFC, ha h−1, calculated knowing the working speed and the width of the header), Effective Field Capacity (EFC, ha h−1, calculated taking into account accessory times) and Material Capacity (MC, Mg h−1, calculated knowing the EFC and the effective seed yield). The percentage ratio between EFC and TFC is named field efficiency (FE, %).

After harvesting operation, the collected material was unloaded onto a trailer and transported to the farm scale in order to be weighted.

#### *2.5. Cost Analysis*

Purchase and operating costs of the machinery were obtained interviewing the contractor, whilst the work productivity of the combine harvester was derived from the results of field tests and standard values for calculation were obtained from CRPA (Research Centre on Animal productions) methodology [45] as reported in Suardi et al. (2020) [46–48].

Hourly costs of harvesting machinery were calculated taking into account the market value of the combine harvester. The price of the combine harvester was discounted to 2019, using the lending rate of 3% provided by Banca d'Italia [49]. The parameter used for cost analysis are given in Table 1.

**Table 1.** Applied parameters for cost analysis.

#### *2.6. Seed Loss Evaluation*

Camelina seed loss was evaluated by counting the number of the seeds lying on the ground after the passage of the combine harvester. Specifically, two different areas behind the machine were selected as shown in Figure 2a: (A) in correspondence of the swath; (B) beside the swath but within the maximum cutting bar width. Ten squared sampling plots

10 cm × 10 cm (Figure 2b) were randomly selected within each region. Thus, in A, the seed loss was due to natural shattering (SS), impact of the header (ISL) and inefficiency of the cleaning shoe (CLS). On the other hand, in B, the seed loss was due to SS and ISL. Consequently, CLS was calculated as difference between the total seeds found in A and B regions. Since the loss due to CLS was concentrated in 1.6 m (the width of the swath), the difference in seed number between A and B was divided by 4.125 (the ratio between the cutting bar width and the swath width). By knowing the 1000-seed weight, the amount of seed loss was calculated in weight and referred to hectares.

**Figure 2.** On the left (**a**), identification of the areas A and B behind the combine harvester. On the right (**b**), example of a sample plot to detect seeds on the ground.

Furthermore, the effective seed loss (ESL) was also estimated by calculating the difference between the potential seed yield (PSY), measured in the pre-harvesting plot, and effective seed yield (ESY), measured by the farm scale after weighing the trailer.

The difference of the two methodologies was used to estimate the SS.

#### *2.7. Statistical Analysis*

The analysis of variance (ANOVA) was performed using the R 3.6.1 software to separate statistically different means among the groups (*p* ≤ 0.05) [50].

#### **3. Results**

#### *3.1. Pre-Harvest Test*

Results of the pre-harvest tests are shown in Table 2. Before harvesting, 424 plants per m2 were standing on the field and the mean plant height was 60 cm. Straw, siliques and seed moisture were 44.40%, 9.91%, and 6.45% respectively.

As reported in Figure 3, the largest aboveground portion was straw (69.62% *w/w*), then siliques and seeds (14.44% *w/w* and 15.94% *w/w* respectively). The harvest index (HI) was 0.223 and the potential seed yield was 1.17 Mg ha−1 FM.

**Figure 3.** Percentage of straw, siliques and seeds of the aboveground biomass.

**Table 2.** Results of pre-harvest test reporting the mean quantity of available aboveground biomass, moisture content, and allocation among siliques, seeds and stalks. Weigh and bulk density of seeds is also reported.

#### *3.2. Work Productivity and Costs*

Working performance of the combine harvester is reported in Table 3. The working speed was estimated being 6.57 km h−1, while TFC and EFC were 4.34 ha h−1 and 4.22 ha h−1, respectively. Considering the effective seed yield (1.10 Mg ha−1 FM), the MC and FE resulted in 4.66 Mg h−1 FM and 97.24%, respectively.

**Table 3.** Evaluation of the work performance of the combine harvester: theoretical and effective field capacity, field efficiency and material capacity.

The analysis of working performance allowed to estimate the harvesting costs which were: 205.17 € h−1, 48.51 € ha−1 and 43.92 € Mg−1 FM.

#### *3.3. Seed Loss Evaluation*

The seed loss calculated for each source of is reported in Table 4. TSL was 0.093 ± 0.033 Mg ha−1, or 7.95 ± 0.28% of PSY. The majority of seed loss (4.87 ± 2.35% *w/w*) is linked to the impact of the header and the natural shattering. The latter further estimated in 1.97% *w/w* of the PSY as difference between TSL and ESL. On the other hand, CLS accounted for 3.08 ± 0.54% *w/w* of the TSL. The effective seed loss measured as the mere difference between the PSY and ESY, was 0.07 Mg ha−1.

**Table 4.** Seed loss assessment according to the two methodologies. Common letters within columns denote the absence of significant difference (*p* < 0.05).

Note: (\*) this value was not replicated since all grains were collected within one trailer and weighted only once at the end of the harvesting.

#### **4. Discussions**

#### *4.1. Aboveground Biomass Yield*

The potential seed yield assessed in the pre-harvest test of 1.17 ± 0.18 Mg ha−1 FM (seed moisture content of 6.45 ± 0.40%) is in line with the findings reported in Mauri et al. (2019) and Stefanoni et al. (2020) for similar experiments conducted in Spain [39,51] as well as in USA as reported by Schillinger et al. 2019 [52]. Higher values of seed yield are reported by Royo-Esnal et al. (2018) in Eastern Spain with seed yield ranging from 0.92 to 2.31 Mg ha−1 FM after a comparison of different sowing rates: 8 kg ha−1 and 11 kg ha−1 [27]. However, the authors did not find a significant effect of the sowing rate upon potential seed yield, nor with the weed coverage. Similarly, Zanetti et al. (2020) reported a negligible effect of the plant density on seed yield, whilst later sowing could improve oil content [22]. In the present study, instead, fertilizer and chemicals were used to both providing nutrients and controlling the weeds. In similar studies, where camelina was grown under conventional farming, the potential seed yield doubled in comparison with not fertilized fields (namely, 0.93 Mg ha−1 FM and 1.81 Mg ha−1 FM) [53]. Comparing with other herbaceous oilseeds, for instance, camelina performs slightly lower than castor (*Ricinus communis* L. up to 4.4 Mg ha−1), canola (*Brassica napus* L. 2.19 Mg ha−1 FM), sunflower (*Heliantus annuus* L. 1.97 Mg ha−1 FM) [33], although it is suitable for cropping in marginal land [54]. After harvesting, seeds usually face some storing which could be also long in time before being processed. This condition can lead to low quality product, or even loss of the entire product if moisture is too high. In the present study, seed moisture was found as low as 6.45 ± 0.4% which is far below the threshold of 8% as reported by [55]. 1000 seed-weight was also recorded and it averaged to 1.04 ± 0.07 g in fresh weight which is consistent with the value found by other authors [39]. If compared with other Brassicaceae family and seed weight, it is rather low. In fact, Kuai et al. (2015) reported 3.3 and 3.5 g in rapeseed (*Brassica napus* L.) [56], Zhu et al. (2016) reported values ranging from 6.0 to 9.5 g per thousand seeds in crambe (*Crambe abyssinica*) [57].

Despite the seeds that find application on both food and non-food sectors, straw and siliques from camelina (5.10 ± 1.15 Mg ha−1 FM and 1.06 ± 0.25 Mg ha−1 FM respectively) can also be attractive for energy industry. In fact, they both are valid feedstocks for bioenergy production via pyrolysis due to the low nitrogen content (0.4–0.5%) and the low char production (approximately 25.5%) [58]. However, the chemical-physical properties of camelina residual biomass can vary according to the growth conditions. For instance, camelina grown in the Central Italy exhibits high cellulose and hemicellulose content in comparison with camelina grown in the Northern Italy while the ash content is not affected by such factor [59]. This implies that different scenarios are opened for the exploitation of camelina residual biomass in a sustainable green chemistry approach. Moreover, the development of a proper value chain of the residual biomass may contribute to the reduction of greenhouse gas emissions that occur during the degradation of the organic matter in the soil as reported in other oil crops [60].

#### *4.2. Work Productivity and Costs*

In the present study, a conventional combine harvester equipped with a cereal header was used. The literature still lacks the knowledge on such kind of strategy for harvesting camelina seeds, therefore a comparison is possible relying on the findings reported in Stefanoni et al. (2020) [39]. Here, the working speed of the machine was 30.1% higher. This caused the increase of TFC, EFC, FE, and MC by 29.29%, 33.12%, 3.54%, and 54.82% respectively. Interestingly, the cutting bars of the combine harvesters measured 6.6 and 6.7 m wide. Such a negligible deviation leads to the conclusion that the difference in the performance are exclusively related to the different working speeds.

Interestingly, comparing the performance of the combine harvester found in the present study with those reported in similar studies but conducted on wheat grains harvesting, here again the findings are higher. Normally, TFC ranges from 2.61 ha h−1 to 3.72 ha h−1, while the EFC is 1.92–2.28 ha h−1 and the FE is as high as 83% [46–48]. However, it is important to underline that such high working performance is related to the dimensions and to the particular shape of the experimental field, which allowed to minimize the turnings, thus decreasing the accessory time and increasing the EFC and FE.

The harvesting cost was assessed in 48.51 € ha−1 and 43.92 € Mg−1 FM which are consistent with the cost shown by Stolarski et al. (2019) [41], but much lower than that calculated in a similar harvesting trial performed in Spain on camelina crop (65.97 € ha−1 and 69.42 € Mg−1 FM) [39]. Other trials performed on wheat and corn grain harvesting with combine harvester showed harvesting costs being 77.98 and 129.51 € ha−1, respectively [46,61].

#### *4.3. Seed Loss Evaluation*

The evaluation of the seeds loss during harvesting stage is an important parameter to take into account since it contributes to reduce the revenue of farmers and contractors therefore, the loss of seeds should be as low as possible. Generally, the amount of seed lost is calculated as the difference between the potential seed yield (1.17 ± 0.18 Mg ha−1 FM) and the effective seed yield (1.10 Mg ha−1 FM) which, in this specific trial, was 0.07 Mg ha−1 FM (5.98% *w/w*). Higher values were found by Stefanoni et al. (2020) and Sintim et al. (2016) which found 7.82 and 11.70% *w/w*, respectively [39,40]. In other herbaceous oil crops, seed loss ranges from 1% as in sunflower [62,63] or in canola [64,65], and 3% as in safflower [33], or even higher as in castor bean harvesting [33]. However, such information only provides evidence regarding the total amount of seed loss, but it fails in pointing out what is responsible for that loss. A combine harvester is a complicated machine which can generate different sources of loss particularly if seeds are small and light in weight (only 1.04 ± 0.07 g FM per 1000 seeds). The main sources of loss are the impact of the header and the inefficiency of the cleaning shoe. If some actions have to be taken against the seed loss in camelina harvesting, their respective contribution to the TSL must be investigated. According to Table 4, the inefficiency of the cleaning shoe of the combine harvester (CSL) triggered the loss of 0.036 ± 0.006 Mg ha−1 FM (3.08 ± 0.54% *w/w*) of the seeds, while 0.057 ± 0.028 Mg ha−1 FM (4.87 ± 2.35% *w/w*) of seeds were lost due SS and ISL. Interestingly, TSL and ESL differed for 0.023 Mg ha−1 FM (1.97% *w/w* of the PSY) which can be partially explained as the loss due to SS (natural pod shattering) which occurs spontaneously in camelina as it ripens. In fact, late harvesting can lead to a loss of seeds due to SS as high as 25% *w/w* in some cultivars [66]. Moreover, pod shattering can be triggered by a minimum external input in completely ripened pods. Therefore, the mechanical disturbance provided by the combine harvester can contribute significantly to increase such phenomenon, particularly as working and rotation speed of the reel (the latter value was not measured in the present study).

#### **5. Conclusions**

Camelina is gathering more and more attention throughout the Europe since its multipurpose oil as well as the aboveground suitability for bioenergy purposes. However, the related value chain is still not well developed, partially because the crucial phase of the

harvest has not been comprehensively investigated so far. Our findings support the hypothesis that a combine harvester equipped with wheat header is suitable for camelina seed harvesting, which is particular convenient for farmers and contractor who use camelina as rotation crop in winter cereals since the same machine is valid for both crops. Furthermore, the cost and the performance are similar. Little concern may arise regarding the seed loss which are mainly linked to impact of the header of the combine harvester, and the inability of the cleaning shoe to efficiently discriminate the seeds from the other portions of the biomass. This latter problem can be partially addressed by simply reducing the speed of the machine. Instead, natural pod shattering contributes marginally to the loss of seeds.

**Author Contributions:** Conceptualization, W.S., F.L., S.B., and L.P.; methodology, W.S., F.L., S.B., and J.P.R.; data curation, W.S., F.L., S.B., N.P., and J.P.R.; writing—original draft preparation, W.S., F.L., S.B., and N.P.; writing—review and editing, W.S., F.L., S.B., L.P., and J.P.R.; supervision, L.P.; funding acquisition, L.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by PRIMA foundation, project 4CE-MED, grant Number 1911 and by Horizon 2020 project Panacea, grant Number 773501.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to restrictions e.g., privacy.

**Acknowledgments:** The authors wish to thank Camelina Company (Camino de la Carrera, 11-11, 28140 Fuente el Saz, Madrid, Spain) for the organization of the tests and the support during the trials and Sandu Lazar for the help in performing the field and laboratory tests.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Inulin Content in Chipped and Whole Roots of Cardoon after Six Months Storage under Natural Conditions**

**Luigi Pari 1, Vincenzo Alfano 1,\*, Walter Stefanoni 1, Francesco Latterini 1, Federico Liuzzi 2, Isabella De Bari 2, Vito Valerio 2 and Anna Ciancolini 3**

**Abstract:** Industries currently rely on chicory and Jerusalem artichoke for inulin extraction but also cardoon is proved to synthetize and store high quantity of inulin in roots as well. Cardoon is a multipurpose crop, well adapted to marginal lands, whose main residues at the end of cropping cycle consist of roots. However, cardoon roots are a suitable source of inulin, that is of high interest for new generation biodegradable bioplastics production. On the other hand, a sustainable supply chain for inulin production from cardoon roots has not been developed yet. In particular, in the inulin supply chain the most critical part is storage, which can negatively affect both cost and inulin quantity. In the present study the effect on inulin content in cardoon roots stored as dried chipped roots (CRt) and dried whole roots (WRt) was investigated in a 6-month storage trial. Our findings suggest that chipping before storage did not affect the inulin content during the storage. Furthermore, it reduced the time needed for drying by 33.3% and increased the bulk density by 154.9% with the consequent reduction of direct cost for drying, transportation and storage.

**Keywords:** Cynara roots; biorefinery; marginal lands; multipurpose crop; fermentable sugars; agricultural residues exploitation

#### **1. Introduction**

Cardoon (*Cynara cardunculus* L.) is one of the most promising feedstocks for biorefinery in the Mediterranean areas since it is a multipurpose perennial crop well adapted to drought environments and low productive marginal lands [1–4]. The cultivation of this species mainly focuses on the exploitation of the aerial biomass as seeds, leaves and stalks [5–7]. The vegetable oil extracted from seeds is rich in monounsaturated fatty acids useful to produce important intermediates such as azelaic acid or pelargonic acid, that are highly demanded by synthetic fertilizer industries as well as cosmetic industries worldwide [8,9]. On the other hand, leaves and stalks represent an important source of lignocellulosic biomass potentially suitable for the production of intermediate compounds, like bioethanol and Bio-butanediol, which are widely used for producing bioplastics [10–12].

However, the potential of cardoon as a multipurpose crop has not been fully exploited yet, in particular regarding the presence in the roots of inulin suitable for nutraceutical, pharmaceutical and other biorefining applications [13–15].

Inulin is a linear fructan, i.e., a polymer of fructose units linked by β (2 → 1) glycosidic bonds with a variable degree of polymerization (DP), between 3 and 60, and usually a glucose molecule at the end [16]. Inulin can be used in food and pharmaceutical industry for several purposes, such as prebiotics to stimulate the growth of probiotic gut bacteria, for nutritional purposes as low caloric soluble dietary fiber and also as a mediate sugar

**Citation:** Pari, L.; Alfano, V.; Stefanoni, W.; Latterini, F.; Liuzzi, F.; De Bari, I.; Valerio, V.; Ciancolini, A. Inulin Content in Chipped and Whole Roots of Cardoon after Six Months Storage under Natural Conditions. *Sustainability* **2021**, *13*, 3902. https:// doi.org/10.3390/su13073902

Academic Editor: Anastasios Michailidis

Received: 12 March 2021 Accepted: 29 March 2021 Published: 1 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and lipid metabolism in diabetic and hypercholesterolemia [17]. In medicine, inulin is also used as a diagnostic agent for the determination of kidney function [18]. In the biorefinery industry, inulin and inulin-rich biomass are gaining interest for the production of fructose by enzymatic hydrolysis of inulin, as alternative way to the current approaches based on acid hydrolysis of sucrose [19,20]. In the biorefinery, moreover, the availability of fermentable sugars is crucial to produce ethanol, and inulin is a good feedstock for bioethanol production by fermentation after hydrolysis [21,22].

Inulin is a reserve carbohydrate accumulated mainly in the roots and tubers of many plants belonging to the Asteraceae family, like Cardoon [16].

Among the Asteraceae family plants, Chicory (*Cichorium intybus* L.) and Jerusalem artichoke (*Helianthus tuberosus* L.) are currently the major industrial sources of inulin [23]. In a comparative study aimed at evaluating different types of inulin, extracted from Cardoon roots, Jerusalem artichoke tubers and Chicory roots, the inulin amount resulted respectively in 115, 390 and 550 g kg−1 of d.m. [24].

In the perspective of new generation biorefineries and the circular bioeconomy concept, the recovery of inulin from cardoon roots at the end of crop cycle, in addition to various high added-value raw materials from seeds and stalks, seems to be an interesting opportunity. However, the full development of an effective value chain for the biochemical industries, implies well-organized logistics. Storage phase, in particular, has a high impact on the quality of the raw material and on the overall costs of the value chain [25].

Effects of storage conditions on inulin content have been investigated in different inulin-containing crops. In the case of the storage of Jerusalem artichoke tubers, inulin composition remained stable under frozen storage (−18 ◦C) during 3 months of study, while a significant degradation of inulin to sucrose and fructo-oligosaccharides was observed after 4 weeks when the storage was performed at 4 ◦C [26]. In another study, inulin hydrolase activity in the tubers of Jerusalem artichoke peaked at the 15th day of storage at ambient conditions: inulin underwent depolymerization causing a decrease in inulin content and an increase in soluble sugars [27]. In a 28 day storage trial a decrease of 70% and 96% was reported for artichoke heads after storage at 4 and 18 ◦C, respectively. Similarly, in storage of sliced artichoke heads at 4 ◦C, about 60% decrease of inulin content was observed during the first 11 days of storage [28].

In addition to the temperature, moisture plays a key role to start the enzymatic hydrolysis of the inulin. For example, it has been observed that the enzymatic hydrolysis of the inulin during storage of chicory roots depends on the moisture content, while the generated sugars favored the loss of material as a result of cell respiration and microbial activity [29]. In fact, microorganisms require minimum thresholds of moisture to maintain and optimize metabolism, that is the breakdown and the consumption of the sugar-based components of dry matter. Moisture below 10% is generally considered to be low enough to prevent microbial degradation and allow for safe long-term storage of biomass [30]. In this framework, the thermal drying technologies have gained interest as effective tools for extending the length of storage as well as reducing the handling cost and ease the transportation that affect the value chain at the industrial level [31,32].

On the other hand, a suitable storage system for a biorefinery has not only to focus on the capacity of keeping a high content of a given product, but there is also the need of finding a suitable solution regarding the economic sustainability, with particular reference to transport costs which can have a substantial impact on the overall value chain [33,34]. Under this point of view biomass chipping is an interesting approach. Indeed, the higher bulk density achieved by the chipped material improves the logistics by reducing the space needed during transport as well as in the storage area [35]. On the other hand, chipping increases the exposed surface area and reduces the air permeability [36] with an expectable opposite effect on drying efficiency as well as on the maintenance of dry matter and inulin content.

In order to set up a suitable value chain for inulin production from cardoon roots biomass, there is the need of investigating a storage system which combines effective longterm maintenance of inulin and economic sustainability. Considering the current lack of knowledge on this particular topic, a specific task of the Italian Project Cometa-Autoctone Mediterranean crops and their valorization with advanced green chemistry technologies (funded by Ministry of Education, Universities and Research), was addressed to develop an effective handling and storage strategy for cardoon roots aimed to inulin production. In this framework the present study aimed to investigate the effect of chipping and drying approach on inulin content of cardoon roots biomass over 6 months of storage.

#### **2. Materials and Methods**

#### *2.1. Location of the Field and Plant Material*

Cardoon roots were taken in May 2020 from three year old plants cultivated in Terni (42.561335 N latitude, 12.62860 E longitude) (Umbria Region, Central Italy) on clay soil. Cardoon (cultivar Trinaseed) were grown from seeds sowed in November 2017 with a precision sowing machine at the rate of 3.0 kg/ha of seed and spacing distance of 0.75 × 0.17 m.

At the sowing, the seedbed was prepared between October and November with ploughing at 20 cm, followed by harrowing at 10–15 cm. During the first year, mineral fertilization with 46 kg of P2O5 and 64 kg/ha of N was added, and a.i. pendimethalin was used to control weeds. Starting from the second year, 46 kg/ha of P2O5 and 18 kg/ha of N were applied during the vegetative growth of the plants in autumn and in the early spring before the stem elongation phase. The fertilization rates were calculated on the basis of the soil fertility and crop nutrient uptake. Crop water requirements were satisfied by rain.

Approximately 500 plants were randomly uprooted using an excavator carefully avoiding damage to both canopy and root systems. The whole plants were put in sealed bags and carried to the laboratory of The Research Centre for Engineering and Agro-Food Processing of the Council for agricultural research and economics (CREA) in Monterotondo, Central Italy (42 10019 N latitude 12 62066 E longitude) for sampling. Firstly, the soil was removed from roots using a cold-water pressure washer. Afterwards, the plants were left to dry naturally for a few minutes. Then the roots were mechanically cut off from the plants.

The bulk density of roots was determined using a box with an internal volume of 0.0064 m3, the value was reported as kg m−3. The box was filled with roots and weighed with a KERN GmbH dynamometer (CH 50K50 model-range of measurements 50 kg and sensitivity 50 g). Three samples were taken for mean value. In 30 randomly chosen plants the fresh weight of canopy and roots were measured using a precision scale (Kern PCB 6000-0). The length of leaves and roots of this plants sample was measured with a ruler. Roots were further investigated by determining the moisture content according to [37].

#### *2.2. Chipping, Drying and Storage*

After cleaning, roots were divided into two groups (treatments) to monitor the inulin content in dried chipped roots (CRt) and dried whole roots (WRt) of cardoon in 6-months storage. CRt was obtained by selecting 15 kg of randomly chosen roots that were fresh chipped using an electric 2.0 kW bio-shredder (Zanon, mod. BIO 3). The particle size distribution (PSD) of the chipped material produced was analyzed according to [38].

Chips were collected and put into the oven for drying at 60 ◦C until they reached constant weight. Simultaneously, a further 15 kg of randomly chosen whole roots were collected and put in the oven for drying. At constant weight, the whole roots were removed to obtain WRt. The apparent bulk density was measured three times in CRt and WRt, respectively, according to [39] for mean value estimation.

Storage of CRt and WRt was performed outside the building, under a farm shed. Specifically, chips from CRt were collected in jute bags while the whole roots from WRt treatment were piled up as shown in Figure 1 after being labeled and weighed individually.

**Figure 1.** Storage under a farm shed of dried chipped roots (CRt) on the left side and dried whole roots (WRt) on the right side.

Monthly, approximately 200 g of chips from CRt and five roots from WRt were sampled and sent to ENEA laboratory for inulin determination. WRt roots were bioshredded before the shipment. A representative subsample from each treatment was kept for dry matter assessment.

#### *2.3. Weather Data Monitoring*

During the entire storage period of 6 months, the main weather-climatic parameters such as temperature, precipitation and air humidity were recorded with a weather station "DAVIS VANTAGE PRO 2" (Davis Instruments, 3465 Diablo Avenue, Hayward, CA 94545- 2778, USA) located in the proximity of the storage site and connected to wireless net. Data are shown in Figure 2.

**Figure 2.** Trend of main climatic parameters: temperature (◦C), precipitation (mm) and air humidity (%) recorded during the storage of cardoon roots from May to November 2020.

#### *2.4. Inulin Content Determination*

The collected samples from CRt and WRt were grinded to 0.5 mm in a ZM200 Retsch® ultracentrifugal mill (Retsch GmbH, Haan, Germany).

After elimination of residual humidity by drying at 50 ◦C for 4 h in a ventilated oven, the inulin content was determined by using a modified Raccuia method [19]. In particular, the quantitative extraction of the inulin from the roots was performed by suspending 1 g of powdered dry root in 20 mL of deionized water at 100 ◦C for 1 h in a Benchmark Scientific (South Plainfield, NJ, USA) Multi-Therm Heat-Shake kept under constant stirring at 500 rpm. Subsequently, the sample was centrifuged at 3500 rpm for 5 min and 4 mL of 0.75 M HCl were added to 2 mL of supernatant. The acid solution containing the inulin was then hydrolyzed in the heat-shake system for 15 min under the same conditions (100 ◦C, 500 rpm).

After centrifugation at 3500 rpm for 5 min, the supernatant was filtered through 0.45 μm PTFE filter (Whatman, USA) and carbohydrates were analyzed by using an HPIC DX 300 cromatographic system (Dionex, Sunnyvale, CA, USA) equipped with a Nucleogel® Ion 300 OA column (Macherey–Nagel, Düren, Germany) and sulphuric acid 10 mN as eluent. The detector was a Shodex RI101 refractive index (Showa Denko, Japan). All reagents and standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). The extraction and hydrolysis processes were conducted in triplicate for each sample.

The inulin content was determined by the following Equation (1):

$$\text{Inulin }\%=\frac{\left(\mathbb{C}\_f + \mathbb{C}\_\\$\right) \times 0.9 \times 3}{\mathbb{C}\_R} \times 100\tag{1}$$

where *Cf* and *Cg* are the concentration in gL−1 of fructose and glucose, respectively; 0.9 is the correction factor applied for the oligomer-to-monomer hydration; 3 is the dilution factor for the HCl hydrolysis; *CR* is the concentration in gL−1 of the initial suspended roots.

#### *2.5. Statistical Analysis*

Statistical analysis was performed to assess significant differences among the mean values of dry matter and inulin content. Normality and homoscedasticity of the data were tested with Shapiro test and F test, respectively. *T*-test was performed to investigate significantly different means (*p* ≤ 0.05) among treatments. Statistical analysis was performed by R 3.6.1 software to separate statistically different means [40].

#### **3. Results and Discussion**

#### *3.1. Characterization of Cardoon Roots and Evaluation of Drying Times*

The growth analysis of sample plants was performed in order to estimate the available aboveground and belowground biomass. The average fresh weight of canopy and roots was, respectively, 0.9 and 0.45 kg per plant (71 and 35 t f.w. ha−1). The moisture content of roots was assessed as 70% *w/w* of fresh weight. Hence, the expected quantity of dry roots per hectare can be estimated in 10.6 t, similarly to 9.8 t DM ha−1 reported by [13]. Results of roots' characterization are given in Table 1.

**Table 1.** Characteristics of roots and canopy of three year old cardoon plants (mean ± sd) sampled in May 2020.

The bulk density of either fresh chips and dried chips was about 2.5 times higher than the bulk density of fresh whole roots and dried whole roots, respectively (Table 2).

**Table 2.** Bulk density of the whole roots and the chipped material, before and after drying

Therefore, chipping represents an advisable option to reduce the volume needed for both transportation and storage. Consequently, the cost of the operations can be reduced as well. Although it was not investigated in the present study, sieving can follow the chipping phase to help removing unwanted debris from chips, which could be detrimental for further industrial processes. With this aim the chipping should be carried out with a forestry chipper able to produce a more homogeneous product.

On the contrary, in our case, as shown by PSD analysis (Figure 3), 90% of the chipped material was less than 8 mm length, making it not possible to separate by sieving unwanted debris from chips.

Moreover, the drying process can also benefit from chipping by reducing time and energy required [41]. According to our results, indeed, chipped roots could reach constant weight after 48 h, whilst whole roots needed 24 h more to dry completely (Figure 4).

**Figure 4.** Reduction of the average weight of whole roots and chipped roots during drying in a thermo-ventilated oven at 60 ◦C.

#### *3.2. Inulin Content*

Inulin content at T0 (beginning of storage, immediately after drying) was 43.5 ± 0.65% and 47.1 ± 1.30% *w/w* in WRt and CRt, respectively (Figure 5). Drying time negatively affected the inulin content in WRt which resulted in 3.54% *w/w* lower than CRt. Conceivably, in WRt treatment, the inner tissues of the roots took longer to dry out than the outmost tissues of the same root. Hence, the metabolic activities naturally occurring in living cells stopped later and this partially explains the loss of inulin in WRt.

**Figure 5.** Inulin content in dry whole and chipped roots of cardoon at T0 and after 6 months of storage (T6). Common letters denote the absence of significant difference (*p* < 0.05).

Regardless of the difference in inulin content found among treatments, the values herein reported are consistent with the literature although some authors also highlight that possible changes in inulin content may be experienced according to the harvest season [14]. Higher values are usually found in spring, particularly between full blossom and fruit ripening. For this reason, root sampling was performed in May when the concentration is supposed to peak.

After 6 months storage (T6), inulin content did not change significantly in comparison with the initial content measured in the respective treatment, namely: 42.3 ± 0.82% *w/w* in WRt and 48.3 ± 1.12% *w/w* in CRt. Therefore, the drying process performed before the storage prevented the degradation of inulin, at least over the following 6 months of storage. This finding highlights the possibility for industries to exploit the drying process at the industrial scale to help storing the cardoon roots for longer time since inulin loss is prevented effectively. Artificial drying is certainly costly in terms of money and energy, but it is surely less costly than freezing. Additionally, the machinery required for drying is easier to run and cheaper to buy (e.g., a ventilated oven) with enormous advantage also for the transportation which does not longer require the ice-chain. Chipping also contributes to enhance the inulin supply chain as the higher bulk density of chipped material would require less space for drying, storage and transportation.

#### *3.3. Moisture Content and Dry Matter Content*

The moisture content at the first month of storage (T1) in WRt and CRt increased significantly up to 12.1% and 10.8%, respectively. This was probably due to the reabsorption of humidity from the external environment. In fact, as shown in Figure 6, the monthly moisture content measured in WRt and CRt traces the air moisture pattern recorded by weather station. The moisture increase in roots was more evident in WRt where 16.3% *w/w*

of moisture was recorded at T6 (i.e., 3.4% higher than CRt). This was probably due to a greater exposure of the whole roots to air humidity with respect to the chipped material stored inside the jute bag.

**Figure 6.** Moisture content (mean ± SD) in WRt and CRt during the 6 months storage in comparison with air moisture recorded by a weather station over the same period.

During the first month of storage, a significant reduction in weight in both treatments was recorded: 10.9% and 3.5% in WRt and CRt, respectively (Figure 7). Despite this, during the following months the roots' dry weight remained constant in both treatments. However, a significant difference of approximately 6% was recorded between the treatments throughout the trial. Both results were probably due to the higher water content recorded in the whole roots—not completely removed after drying (less drying efficiency) or greatly reabsorbed from the external environment (higher exposure to air humidity)—which promoted microbial activity.

**Figure 7.** Dry matter content in dried whole roots (WRt) and dried chipped (CRt) roots at the first month of storage (June, T1) and after 6 months of storage (T6). Common letters denote the absence of significant difference (*p* < 0.05).

#### **4. Conclusions**

In the perspective of new generation biorefineries and the circular bioeconomy framework, the exploitation of cardoon also for inulin production is rather appealing, particularly if plants have been previously exploited for the production of further high added-value raw materials like seeds and stalks. Due the limited favorable period for harvesting the roots when inulin content is maximum, industries need to store enormous quantities of roots and process them gradually. Hence, storage plays a fundamental role in supply chain. Our findings suggest that during a 6-month storage inulin loss is negligible if roots are previously dried. Furthermore, chipping could also be a good practice since it is possible to reduce the volume required for storage (and also transportation) while it promotes a quicker drying; thus, less energy is required to dry out the roots.

In conclusion, our results highlight the possibility to chip cardoon roots meant for inulin extraction to ameliorate the supply chain of such a material. Although drying remains a costly strategy, chipping would help to reduce such cost by reducing the time required. However, further studies should provide clues to improve also the harvesting and cleaning process.

**Author Contributions:** Conceptualization, L.P. and V.A.; methodology, V.A., W.S. and F.L. (Francesco Latterini); validation, A.C.; formal analysis, V.A., W.S., F.L. (Francesco Latterini), F.L. (Federico Liuzzi) and V.V.; investigation, V.A., W.S., F.L. (Francesco Latterini), F.L. (Federico Liuzzi) and V.V.; resources, A.C.; writing—original draft preparation, V.A., F.L. (Federico Liuzzi), V.V.; writing—review and editing, V.A., W.S. and F.L. (Francesco Latterini).; supervision, L.P. and I.D.B.; funding acquisition, L.P. and I.D.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was performed within the Italian Project COMETA-Autoctone Mediterranean crops and their valorization with advanced green chemistry technologies. The project was funded by Ministry of Education, Universities and Research in the frame of PON "Ricerca e Innovazione" 2014–2020 and FSC/ARS01\_00606 COMETA/CUP. B2G18000180004/Azione II.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data is not publicly available, though the data may be made available on request from the corresponding author.

**Acknowledgments:** The authors would like to thank Salvatore Antonino Raccuia for providing suggestions and sharing his knowledge on cardoon roots and storage methods.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Edited by Mohammed A. A. Khalid*

Biophysical chemistry is one of the most interesting interdisciplinary research fields. Some of its different subjects have been intensively studied for decades. Now the field attracts not only scientists from chemistry, physics, and biology backgrounds but also those from medicine, pharmacy, and other sciences. We aimed to start this version of the book Biophysical Chemistry from advanced principles, as we include some of the most advanced subject matter, such as advanced topics in catalysis applications (first section) and therapeutic applications (second section). This led us to limit our selection to only chapters with high standards, therefore there are only six chapters, divided into two sections. We have assumed that the interested readers are familiar with the fundamentals of some advanced topics in mathematics such as integration, differentiation, and calculus and have some knowledge of organic and physical chemistry, biology, and pharmacy. We hope that the book will be valuable to graduate and postdoctoral students with the requisite background, and by some advanced researchers active in chemistry, biology, biochemistry, medicine, pharmacy, and other sciences.

Published in London, UK © 2020 IntechOpen © Sinhyu / iStock

Biophysical Chemistry - Advance Applications

IntechOpen Book Series

Physiology, Volume 7

Biophysical Chemistry

Advance Applications

*Edited by Mohammed A. A. Khalid*


**8. Future Perspectives**

AN is a particularly hard-to-treat population, and pharmacological trials in AN often suffer from a high dropout rate. People with AN have reported specific concerns around pharmacological interventions, such as fears around drug-instigating weight gain [185]. However, patients also report wanting medication to alleviate anxiety, eating disorder thoughts, poor concentration and sleep problems [185]. A recent survey conducted on 200 participants with eating disorders (*n* = 105 with AN) investigated views on psychedelic drugs as a treatment for eating disorders [186]. Approximately half of the participants expressed interest in participating in psychedelic interventional research in the context of various concerns. The concerns were mitigated when the participants were informed that esketamine was licensed for the treatment of treatment-resistant depression. Importantly, this survey highlighted important methodological considerations, including the need for collaboration with service users and those with lived experience in the design of trials. The participants emphasised the need for a safe, professional and controlled environment during the dosing, a good rapport with the research team, trust in the trial and trial team and the provision of psychoeducation about psychedelic drugs. They also emphasised the need for psychological preparation before the session and an assessment of the "set". Importantly, a third of the overall group reported that they would never take part in such research. Thus, establishing an ongoing dialogue with service users will be an essential consideration in the design of clinical trials.

Overall, it is apparent that future studies should first aim to establish a safety profile of ketamine for AN through well-controlled feasibility and pilot studies. The outcome measures should be agreed upon with service users. Investigational studies of neuromodulation (e.g., transcranial magnetic stimulation and deep brain stimulation) in severe-enduring AN have found proximal improvements in depression, with improvements in eating disorder psychopathology and weight taking 12–18 months to emerge [187–189]. Therefore, whilst the BMI is often the metric of success in interventional trials in AN, for patients with severe-enduring AN where weight gain is slow, other metrics of improvement such as

depression scores, social connectedness and quality of life should be considered, and long follow-up periods should be implemented. Given the hypothesised role of ketamine in providing a window of neuroplasticity, it will be essential that ketamine is combined with psychotherapy in future trials. Whilst the antidepressant effects of ketamine are transient, there is emerging evidence that psychotherapies (such as CBT) can extend the duration of antidepressant effects [190]. Additionally, the experiential components of ketamine may facilitate the therapeutic bond between patient and clinician [160], which is known to be a predictor of response to treatments [191].

Ketamine-assisted therapy generally follows a three-stage process: (1) preparation, where the patient and client discuss the mindset going into the experience, treatment goals and expectations and set intentions; (2) dosing, where patients are given the ketamine administration; and (3) integration, where the ketamine experience is reflected upon, and insights/lessons gained during the experience are integrated into the self. In the treatment of AN, it may be useful to combine specific therapeutic strategies with ketamine, such as compassion-based meditation exercises, yoga, cognitive bias modifications and exposure therapies. Similarly, specific therapeutic approaches may work well with ketamine, such as compassion-focused therapy, the Maudsley Model of Anorexia Treatment for Adults (MANTRA), family-based interventions and other psychotherapies. However, it is important to note that a "one size fits all" approach is unlikely to be successful, and treatments should be tailored to the individual.