*Agroforestry DOI: http://dx.doi.org/10.5772/intechopen.106432*

Young branches and foliage structure a fantastic grub. The blossoms are the helpful wellspring of honey. Wood is utilized for excellent furnishings, cupboards, beautifying facades, marine and aeroplane grade compressed wood, fancy turnery, cutting, etching, apparatus handles and outdoor supplies. The revolution age for wood is around 30 years (**Figure 7**).

**Figure 8.** *Neem tree under banana cultivation.*

**Figure 9.** Salix alba*.*

organic products mature in midsummer. It is engendered through cuttings. The wood is extreme, solid, and light in weight yet has negligible protection from rot. Coppice shoots and branches from pollarded plants are utilized for making. Its lightweight wood is utilized for making cricket bats as it does not fragment without any problem (**Figure 9**).


*4.5. Impact of Workplace Accidents and Occupational Hazards in the Food-Handling Industry*

At this juncture, it is essential to understand how company size can influence the number of accidents. Thus, information was collected from a sample set of 25 companies in the sector (calculating the average over the years 2019 and 2018), where NA = number of accidents, TC = tons sold, and NW = number of workers. First, asimple correlation analysis of the variables (first panel of Table 4) was conducted. As shown, all variables are closely related. It is evident that the number of accidents is correlated with the trade total (r = 0.78) and the size of the business. This is to be expected, although it also indicates that a bigger company demands a higher level of organisation and greater control in order to reduce the NA. However, this has not been verified, as can be confirmed by looking at the correlation coefficient between NW and NA (0.94). Using ratios helps deepen the analysis. The *productivity index* (PI = TC/NW) and *incidence rate* (IR = NA/NW) are two variables used in the analysis. One might think that the company with the highest productivity (PI) would use the resources more intensively, thus affecting a larger NA, a hypothesis that is not supported by the data.

**Table 4.** Correlation between variables.

Sample of 25 companies. Source: Compiled by the authors.

The companies studied employ approximately 40% of the total number of women who work in the food-handling sector of the three main areas: La Cañada, Campo de Níjar, and Campo de Dalías.

According to data from the Andalusian Regional Government's occupational-risk prevention service, there has been a considerable decrease in the incidence rate over the last five years (Table 5).

1 The firms represented are all social enterprises and fall into two categories: cooperatives and agricultural transformation societies.  Source: [29].

> The average accident rate for the period 2000–2014 was 26 accidents per company, and the incidence rate was almost 20%. In contrast, the period 2015–2019 was characterised by a decrease in both rates, with the average number of accidents per company falling to 23 and the average rate of incidents decreasing to 16.76%. Therefore, the period 2015–2019 evinced the efforts made to increase the occupational safety of workers.

> An analysis of women's empowerment and job satisfaction should include an understanding of the degree of risk involved in their work. The workplace incidents with the highest index of danger are overexertion and falling objects. (Table 6).

**Table 6.** Main position and risk analysis.

Most of the injuries suffered by women are specific to the lower and upper limbs (Figure 5).

To understand the recent improvements in the workplace, it is important to consider that the fruit- and vegetable-handling companies are applying the Hazard Analysis and Critical Control Points (HACCP), as well as occupational-risk prevention. This widespread use is due, among other reasons, to the market (customers), which demands their application. It is an excellent example of private initiatives demanding stricter limits that have surpassed public regulatory capacities.

**Figure 5.** Accidents according to the anatomical region of the body affected, 2019. Source: Compiled by the authors.


**4.2. Metabolic drug interactions between immunosuppressants and post-transplant medication**

#### *4.2.1. Treatment and prevention of infections*

Environmental circumstances and immune deficiencies due to immunosuppression therapy make recipients susceptible for infections that are one of the leading complications after organ transplantation; therefore, prevention and management of infections is a major task primarily in the early postoperative period. Since fungal infections are a threatening cause of morbidity and mortality, the antifungal prophylaxis is an important element of posttransplant medication. The antifungal azole-derivatives are potent (some of them are very strong) CYP3A inhibitors, predicting potential metabolic drug interactions with calcineurin inhibitors, mTOR inhibitors, or corticosteroids. The most potent CYP3A inhibitor is ketoconazole, able to increase blood concentrations (AUC) of ciclosporin (> 4-fold), tacrolimus (> 2-fold), sirolimus (11-fold), everolimus (15-fold), and methylprednisolone (> 2-fold) [73, 74]. Because of the substantial increase in blood concentrations of several immunosuppressants that can be avoided by drastic reduction of immunosuppressant doses and because of other adverse effects of ketoconazole, the concomitant medication is discouraged. Fluconazole, itraconazole, and voriconazole are alternative regimens for antifungal therapy or prophylaxis; however, all three drugs are azole derivatives and have the capability to inhibit CYP3A function, albeit at a lower extent than ketoconazole [75–77]. Although the continuous immunosuppressant monitoring is highly recommended and dose adjustment (reduction) is generally required, the antifungal treatment with fluconazole, itraconazole, or voriconazole can be safely applied except for voriconazole-sirolimus combination [78]. Because of an extreme (7-fold) increase of sirolimus blood concentrations as a consequence of concomitant use of voriconazole, this combination is contraindicated. Amphotericin B, the nonazole type antifungal agent, does not influence CYP activities; therefore, no metabolic drug interactions can be expected in concomitant treatment with immunosuppressants. However, the widespread use of amphotericin B is limited because of its toxicity profile, primarily because its nephrotoxic side-effect can contribute to the renal injury by ciclosporin or tacrolimus.

Organ transplant patients are at high risk for developing bacterial infections that occur in 20–40% of transplants. Potential sources of infection are from hospital and community exposures, as well as from endogenous flora of patients. Among the antibiotics used for treatment of infections, the macrolide erythromycin and clarithromycin have been reported to interact with immunosuppressive agents. These macrolides are CYP3A substrates and bind to CYP3A4 enzymes, leading to a complex formation that completely inactivates CYP3A4 enzyme [79–82]. The *in vitro* findings were confirmed by clinical observations that blood concentrations of ciclosporin/tacrolimus or sirolimus/everolimus increased as a consequences of concomitant treatment with erythromycin or clarithromycin [73, 83–86]. Page et al. [87] and Mori et al. [88] have reported some potential of azithromycin for drug interaction with ciclosporin and tacrolimus; however, *in vitro* experiments demonstrated that azithromycin poorly interfere with CYP3A4 [89]. When concomitant therapy with these macrolides is necessary, blood concentrations of calcineurin inhibitors or mTOR inhibitors should be carefully monitored, and the immunosuppressant doses should be adjusted. In contrast, the macrolide rifampicin is a potent CYP3A4 inducer and can activate PXR, resulting in a substantial increase in CYP3A4 expression [90]. The increased CYP3A4 activity consequently enhances the metabolism and elimination of calcineurin inhibitors, mTOR inhibitors, and corticosteroids [91–93]. However, blood concentration–guided dose-adjustment of immunosuppressants should be applied carefully because increased metabolism can evoke elevation of toxic metabolite formation (e.g., ciclosporin).

adjustment [68]. Interestingly, CYP3A5 nonexpressers with *CYP3A5\*3/\*3* genotype are more susceptible to glucocorticoid induction than *CYP3A5\*1* carriers [69]; thus, more pronounced increase in tacrolimus exposure can be expected in CYP3A5 nonexpressers after glucocorticoid

ganciclovir, valganciclovir Mycophenolate-glucuronide inhibits renal tubular

Inhibition of CYP3A4

rifampicin CYP3A4 induction; enhanced metabolism of corticosteroids

ritonavir Irreversible inhibition of CYP3A4; increased blood levels of corticosteroids

secretion of ganciclovir; increased blood levels of ganciclovir and increased risk of toxicity (nephrotoxicity, neutropenia, leukopenia)

Increased blood levels of corticosteroids

Clinically significant interaction between mycophenolic acid, the active metabolite of mycophenolate mofetil, and ciclosporin has been reported [70]. The mycophenolate-glucuronide metabolite eliminated into bile undergoes enterohepatic cycling because of intestinal bacterial metabolism and reabsorption of mycophenolic acid. The enterohepatic circulation, contributing to overall pharmacokinetics of mycophenolic acid by 37% in human, is inhibited by concomitant administration of ciclosporin but does not interfere with tacrolimus or sirolimus [71, 72]. In ciclosporin-mycophenolate combination therapy, the reduced blood concentration of mycophenolic acid is necessary to ameliorate by increasing dose of mycophenolate mofetil. Furthermore, special attention on optimal dosing is required during switching ciclosporin-

**4.2. Metabolic drug interactions between immunosuppressants and post-transplant**

Environmental circumstances and immune deficiencies due to immunosuppression therapy make recipients susceptible for infections that are one of the leading complications after organ transplantation; therefore, prevention and management of infections is a major task

mycophenolate to tacrolimus-mycophenolate therapy and *vice versa*.

**Immunosuppressant Drug interactions Consequences** *Antiviral agents*:

420 Organ Donation and Transplantation - Current Status and Future Challenges

*Antifungals*:

*Antibiotics*:

*Antiviral agents*:

ketoconazole, fluconazole, voriconazole, itraconazole

**Table 2.** Clinically relevant pharmacokinetic drug interactions with immunosuppressants.

withdrawal.

Prednisolone Methylprednisolone

**medication**

*4.2.1. Treatment and prevention of infections*

A significant cause of graft failure still remains viral infections, which are acquired as new infection or reactivation of latent viruses. After transplantation, cytomegalovirus (CMV) is the most common viral infection in recipients, primarily in those CMV-seronegative patients who were transplanted with graft from CMV-seropositive donors, resulting in viral reactivation. For prophylaxis and treatment of CMV infection, aciclovir, ganciclovir, and valganciclovir (the prodrug of ganciclovir) are generally applied. None of these antiviral drugs influences the function of drug-metabolizing CYPs or UDP-glucuronyl transferases, and consequently, they do not modify the pharmacokinetic properties of immunosuppressants. Aciclovir and ganciclovir are eliminated primarily in the urine as unchanged compounds. Increased risk of nephrotoxicity and leukopenia has been reported in patients who were co-medicated with a drug that can reduce renal clearance of aciclovir or ganciclovir. During co-administration with mycophenolate or mycophenolate-mofetil, mycophenolate-glucuronide and aciclovir or ganciclovir can significantly compete for renal tubular secretion, resulting in an increase in aciclovir/ganciclovir and mycophenolate-glucuronide exposure, as well as the risk of nephrotoxicity or leukopenia [94–96]. Management of potent metabolic drug interactions between antiviral protease inhibitors and immunosuppressants is a major challenge because most of the protease inhibitors are clinically significant CYP3A4 inhibitors. Ritonavir-boosted therapies require substantial reduction of immunosuppressant doses (to 5–20% for ciclosporin; to 1–3.5% for tacrolimus) with continuous monitoring of blood concentrations [97–101].

frequent monitoring of immunosuppressant blood concentrations. The metabolism of all three Ca-channel blockers is primarily catalyzed by CYP3A4, anticipating potential drug interactions with immunosuppressants. Furthermore, significant inhibition of CYP3A4 by diltiazem, verapamil, and amlodipine has been demonstrated with an additional inhibitory property of metabolite intermediate complex formation that catalytically inactivates CYP3A4 and CYP3A5 enzymes [79, 80, 82, 110–114]. The inactivation of CYP3A enzymes by comedication with these antihypertensive drugs consequently leads to a permanent increase in blood concentrations of calcineurin inhibitors or mTOR inhibitors [115–122]. In transplant recipients comedicated with sirolimus and verapamil, an increase of blood concentrations of both sirolimus and verapamil was observed [123]. Furthermore, in patients carrying wild-type *CYP3A5\*1* allele, concomitant treatment with amlodipine significantly decreased tacrolimus clearance, and along with the changes in tacrolimus pharmacokinetics, an increase in amlodipine blood concentrations was also observed [124]. The metabolism of the Ca-channel blocker nifedipine is catalyzed almost exclusively by CYP3A enzymes, and competition for the active site of CYP3As may be expected if nifedipine and CYP3A substrate calcineurine inhibitors or mTOR inhibitors are concomitantly applied. In contrast, no evidence for pharmacokinetic drug interactions has been provided in transplant recipients treated with nifedipine and ciclosporin/tacrolimus or sirolimus/ everolimus. Carvedilol is often used for treatment of hypertension in transplant patients, and pharmacokinetic drug interaction between carvedilol and ciclosporin has been observed that required 10–20% reduction of ciclosporin doses to maintain the blood concentrations within the therapeutic range [125, 126]. The major metabolic pathways of carvedilol are catalyzed by CYP2D6 and CYP1A2 rather than by CYP3A4 [127]; however, inhibition of CYP3A enzymes by carvedilol does not account for pharmacokinetic drug interaction with ciclosporin. Carvedilol has been demonstrated to block the function of the ABCB1 transporter protein (ATP-binding cassette B1; previously called as Pgp) [128]. In the intestinal wall, ABCB1 transporter pumps pharmacons or other xenobiotics passed into the enterocytes back into the gut lumen. The inhibition of ABCB1-mediated transcellular transport in the intestine by carvedilol is responsible for the increased absorption of ciclosporin. Under careful monitoring of ciclosporin blood concentration, the ABCB1 inhibition by carvedilol can be beneficial in ciclosporin-sparing therapy for transplant patients. Since the absorption of tacrolimus and mTOR inhibitors is also mediated by ABCB1, similar pharmacokinetic drug interactions between these immunosuppres-

Metabolic Drug Interactions with Immunosuppressants http://dx.doi.org/10.5772/intechopen.74524 423

sants and carvedilol are presumably developed as with ciclosporin.

Hyperglycemia developing posttransplant diabetes mellitus is generally medication related. Corticosteroids can evoke reduction of glucose tolerance, whereas ciclosporin and tacrolimus directly block insulin-release by islet cells. The metabolism of the sulfonylurea type antidiabetic agents (e.g., tolbutamide, glipizide, glibenclamide, and glimepiride) is mediated by CYP2C9; therefore, metabolic drug interactions with immunosuppressants are not expected in patients treated with any of these oral hypoglycemic drugs. Although the thiazolidinedione type troglitazone and rosiglitazone are not CYP3A substrates, they can induce the expression of CYP3A enzymes by activation of the nuclear receptors, PXR and CAR [129–132]. Enhanced transcription results in an increase in CYP3A activities and the metabolism of calcineurin inhibitors,

*4.2.4. Antihyperglycemic therapy*


**4. Data**

The hourly WS dataset for Gda ´nsk (latitude: 54.352◦ N, longitude: 18.646◦ E, 10 m. height) over the last seven years between 1 January 2015 and 26 July 2021 was gathered from Open Weather Map [82]. Then, using MLE, the TPWD was fitted to the WS data for each month and the annual data. For the purpose of determining the optimal parameters of the WD that maximize the likelihood function, the SA, GA, DE, and EGO were applied and performance of the techniques was compared. Following the obtaining of the parameters, potential WE and wind power for Gda ´nsk, the capital of the Pomerania Voivodeship in Poland, were calculated.

The dataset contains 60,100 rows and 25 columns and it provides information about WS, minimum temperature, maximum temperature, pressure, wind angle, amount of rain, amount of snow, information about how the weather looks (rainy, snowy, etc.).

Table 3 shows summary statistics regarding monthly average of WS and minimum and maximum temperatures in Celciusº. As shown in the table, the warmest month for Gda ´nsk is August and the coldest month is January. Table 3 also presents the monthly average of WS (m/s) in Gda ´nsk. As shown in the table, the monthly average WS does not differ dramatically between months within a year. According to the table, it can be concluded that the months in which the average WS is higher than others are April, December, and May. The lowest WS average is observed in August.

**Table 3.** Average WS, minimum temperature, maximum temperature per month in Gda ´nsk between 1 January 2015 and 26 July 2021.

Since obtaining the dataset from the third-party vendor was easy and quick and since the dataset contains hourly WS information for almost seven years, it was preferred to be used in the study. However, variables, including WS, in the dataset were collected by a single sensor located near the old town in Gda ´nsk. In conclusion to this, estimations for potential wind power were made only for this location. There was also no possibility to get WS information for different heights or for different parts of the city (e.g., parts of the city where long and tall buildings are located). For future studies, researchers plan to obtain datasets from different sources, such as local authorities or any other official sources, to be able to avoid the limitations mentioned above.


**Mechano-Enzymatic Deconstruction with a New Enzymatic Cocktail to Enhance Enzymatic Hydrolysis and Bioethanol Fermentation of Two Macroalgae Species**

#### **Sameh Amamou 1 , Cecilia Sambusiti 1 , Florian Monlau 1,2, Eric Dubreucq 1 and Abdellatif Barakat 1,3,\* ID**

Received: 14 November 2017; Accepted: 7 January 2018; Published: 17 January 2018

**Abstract:** The aim of this study was to explore the efficiency of a mechano-enzymatic deconstruction of two macroalgae species for sugars and bioethanol production, by using a new enzymatic cocktail (Haliatase) and two types of milling modes (vibro-ball: VBM and centrifugal milling: CM). By increasing the enzymatic concentration from 3.4 to 30 g/L, the total sugars released after 72 h of hydrolysis increased (from 6.7 to 13.1 g/100 g TS and from 7.95 to 10.8 g/100 g TS for the green algae *U. lactuca* and the red algae *G. sesquipedale*, respectively). Conversely, total sugars released from *G. sesquipedale* increased (up to 126% and 129% after VBM and CM, respectively). The best bioethanol yield (6 geth/100 g TS) was reached after 72 h of fermentation of *U. lactuca* and no increase was obtained after centrifugal milling. The latter led to an enhancement of the ethanol yield of *G. sesquipedale* (from 2 to 4 g/100 g TS).

**Keywords:** bioethanol; enzymatic hydrolysis; macroalgae; mechanical pretreatment

#### **1. Introduction**

Over the last decades, the world has been facing critical economic and environmental issues, such as the exhaustion of fuels, environmental pollution and climate change, combined with the increase of the world population. These issues led to the expansion of research and development on renewable and sustainable biofuels [1,2]. Both lignocellulosic biomasses and algae constitute sustainable sources of bioenergy and biomolecules (i.e., surfactant, bioethanol, biogas and biodiesel) and they represent promising alternative sources to petroleum-based fuels and chemicals.

In particular, macro- and/or micro-algae permit us to overcome the major limitations associated with lignocellulosic plants [3]. Macroalgae, also called seaweeds, represent renewable abundant biomasses, which could be easily cultivated in aquatic environment. Thus, they do not compete with land use, and water consumption necessary for terrestrial plants.

Furthermore, macroalgae are characterized by a higher growth rate than lignocellulosic biomasses and higher hydrolysable sugar contents than crops with almost no lignin [4–6]. Macroalgae are multicellular photosynthetic organisms divided into three major groups: green, red and brown algae, according to the thallus color derived from natural pigments and chlorophylls [4,7]. Generally, the amounts of carbohydrates vary between 25–60%, 30–60% and 30–50% dry wt. for green, red, and brown algae, respectively. In particular, green algae are mainly composed of mannan, ulvan, starch and cellulose, while red algae are mainly composed of carrageenan (up to 75% dry wt.) and agar (up to 52% dry wt.) as polysaccharides [6].

Several researches have reported the use of marine alga biomass as bioethanol feedstock. Different macroalgae groups such as *Gelidium amansii* [8], *Gracilaria salicornia* [9] and *Kappaphycus alvarezii* belonging to red seaweed and green algal species such as *Ulva* spp. have been considered as potential sources for conversion to bioethanol. As the interests in seaweeds were expanding, intense research was required for an efficient use of this biomass. However, it still faces technical and economic challenges and still depends on the development of eco-friendly pretreatment and conversion methods [10]. Since this step is often required to facilitate the enzymatic hydrolysis of macroalgae and their further sugars and bioethanol conversion. So far, the most common pretreatments used to enhance the hydrolysis and thus bioethanol production of macroalgae are physical (wet oxidation, thermal, milling and oven drying), chemical (acidic and alkaline), and thermo-chemical pretreatments [10]. However, one of the major drawbacks of using thermal and thermo-chemical pretreatments is the possible formation of organic acids and furan derivatives, which can inhibit bioethanol fermentation [11,12]. In addition, to find the most sustainable and cost-effective pretreatment, another challenge of producing bioethanol and interesting molecules from *G. sesquipedale* (red) and green *U. lactuca* (green) macroalgae is to find the specific enzymes able to efficiently hydrolyze their polysaccharides (Figure 1). Mechanical fractionation of biomass is one promising route that can contribute to a future sustainable dry biorefinery without water consumption and without waste production. Grinding or dry fractionation can be easily introduced in a biorefinery scheme improving the overall sustainability process [13]. Thus, coupling mechanical fractionation with enzymes is a promising biorefinery scheme of algae biomass valorization. In addition, the use of this natural enzymatic cocktail (i.e., Haliatase) coupling to mechanical fractionation has never yet been investigated in algae biomass biorefineries. Thus, the main objectives of this study were the following:

**Figure 1.** Mechano-enzymatic pretreatment and deconstruction of macroalgae developed in this study.

#### **2. Results and Discussion**

#### *2.1. Chemical Composition*

The chemical compositions of both red *G. sesquipedale* and green *U. lactuca* macroalgae are shown in Table 1. *U. lactuca* had higher ash content (32 g/100 g TS) than *G. sesquipedale* (11 g/100 g TS). These values are in agreement with literature studies that reported ash values ranging from 11 to 34 g/TS and from 9 to 20 g/100 g TS, for green and red macroalgae, respectively [8,14,15]. Similar protein content (13 and 16 g/100 g TS) was observed for *U. lactuca* and *G.sesquipedale*, in accordance with literature data for red (10–16 g/100 g TS) and green (12–21 g/100 g TS) macroalgae [14–16].

**Table 1.** Chemical composition of red *G. sesquipedale* and green *U. lactuca* Values correspond to mean ± SD (standard deviation) of measurement performed in duplicate.

n.d.: Not detected; \* Monosaccharide profile of polymeric carbohydrates determined after acid hydrolysis and HPLC quantification; SD: standard deviation.

Total sugar content of *U. lactuca* and *G. sesquipedale* were 25.8 and 30.9 g/100 g TS, respectively. Similar sugar content was reported by Jard et al. [16] for red and green macroalgae. As reported by Jung et al. [7], the red alga *G. sesquipedale* was mainly composed of glucose, galactose, and agar, while *U. lactuca* consisted of glucose, xylose, and rhamnose. Glucuronic and galacturonic acids were also detected in both algae with values of 5.01 and 3.32 g/100 g TS for the green and red algae, respectively. However, it is noteworthy that the chemical composition of macroalgae presents a great variability in the literature, which is related to several factors, such as species, geographical origin, season, environmental, and physiological variations, but also to the analytical method used for their characterization.

#### *2.2. Particle Size of Macroalgae*

Particle size of the untreated and milled macroalgae is reported in Figure 2. After milling, a lower mean particle size was obtained for the green alga *U. lactuca* (147–161 µm) than the red alga *G. sesquipedale* (201–355 µm), which can be explained by the lower particle size distribution of the untreated green algae biomass (289 µm) than the red one (472 µm). Furthermore, it was observed that for the red alga, CM was more effective than VBM in particle size reduction (Anova *p*-values < 0.05) (Figure 2); while for the green alga similar effect of VBM and CM was noticed with a slightly higher effect of VBM but not significant with an Anova *p*-values of 0.59. This could be explained by the

high ash content (around 30%) and a possible synergistic impact between the mineral ash and the mechanical process (i.e., VBM) as previously mentioned by Motte et al. [13]. Indeed, they highlighted in their study that a mineral-vegetal co-milling in a VBM could significantly reduce the final particle size of the lignocellulosic biomass compared to a simple milling of lignocellulosic biomass.

**Figure 2.** Mean particle sizes of untreated and milled algae biomass. Values correspond to mean ± SD (standard deviation) of measurement performed in duplicate.

#### *2.3. Effect of Haliatase Cocktail Activity on Sugars Yield of Untreated Macroalgae*

The effect of Haliatase dosage on total sugars released during enzymatic hydrolysis of untreated *U. lactuca* and *G. sesquipedale* was investigated (Figure 3). For both macroalgae, the increase of the enzymatic concentration led to higher total sugars released. By increasing the enzymatic concentration from 3.4 to 30 g/L, the total sugars released after 72 h of hydrolysis varied from 6.7 to 13.1 g/100 g TS and from 7.9 to 10.8 g/100 g TS for *U. lactuca* and *G. sesquipedale*, respectively (significant difference with Anova *p*-values < 0.05) (Table 2).

**Figure 3.** *Cont.*

**Figure 3.** Total sugars released after 72 h of enzymatic hydrolysis for various enzyme dosages (3.4, 10 and 30 g/L) for both untreated (**A**) *Ulva lactuca* and (**B**) *Gelidium sesquipedale*.

**Table 2.** Total sugar yields (g/100 g TS) obtained after 72 h of enzymatic hydrolysis of untreated and milled algae with an enzyme dosage of 3.4, 10 and 30 g/L.

β However, the increase of enzyme concentration from 10 to 30 g/L did not lead to a further enhancement of total sugar yield of both red and green algae witch is assumed with Anova *p*-values of 0.13 and 1.55, respectively, for green and red algae. Finally, for both algae species, the major soluble sugar released during the enzymatic hydrolysis was glucose which is very pertinent in the case of ethanol production using the *Saccharomyces cerevisae* strain. Glucose released after 72 h of enzymatic hydrolysis varied from 5.8 to 10.5 g/100 g TS for *U. lactuca* and from 1.5 to 4.1 g/100 g TS for *G. sesquipedale* after increasing the enzymatic dosage from 3.4 to 30 g/L (significant difference with Anova *p*-values < 0.05) (Table 3). If the results of glucose yields were not significantly different from 10 g/L to 30 g/L for red algae (Anova *p*-values of 0.95), they were for green algae (Anova *p*-values < 0.05). Cho et al. [17] reported an enzymatic saccharification of *Gelidium amansii* using Celluclast 1.5 L (endoglucanase: (8.4 U/mL), Viscozymes L (β-glucanase: 1.2 U/mL) and a mixture of both enzymes. They obtained a glucose concentration of 5.5 g/L after an enzymatic hydrolysis using Celluclast 1.5 L. Interestingly, the glucose concentration released was improved to 7.6 g/L by using the mixture of "Celluclast + Viscozymes" enzymes after 48 h of hydrolysis. Nonetheless, in both assays, only the fibers rich in cellulose were hydrolyzed whereas agar was not solubilized [17].

**Table 3.** Glucose yield (g/100 g TS) obtained after 72 h of enzymatic hydrolysis of untreated and milled algae with an enzyme dosage of 3.4, 10 and 30 g/L.

#### *2.4. Effect of Mechanical Pretreatments on Enzymatic Hydrolysis of Macroalgae*

The effect of mechanical pretreatments (i.e., centrifugal milling, vibro-ball milling) on total sugars released during enzymatic hydrolysis of untreated *U. lactuca* and *G. sesquipedale* was investigated (Figure 4). The results revealed that, whatever the enzymatic dosage, mechanical pretreatments did not have any effect on the total sugars released from green alga *U. lactuca* (Anova *p*-values > 0.05) Conversely, total sugars released from *G. sesquipedale* increased after mechanical pretreatments (up to 126% and 129% after vibro-ball and centrifugal milling fractionation, respectively, Anova *p*-values < 0.05). Otherwise, fractionation was more effective in glucose releasing (up to 214% and 261% after vibro-ball and centrifugal milling, respectively). It is important to note also that mechanical fractionation seems to be less effective after increasing the enzymatic dosage from 3.4 g/L to 30 g/L. Thus, glucose released from *G sesquipedale* increased after CM by 161% and 62% for 3.4 and 30 g/L of enzymatic dosage, respectively. Furthermore, whatever the enzymatic dosage, CM was more effective than VBM in improving the total sugars and glucose released. Moreover, it is noteworthy that the high-energy requirement is one of the drawbacks of mechanical treatments. In a previous study, [18–20] reported that the energy requirement for CM (100 kWh t−1 TS) was lower than that of VBM (2000 kWh t−1 TS). Thus, CM was chosen for the performance of experimentation. − −

**Figure 4.** Total sugars released after 72 h of enzymatic hydrolysis for untreated and milled (G: Green, R: Red) macroalgae with an enzyme dosage of (**A**) 3.4; (**B**) 10 and (**C**) 30 g/L. Values correspond to mean ± SD of measurement performed in duplicate.

#### *2.5. Bioethanol Fermentation of U. lactuca and G. sesquipedale*

Taking into account the previous results, CM was chosen as mechanical treatment prior to a simultaneous saccharification and fermentation (SSF). Also, 10 g/L was selected as the optimal enzymatic dosage of Haliatase and, thus, used in saccharification process. Bioethanol yields of untreated and centrifugal milled macroalgae were evaluated and compared through SSF experiments (Figure 5). CM treatment did not affect the ethanol production of *U. lactuca* with values around 6 geth/100 g TS (Anova *p*-values of 0.35), confirming the enzymatic hydrolysis data (Figure 4). Conversely, the CM led to an enhancement of the ethanol yield of the red algae (from 1.95 to 3.51 g/100 g TS, significantly different, with Anova *p*-values < 0.05). However, although bioethanol yield obtained with *U. lactuca* was higher than that of *G. sesquipedale*, the bioethanol conversion efficiency of the red one was higher, because the glucose content of red algae (9.62%) is lower than that (15.2%) of green algae (Table 1). Thus, after CM fractionation, 64% and 69% of bioethanol conversion efficiency (expressed in % of the theoretical yield) (Table 4) was obtained for green and red algae, respectively. Furthermore, for algae strains, the galactose and xylose were not consumed. Such observation could be attributed to a diauxic effect commonly observed but it is not totally satisfactory, as the galactose consumption of the red algae did not start even after a total depletion of the glucose, so probably 72 h would be too short to initiate diauxic effect since Berlowska et al. (2017) [21] Berlowska et al., 2017 have demonstrated that actually, *S. cerevisae* ethanol red was capable to metabolize galactose but in absence of glucose. Nevertheless, despite the fact that a large number of yeast species can metabolize xylose, only 1% of strains convert xylose to ethanol [22]. Thus, it is important to find the most active yeast species for bioethanol fermentation of hexoses (other than glucose) and pentose sugars, in order to achieve higher ethanol yield.

**Figure 5.** Simultaneous saccharification and fermentation of untreated and milled macroalgae (G: Green, R: Red) at an enzyme dosage of 10 g/L. (**A**) Glucose content (g/100 g TS); (**B**) Ethanol yield (g/100 g TS). Values correspond to mean ± SD of measurement performed in duplicate.

β

−

**Table 4.** Ethanol yield (g/100 g TS) obtained after 72 h of SSF of untreated and milled algae with an enzyme dosage of 10 g/L.

\* Theoretical ethanol yield: 8 g/100 g TS and 5 g/100 g TS for *U. lactuca* and *G. sesquipedale* respectively.

In this matter, Cho et al., 2014 [17], reported that glucose causes the repression of galactose uptake which decreased ethanol yield. The acclimation of galactose was then reported as the key of a fermentation process since it has allowed simultaneous utilization of glucose and galactose. In fact, ethanol yield doubled (from 0.21 to 0.44 g/100 g TS) after using *S. cerevisiae* acclimated to high concentration of galactose.

Regarding treatment effects on ethanol fermentation, Schultz-Jensen et al. [10] investigated the ethanol fermentation of the green macroalga *Chaetomorpha linum* after wet oxidation, hydrothermal treatment, plasma, and ball milling for 48 h at 40 ◦C. Interestingly, the best ethanol recovery was obtained after ball milling with an ethanol yield of 18 g/100 g TS, corresponding to 78% of the theoretical ethanol yield.

#### **3. Materials and Methods**

#### *3.1. Macroalgae*

Red alga *Gelidium sesquipedale* and green alga *Ulva lactuca* were obtained from the Morocco coast. Once collected, samples were washed with tap water and further air-dried (8% DM) and milled using a cutting mill to a particle size less than 2 mm (SM100 Retsch, Haan, Germany). Then, they were further milled using two equipments, characterized by different mechanical stresses, such as impact, compression, friction, and shear: (i) a centrifugal mill "CM" (Retsch ZM 200, Haan, Germany) with 0.25 mm screen size, operated at ambient temperature with a speed of 12000 rpm; (ii) a vibratory ball mill "VBM" (Retsch MM400, Haan, Germany) operated at ambient temperature, at a frequency of 15 s−1 for 5 min.

### *3.2. Enzymatic Cocktail*

The enzymatic cocktail (i.e., Haliatase enzyme) has been obtained from KURA BIOTECH SPA, (Puerto Varas, Chile) and it is derived from the hepatopanchreas of cultured abalone (Haliotis rufescens). It is a multi-enzymatic cocktail capable of degrading the cell walls of macroalgae by hydrolyzing most of their polysaccharides components. It is composed of mainly β-glucanase (1875 U/g), carragenase (315 U/g) and agarase (440 U/g).

#### *3.3. Enzymatic Hydrolysis*

Enzymatic hydrolysis of untreated and milled samples was performed in 40 mL of closed flasks (working volume of 20 mL). An amount (1 g) of each sample (solid loading of 50 g/L), 2 mL of acetate buffer (500 mM) and 15.8 mL of ultra-pure water were added to each flask. The pH was then adjusted to 5.5 with NaOH (1 N) or HCl (2 N). Finally, 1.2 mL of sodium azide (final concentration 1 g/L) and 1 mL of concentrated Haliatase enzyme were added to have final enzymatic concentrations of 3.4, 10 and 30 g/L, respectively. Flasks were kept at 37 ◦C for 72 h with stirring 500 rpm. Samples were withdrawn at 0, 2, 4, 6, 24, 48 and 72 h and the corresponding supernatants were analyzed by HPLC (Waters corporation, Milford, CA, USA), equipped with a BioRad HPX-87H column (Biorad, Hercules, CA, USA) at 40 ◦C, a refractive index detector at 40 ◦C and a 0.005 M H2SO4 solvent at

0.3 mL/min. Tests were performed in duplicate, to evaluate the amount of C5-C6 sugars released during the enzymatic hydrolysis.

Sugar yields (g*i*/100 g TS) were calculated according to Equation (1):

$$\text{Sugar } \text{''}i\text{'' yield}\_{\text{t}} = \left[\mathbb{C}\_{\text{t}} \text{ sugar}\text{''}i\text{''/C} \text{solid}\right] \times 100\tag{1}$$

where: *C*t sugar "*i*" (gsugar "*i*"/L) is the concentration of C5 and C6 sugars produced during hydrolysis, at time t; *C* solid (g TS/L) is the total solids concentration in the flask.

The analysis of variance (Anova) method was used to analyse the impact of the enzymatic dose and mechanical fractionation (CM and VBM) on both red and green algae, the confidence level considered was 95%.

#### *3.4. Bioethanol Fermentation*

Bioethanol yields of untreated and milled (CM) macroalgae were evaluated and compared through simultaneous saccharification and fermentation (SSF) experiments. Tests were performed by using unsterilized samples, into 40 mL flasks (working volume of 20 mL) closed with rubber septa and equipped with an air vent system, constituted of sterilized needle and filter, in order to evacuate the CO2 produced during the bioconversion. A lyophilized *S. cerevisiae* yeast strain (Ethanol Red®, FERMENTIS, a division of S. I. LESAFFRE, Lille, France) was used as inoculum. For this purpose, lyophilized cells were previously washed and then suspended in sterilized distilled water to a concentration of 30 g TS/L. Each flask contained: 1 g TS of sample (solid loading of 50 g/L), 1 mL of concentrated Haliatase enzyme, to have an enzymatic concentration of 10 g/L in each flask, 1 mL of yeast (30 g TS/L), 2 mL of nutrients, containing: 50 g TS/L yeast extract (Difco®), 4 g TS/L urea, 0.5 g TS/L chloramphenicol and 50 mM acetate buffer (pH = 5). Flasks were incubated at 37 ◦C for 72 h under stirring. Samples were withdrawn at 0, 2, 4, 6, 24, 48 and 72 h and the cell free supernatants were evaluated for ethanol and C6 sugars (i.e., glucose, galactose, fucose and rhamnose) concentrations by HPLC as previously mentioned.

Ethanol yields (gethanol/100 g TS) were calculated according to Equation (2):

$$\text{Ethanol yield}\_{t} = \left[ \mathcal{C}\_{t} \text{ethanol/C solid} \right] \times 100 \tag{2}$$

where *C*t ethanol (gethanol/L) is the concentration of ethanol produced during SSF, at time t; *C* solid (g TS/L) is the total solids concentration in the flask.

The analysis of variance (Anova) method was used to analyze the impact of mechanical fractionation (CM and VBM) on both red and green algae bioethanol fermentation, the confidence level considered was 95%.

#### *3.5. Analytical Determinations*

Particle size distribution of untreated macroalgae was determined by a vibratory sieving apparatus (Analytical Sieve Shaker AS 200, Retsch®, Haan, Germany) equipped with six sieves of different sizes (1, 0.8, 0.71, 0.56, 0.32 and 0.2 mm). Particle size distribution of milled macroalgae was analyzed by a laser granulometry (MASTERSIZER 2000, Malvern Instrument, Orsay, France). Total Solids (TS), Volatile Solids (VS) and ash contents were determined according to APHA methods [22]. Ultimate analysis (C, N, H and S) was accomplished with an elemental analyzer (Elementar "VarioMacroCube", Elementar group, Langenselbold, Germany). Proteins content was estimated by multiplying N by 6.25. Carbohydrates and uronic acids were determined according to a reduced scale hydrolysis procedure, based on the NREL Laboratory Analytical Procedure [23]. Briefly, 80 ± 1 mg of milled algae biomass was subjected to a two-stage sulfuric acid hydrolysis (1 h at 30 ◦C in 72 wt % H2SO4, followed by 1 h at 121 ◦C in 4 wt % H2SO4 for red algae and 3 h at 120 ◦C in 6 wt % H2SO4 for the green algae). Samples were withdrawn at 1 h, 2 h and 3 h and the cell free supernatants were evaluated for sugars (i.e., glucose, xylose, arabinose, galactose, fucose, rhamnose) and uronic acids (galacturonic acid and

glucuronic acid) concentrations by high-performance liquid chromatography by HPLC system (Waters corporation), equipped with a BioRad HPX-87H column at 40 ◦C, a refractive index detector at 40 ◦C and a 0.005 M H2SO4 solvent at 0.3 mL/min. It is noteworthy that analytical determinations were performed in duplicate.

#### **4. Conclusions**

A comprehensive study was performed on the efficiency of a new natural enzymatic cocktail to hydrolyze polysaccharides of two types of seaweeds and produce bioethanol. The effects of two mechanical pretreatments were also tested for increasing bioethanol fermentation. The most effective enzymatic dosage for the saccharification process of green *U. lactuca* and red *G. sesquipedale* was 10 g/L and the highest values of glucose released were obtained with green algae after 72 h of enzymatic hydrolysis. Centrifugal milling was more effective in hydrolyzing red *G. sesquipedale* compared to vibro-ball milling, while the mechanical pretreatments applied did not show any effect on green *U. lactuca*. However, green *U. lactuca* showed the highest bioethanol yield compared to red *G. sesquipedale.*

**Acknowledgments:** The authors are grateful to the LipPolGreen Platform of UMR IATE and (INRA Montpellier) for her help in biochemical analysis and fermentation and INRA for the financial support. The authors are grateful to KURA BIOTECH SPA (Chile) for giving us the Haliatase enzyme cocktail.

**Author Contributions:** A.B. and F.M. conceived and designed the experiments; S.A. and C.S. performed the experiments; E.D., A.B. and F.M. analyzed the data; S.A. contributed analysis tools; all authors wrote the paper.

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

#### **Abbreviations**

#### **References**

**Sample Availability:** Samples of the compounds are not available from the authors.

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**1. Introduction**

Numerous applications of heat transfer liquids or coolants can be found in a variety of fields, such as automobiles, industry, electronics, and cooling processes. In all such industrial applications, cooling by liquids has been used for years. The process of cooling by fluids may be the single phase (where there is no phase change in the coolant) or two-phase (where coolant liquid will experience a phase change). In the latter, latent heat influences the cooling efficiency [1]. Several coolants, such as water, ethylene glycol, blend of water and glycol, propylene glycol and amalgamation of water, and propylene glycol, are used as coolants in automobiles and industrial cooling processes. In the last two decades, several researchers have devoted their efforts to increasing thermal conductivity of coolants, thereby improving heat transfer capabilities. The pioneering work of Choi et al. [2] introduced nanofluids by insertion of solid nanoparticles into liquids, thus enhancing the thermal properties of these liquids. This pioneering work has remarkably revolutionized modern engineering and the industrial world.

Nanofluids are an amalgamation of suspended solid material particles and customary liquids (ethylene glycol, water). This new type of advanced material possesses amazing capabilities that trigger the process of heat transfer and augments the thermal conductivity of the base fluid. Enhancement in the thermal conductivity and heat transfer is visualized once ferrite nanoparticles are added into the base liquid. Several examples featuring heat transfer can be quoted, including chemicals, cooling and heating system of buildings, and avionics cooling systems. Nanofluids exhibit potentially exceptional features in comparison to macrometer-sized particles. This is because nanoparticles have sufficiently larger surface area compared to micrometer-sized particles; this is the reason nanofluids possess incomparable capabilities of heat transfer [3].

In several electromagnetic applications with high permeability, e.g., electromagnetic wave absorbers and inductors, usage of nickel–zinc ferrite can be noticed. To minimize energy losses related to bulk powders, usage of nickel–zinc nanoparticles has been recommended by a number of researchers [4–6]. In addition, a majority of electronic gadgets require such materials to be compressed into outsized shapes with the required thickness, which is reasonably challenging if the size of these particles is large enough. Several methods have been proposed to get nickel–zinc ferrite, including ball milling, precipitation, and hydrothermal. Ferrofluids are colloidal fluids comprising ferromagnetic or ferrimagnetic nanoparticles suspended in an electrically insulated hauler fluid. In the current examination, ethylene glycol (C2H6O2) was taken as a carrier fluid. The assumed ferrite nanoparticle was nickel–zinc ferrite (NiZnFe2O4) crystallize in the normal spinal structure. Typically, at room temperature, the inverted spinals are ferromagnetic and normal spinals are paramagnetic. Moreover, zinc ferrites act like antiferromagnetic in nature at low temperature. This feature makes ferromagnetic nanofluids more relevant in different real-world applications [7,8]. The ferrofluid's flow with the effect of thermal gradients and the magnetic field was discussed by Neuringer [9]. Majeed et al. [10] demonstrated the heat transfer investigation in a ferromagnetic fluid flow.

The subject of fluid flow past stretched surfaces has diverse engineering and industrial applications, including paper production, glass blowing, crystal growing, hot rolling, manufacturing of rubber sheets, annealing of copper wires, etc. The coined work of Crane [11] discussing the flow past a linearly stretching surface urged fellow researchers to discover more avenues in this exciting and interesting subject. This was followed by the remarkable work of Gupta and Gupta [12] who pondered on the flow past a spongy surface. Then, Chakrabarti and Gupta [13] examined the hydromagnetic flow past a stretched surface. Andersson et al. [14] considered the flow of power-law fluid past a surface, which was linearly stretched under the influence of magnetic forces. The flow of an Oldroyd-B fluid with the impact of generation/absorption was deliberated by Hayat et al. [15]. Muhammad et al. [16] discussed the effect of thermal stratification in the ferromagnetic fluid on a stretching sheet. Ramzan and Yousaf [17] demonstrated that the elastic viscous nanofluid finished a bi-directional stretching surface in view of Newtonian heating. Hussain et al. [18] utilized the exponentially stretching sheet to scrutinize the flow of Jeffrey nanofluid with radiation effects. Some recent explorations highlighting various fluid flows past stretched surfaces with coatings can be found in references [19–22].

In today's cutting-edge engineering technology, curved stretching has a broad relevance because of its different uses in industry, for example, in transportation and electronics. Sanni et al. [23] attained a numerical solution for the viscous fluid flow on a curved stretched channel. Sajid et al. [24] inspected the ferrofluid (Fe3O4) flow on a curved sheet with effects of Joule heating and magnetic forces. Rosca and Pop [25] studied time-dependent flow along a spongy curved surface. Imtiaz et al. [26] introduced the effect of homogeneous/heterogeneous reactions in ferrofluid embedded in a stretching surface. Naveed et al. [27] calculated heat transfer and used the micropolar fluid to analyze the effects over a curved surface with thermal radiation.

A literature review has specified that copious studies are available relating to nanofluids with linear/nonlinear/exponential stretching surfaces but comparatively less research work is available highlighting curved stretched surfaces. This gets even narrower when we talk about the study of hybrid nanoliquid with entropy optimization past curved surfaces. Therefore, our task here is to discuss hybrid nanoliquid flow comprising ferromagnetic nanoparticle, i.e., nickel–zinc ferrite (NiZnFe2O4), and the base fluid, i.e., ethylene glycol (C2H6O2), over a curved surface with entropy optimization coating. The whole analysis was performed with added impressions of nonlinear thermal radiation with entropy optimization coatings. The analysis was supported by the convective heat and mass flux boundary conditions. Numerical solution of the envisioned model was obtained by utilizing bvp4c from MATLAB. The traits of the sundry parameters on involved distributions were thoroughly discussed keeping their physical justification in mind.


2.2.1. Powders

Biological-derived hydroxyapatite (BHA) powders were obtained from the cortical part of bovine femoral bones, according to the protocol described in [29]. Concisely, heads of femoral bones were cut-off and shafts were further processed. The bone marrows were then extracted and the unwanted soft tissue residues or macroscopic adhering impurities and substances were gently removed from shafts, which were sliced, cleaned and washed with distilled water and deproteinized for 14 days in an alkali media of 1% sodium hypochlorite. The elimination of any potential hazardous contaminants was achieved by a calcination process (at 850 ◦C, for 4 h, in air) of the resulting dry bone fragments [36,37]. Prior to undergoing ball-milling to fine powders, the calcined bone specimens were crushed with a mortar and pestle. It should be emphasized that, for the fabrication of BHA powders, European Union (EU) regulations [38] and ISO 22442-1 [39] were followed. Batches of BHA fine powders were admixed with 1 wt.% of Li-C and Li-P (Sigma-Aldrich GmbH, St. Louis, MO, USA).

### 2.2.2. Target Preparation

The as-obtained Li-C and Li-P mixed powders were pressed at ~6 MPa in a 20 mm diameter mold. The resulting pellets were thermally treated in air, using an oven, for 4 h, at 700 ◦C. A heating rate of 20 ◦C/min and a cooling ramp of 5 ◦C/min were applied. Following this protocol, the fabrication of hard and compact targets was carried out.

### 2.2.3. Coating Fabrication

PLD experiments were conducted inside a stainless-steel deposition chamber, in an ambient water vapor pressure of 50 Pa. The target-to-substrate separation distance was of 5 cm. Coatings were synthesized using a KrF\* excimer laser source (COMPexPro 205, Coherent, Santa Clara, CA, USA, λ = 248 nm, τFWHM ≤ 25 ns). The incident laser fluence was set at 3.5 J/cm2 (with a corresponding pulse energy of 360 mJ). The laser beam was incident at 45◦ on the target surface. For the growth of one film, 15000 consecutive laser pulses were applied. During the multi-pulse laser irradiation, the target

was continuously rotated with 0.3 Hz and translated along two orthogonal axes, to avoid piercing and to obtain unidirectional plasma.

Prior to introduction into the deposition chamber, all substrates were successively cleaned following a three-step protocol [34]. In addition, to avoid any possible micro-contamination, targets were submitted to a "cleaning" process with 1000 laser pulses. To collect the flux of expulsed micro-impurities, a shutter was interposed between the target and the 3D metallic substrates.

To ensure the complete deposition of the 3D Ti implants, a rotating stainless-steel support flange and four clamping pins [40] were used. During depositions, the substrates' temperature was kept constant at (500 ± 5%) ◦C, using a heater connected to a PID-EXCEL temperature controller (EXCEL Instruments, Mumbai, India). A heating rate of 25 ◦C/min and a cooling ramp of 10 ◦C/min were applied.

### 2.2.4. Thermal Treatments

Both the temperature used for heating the substrates during experiments and post-deposition thermal treatments, were demonstrated to have an important influence on the coatings' characteristics and, in particular, on their composition and stoichiometry. Therefore, all synthesized structures were submitted to post-deposition thermal-treatments in water-vapors enriched atmosphere. A removable flange, positioned on the heater so as to ensure an optimal contact with the substrates, was used.

### *2.3. Animals and Surgical Experimental Protocol*

The animal spectrum generally used to test CaP coatings synthesized by the PLD technique is limited to rats, mini-pigs, dogs, goats, sheep and rabbits [17]. Both the advantages and disadvantages of using different animal models have been widely discussed in the study reported by Lu et al. [41]. It should be emphasized that, the rabbit represents one of the most used animals for medical studies [42], due to its size and ease of handling. Moreover, its skeleton reaches maturity in a rather short period of time (approximatively six months) [43].

For the in vivo experiments reported in this study, that took place in the biobase of the University of Medicine and Pharmacy (UMF), Craiova, Romania, a total of 26 skeletally-matured New Zealand White rabbits, aged six months and weighing between 3 and 3.5 kg were used. One should note that, animals were weighed at the beginning of the study and before sacrifice, and no significant weight changes were observed. All animals were free of disease. They were housed in individual plastic cages, in a climate-controlled environment at 22 ◦C, 45% humidity, and 12-h alternating light–dark cycles. During the experimental period, free access to a standard laboratory diet and tap water were provided. All animals were acclimatized for 10 days before use in this study.

Before surgery, the control and functionalized 3D Ti implants were sterilized by autoclaving (at 120 ◦C, for 1 h).

The 26 rabbits were randomly assigned to two groups (*n* = 13 each). The 3D metallic implants were introduced into the femoral condyles [44] (two implants in each rabbit), using the following sequence: at the level of the right femur, the 3D Ti implants functionalized with Li-C and/or Li-P coatings, and at the level of the left femur, the simple Ti implants (controls).

Antibiotic prophylaxis was performed pre-operatively, by intramuscular administration of a cephalosporin (sulfate diluted in physiological serum, at a dose of 40 mg/kg body). The antibiotic treatment continued for up to seven days (two doses/day).

All surgeries were performed under general anesthesia, following a three-step procedure: (i) sedation by subcutaneous administration of fentanyl (0.1 mL/Kg) and midazolam (2 mg/Kg), (ii) maintenance (during the surgical act) of the anesthesia by administration of diluted fentanyl with physiological serum (1 mL fentanyl to 9 mL physiological serum), and (iii) injection at the incision site (under sterile conditions), of 1% xylin (5 mL), as an adjuvant.

At the beginning of the surgical procedure, the incision site was shaved and washed well with water and soap, and disinfected with betadine solution, followed by covering the animal with sterile overlays. An incision of ~3 cm was performed on the lateral face of the distal femoral epiphysis. This interested the epidermis, the dermis, and the facial layers. The muscle present at this level was dissociated and the periosteum was incised longitudinally, exposing the bony lateral face of the distal femoral epiphysis.

Using a dental burr (Stryker Core Reamer), with adjustable rotational drill speed, under continuous saline irrigation, to which burr drills (Osstem surgical kit for dental implants, model TS III SA) with progressive diameter were attached, an opening hole was drilled through the cortical bone. Later, this hole was enlarged progressively (to avoid a possible cortical fracture), to a diameter slightly inferior to the implant's dimensions. Into the bone socket thus created, the 3D metallic implant was inserted in a press-fit manner, ensuring both complete coverage of its surface and stability. The implant was then carefully covered and protected by soft tissues and the muscle fascia and epidermis were closed with sutures (Figure 2).

**Figure 2.** Surgical steps for the correct positioning of the 3D Ti implant: (**a**) exposure of lateral side of the distal femur, with visualization of the cortical bone; (**b**) opening hole drilled through the cortical bone using a dental burr; (**c**) insertion of the implant into the bone socket in a press-fit manner; (**d**) cover and protection of the implant by soft tissues and closing of the wound.

Immediate postoperative radiological examinations by computed tomography (CT) were performed both to confirm the correct position of the implants and to rule out any bone fractures or other possible surgical complications.

After the surgery, all animals were allowed to move freely in their cages, being monitored for general health conditions, on a daily basis. The operative wound was carefully checked and bandaged until complete healing. At the end of the implantation procedures, all rabbits were permitted free access to normal pellet food and water. No post-operative complications were reported.

The surgical protocol used for introducing the 3D metallic implants into the rabbits' femoral condyles was approved by the "Committee of Ethics and Academic and Scientific Deontology" at the UMF in Craiova, Romania (document no. 135/20.12.2019), and the entire experimental process and the surgical technique complied with regulations and precautions of the EU Council Directive of 22 September 2010, regarding the care and use of laboratory animals for scientific purposes (2010/63/EU).

### *2.4. Characterization of Control and Functionalized Three-Dimensional (3D) Ti Implants*


**4. Effect of different protein sources on animal performance**

Several researches have been carried out in order to compare the nutritional characteristics of soybean solvent extract and legume grains, as faba bean, peas and lupine, for ruminant feeding.

Di Francia et al. (2007) evaluated the effect of partial replacement of soybean cake with extruded peas in the diet of lactating buffalo cows on milk yield and quality over the first 100 days of lactation. Their results showed that peas could represent an attractive GMO free

Protein Sources in Ruminant Nutrition 205

No significant differences between the groups were found for carcass measurements. In each case the carcass measurements of this trial ranged into the interval indicated by Keane

Protein source influenced neither body and carcasses conformations nor dressing out with the exception of the incidence of long bones showed a significant difference (6.2 vs 6.7 for

The first quality meat cuts were acceptable in both groups (58.1 and 57.8%, for faba and soybean, respectively). Concerning the comparison between the two protein sources, only the incidence of long bones showed a significant difference (6.2 vs 6.7 for faba and soybean, respectively; P<0.05). It is important to underline that the sample cut measurements were contradictory and conflicted with the data obtained from total carcass dissection. The results of the sample cut dissection indicated a significant (P<0.05) difference between groups faba and soybean in meat incidence (69.4 vs 66.9, for faba and soybean, respectively); while no differences were found between groups at carcass dissection. Moreover, sample cut of soybean group showed in the meantime the smallest meat incidence and the highest LT area

The animals fed faba bean showed significantly higher water losses, measured with the compression method (WHC 7.6 vs. 5.7% for faba and soybean group, respectively; P<0.01). Our grilling loss data (31.7 vs. 28.3 % for faba and soybean group, respectively; P>0.01) were higher than those reported by Sami *et al.* (2004) in Simmental young bulls but they were in agreement with those reported by Pen *et al.* (2005) cooking ST samples of Holstein steers in an oven, on the contrary our drip loss data resulted higher than that reported by these

The chemical composition of LT was not statistically different between groups. Meat from both groups showed a very low fat content (<3%) and higher protein concentrations than the Holstein steers (Pen *et al.*, 2005), confirming the high quality of the Marchigiana meat. Hydroxyproline (60.0 vs. 62.6 mg 100g-1 of meat, in group faba and soybean, respectively) contents were not influenced by protein sources. Regarding the differences registered for this parameter among the texted muscles (LT: *Longissimus thoracis*; ST: *Semitendinosus*; IP: *Iliopsoas* plus *Psoas minor*) the IP samples, which correspond to the tenderloin, showed in all the groups significantly (P<0.01) lower hydroxyproline concentrations than the other two muscles; also between the LT and ST muscles the differences were statistically significant

Cholesterol content (56.3 vs.55.1 mg 100g-1 of meat, in group faba and soybean, respectively) was not influenced by protein sources according to the observations of Cutrignelli (2000) on

Considering the differences among the muscles, cholesterol values were significantly (P<0.05) lower for IP. Cifuni *et al.* (2004) found no differences in cholesterol contents among muscles, while Rusman *et al*. (2003) found significant differences. This contradiction is probably due to the different muscles analysed in each experiment. As theorised by Wheeler *et al.* (1987) the cholesterol content may be affected by the different physiological function of the muscles. In both groups, and especially for LT and ST muscles, the cholesterol contents were slightly higher than the value (less than 50 mg100g-1 of muscle) indicated by the Protected Geographical Indication (PGI) of the "Vitellone Bianco dell'Appennino Centrale" (Council Regulation EEC No 2081/92; Floroni, 2002). Nevertheless, our results are very close

Podolian young bulls and by Poli *et al.* (1996) on Chianina young bulls.

No differences were found in dressing out, organs and tare incidence on net weight.

(2003) for European/North American breeds.

(88.4 vs. 84 for soybean and faba group, respectively).

faba and soybean, respectively; P<0.05).

(P<0.05) being lower for the former.

authors.

protein feed when approaching the problem of the choice of a protein source alternative to soybean in diet formulation for buffalo cows raised in organic farms.

On the other hand Morbidini et al (2005) investigating on the effect of two different fattening diets with different protein sources - soybean meal and flaked faba bean - on slaughtering performance and carcass quality in light Apennine and Italian Merino lambs, observed that the use of faba beans lightly depressed growth performance (Morbidini et al., 2004) and slaughtering weight, even if did not affect the carcass quality.

In order to make a contribution on this issue, our research group conducted a trial on the effect of protein source on growth performance and meat quality of Marchigiana young bulls (Cutrignelli et al 2008 a, b).

The trial was carried out on a farm situated at 700 m a.s.l. in Campania Region (Southern Italy), where 12 weaned young bulls (129 d of age) were equally divided into two groups. Each animal was placed in individual box up to the slaughtering weight (620 kg).

The groups were fed diets with the same protein and energy concentrations and the same forage/concentrate ratios (F/C), but differing in protein source: faba bean (*Vicia faba minor* L.) vs soybean meal (*Soja hispida*).

All the animals were regularly weighed until the body weight (BW) of 620 kg fixed in advance as slaughter weight, was reached. All animals were slaughtered in an authorized slaughterhouse according to EU legislation (EU Regulation EC No 882/2004).

Live animals and carcasses were weighed and measured according to ASPA (1991). After 9 days of refrigeration at 4 ± 1 °C, dissection of the carcasses was carried out.

Samples of *Longissimus thoracis* (LT), *Semitendinosus* (ST), *Iliopsoas* plus *Psoas minor* (IP) muscles, perirenal (PF) and subcutaneous (SF) adipose tissues were collected and rapidly transported, upon refrigeration temperature, to the laboratories for the chemical analysis in order to evaluate the rheological (water holding capacity) and nutritional (chemical composition fatty acid profile, cholesterol and hydrossiproline contents) characteristics of meat.

The protein source did not affect any *infra vitam* parameters except the body weight (BW) at 180 d of age ( 173 vs. 186 kg, for group FB and SB, respectively; P<0.01).

The difference was probably due to the higher non-protein nitrogen (NPN) concentration of the faba bean than the soybean meal (about 12 vs 1.3% of crude protein, respectively). Indeed, in the months immediately following the weaning it should be preferable to administer diets with higher rumen undegradable protein content because at this age the rumen is not yet perfectly functional and microbial protein synthesis is less efficient. Furthermore, for the same reason, the animals in this period are probably unable to neutralize possible anti-nutritional factors of faba beans.

As respects the influence of the replacement of soybean meal solvent extract with legumes seeds, the literature results are contrasting. Moss et al. (1997) found no significant effects on weight gain and feed intake when soybean meal was replaced on an iso-protein basis by lupin seeds in diets for growing bulls (BW from 182 to 243 kg); similar results are reported by Kwak and Kim (2001) on Korean native bulls (BW from 247 to 427 kg) utilising two different concentrations (15 and 30%) of flaked lupin. Instead, according to our results, Murphy and McNiven (1994) found significantly higher weight gain in growing steers (BW from 235.2 to 343.7 kg) fed soybean meal vs raw or roasted lupin although the differences were not significant in the finishing phase (final BW 503.4 kg).

protein feed when approaching the problem of the choice of a protein source alternative to

On the other hand Morbidini et al (2005) investigating on the effect of two different fattening diets with different protein sources - soybean meal and flaked faba bean - on slaughtering performance and carcass quality in light Apennine and Italian Merino lambs, observed that the use of faba beans lightly depressed growth performance (Morbidini et al., 2004) and

In order to make a contribution on this issue, our research group conducted a trial on the effect of protein source on growth performance and meat quality of Marchigiana young

The trial was carried out on a farm situated at 700 m a.s.l. in Campania Region (Southern Italy), where 12 weaned young bulls (129 d of age) were equally divided into two groups.

The groups were fed diets with the same protein and energy concentrations and the same forage/concentrate ratios (F/C), but differing in protein source: faba bean (*Vicia faba minor*

All the animals were regularly weighed until the body weight (BW) of 620 kg fixed in advance as slaughter weight, was reached. All animals were slaughtered in an authorized

Live animals and carcasses were weighed and measured according to ASPA (1991). After 9

Samples of *Longissimus thoracis* (LT), *Semitendinosus* (ST), *Iliopsoas* plus *Psoas minor* (IP) muscles, perirenal (PF) and subcutaneous (SF) adipose tissues were collected and rapidly transported, upon refrigeration temperature, to the laboratories for the chemical analysis in order to evaluate the rheological (water holding capacity) and nutritional (chemical composition fatty acid profile, cholesterol and hydrossiproline contents) characteristics of

The protein source did not affect any *infra vitam* parameters except the body weight (BW) at

The difference was probably due to the higher non-protein nitrogen (NPN) concentration of the faba bean than the soybean meal (about 12 vs 1.3% of crude protein, respectively). Indeed, in the months immediately following the weaning it should be preferable to administer diets with higher rumen undegradable protein content because at this age the rumen is not yet perfectly functional and microbial protein synthesis is less efficient. Furthermore, for the same reason, the animals in this period are probably unable to

As respects the influence of the replacement of soybean meal solvent extract with legumes seeds, the literature results are contrasting. Moss et al. (1997) found no significant effects on weight gain and feed intake when soybean meal was replaced on an iso-protein basis by lupin seeds in diets for growing bulls (BW from 182 to 243 kg); similar results are reported by Kwak and Kim (2001) on Korean native bulls (BW from 247 to 427 kg) utilising two different concentrations (15 and 30%) of flaked lupin. Instead, according to our results, Murphy and McNiven (1994) found significantly higher weight gain in growing steers (BW from 235.2 to 343.7 kg) fed soybean meal vs raw or roasted lupin although the differences

Each animal was placed in individual box up to the slaughtering weight (620 kg).

slaughterhouse according to EU legislation (EU Regulation EC No 882/2004).

days of refrigeration at 4 ± 1 °C, dissection of the carcasses was carried out.

180 d of age ( 173 vs. 186 kg, for group FB and SB, respectively; P<0.01).

neutralize possible anti-nutritional factors of faba beans.

were not significant in the finishing phase (final BW 503.4 kg).

soybean in diet formulation for buffalo cows raised in organic farms.

slaughtering weight, even if did not affect the carcass quality.

bulls (Cutrignelli et al 2008 a, b).

L.) vs soybean meal (*Soja hispida*).

meat.

No significant differences between the groups were found for carcass measurements. In each case the carcass measurements of this trial ranged into the interval indicated by Keane (2003) for European/North American breeds.

No differences were found in dressing out, organs and tare incidence on net weight.

Protein source influenced neither body and carcasses conformations nor dressing out with the exception of the incidence of long bones showed a significant difference (6.2 vs 6.7 for faba and soybean, respectively; P<0.05).

The first quality meat cuts were acceptable in both groups (58.1 and 57.8%, for faba and soybean, respectively). Concerning the comparison between the two protein sources, only the incidence of long bones showed a significant difference (6.2 vs 6.7 for faba and soybean, respectively; P<0.05). It is important to underline that the sample cut measurements were contradictory and conflicted with the data obtained from total carcass dissection. The results of the sample cut dissection indicated a significant (P<0.05) difference between groups faba and soybean in meat incidence (69.4 vs 66.9, for faba and soybean, respectively); while no differences were found between groups at carcass dissection. Moreover, sample cut of soybean group showed in the meantime the smallest meat incidence and the highest LT area (88.4 vs. 84 for soybean and faba group, respectively).

The animals fed faba bean showed significantly higher water losses, measured with the compression method (WHC 7.6 vs. 5.7% for faba and soybean group, respectively; P<0.01).

Our grilling loss data (31.7 vs. 28.3 % for faba and soybean group, respectively; P>0.01) were higher than those reported by Sami *et al.* (2004) in Simmental young bulls but they were in agreement with those reported by Pen *et al.* (2005) cooking ST samples of Holstein steers in an oven, on the contrary our drip loss data resulted higher than that reported by these authors.

The chemical composition of LT was not statistically different between groups. Meat from both groups showed a very low fat content (<3%) and higher protein concentrations than the Holstein steers (Pen *et al.*, 2005), confirming the high quality of the Marchigiana meat.

Hydroxyproline (60.0 vs. 62.6 mg 100g-1 of meat, in group faba and soybean, respectively) contents were not influenced by protein sources. Regarding the differences registered for this parameter among the texted muscles (LT: *Longissimus thoracis*; ST: *Semitendinosus*; IP: *Iliopsoas* plus *Psoas minor*) the IP samples, which correspond to the tenderloin, showed in all the groups significantly (P<0.01) lower hydroxyproline concentrations than the other two muscles; also between the LT and ST muscles the differences were statistically significant (P<0.05) being lower for the former.

Cholesterol content (56.3 vs.55.1 mg 100g-1 of meat, in group faba and soybean, respectively) was not influenced by protein sources according to the observations of Cutrignelli (2000) on Podolian young bulls and by Poli *et al.* (1996) on Chianina young bulls.

Considering the differences among the muscles, cholesterol values were significantly (P<0.05) lower for IP. Cifuni *et al.* (2004) found no differences in cholesterol contents among muscles, while Rusman *et al*. (2003) found significant differences. This contradiction is probably due to the different muscles analysed in each experiment. As theorised by Wheeler *et al.* (1987) the cholesterol content may be affected by the different physiological function of the muscles. In both groups, and especially for LT and ST muscles, the cholesterol contents were slightly higher than the value (less than 50 mg100g-1 of muscle) indicated by the Protected Geographical Indication (PGI) of the "Vitellone Bianco dell'Appennino Centrale" (Council Regulation EEC No 2081/92; Floroni, 2002). Nevertheless, our results are very close

Protein Sources in Ruminant Nutrition 207

taste panel evaluation (Westerling and Hedrick, 1979) and food with high UFA, especially PUFA, is good for human health (Rusman *et al.*, 2003). The ω-6/ω-3 ratio was higher than the value (less than 3) reported by Scollan *et al.* (2006) but lower than that registered by

It has to be underlined that the fatty acid profile o the food arouse high interest in human medicine due to their influence on the functionality of the cardio-circulatory apparatus. A number of epidemiological researches put in evidence that diets with high content of saturated fatty acids (SFA) were associated with high levels of serum cholesterol (especially of low density lipoprotein, LDL) which appear important in atheroma. Successively,

2. short-chain SFA (C 10 and below) likewise do not raise blood cholesterol, so the putative atherogenic SFA are C12:0 (lauric), C14:0 (myristic) and C16:0 (palmitic). Myristic acid is the most atherogenic, with about four times the cholestrol-raising

PUFA are considered protective factors: ω-6 fatty acids show mainly anti-atherogenic activity while ω-3 fatty acids have anti-thrombogenic activity. More recently, high prominence is attributed to the role developed by the MUFA, and particularly by the oleic acid that, reducing the oxidation of the cholesterol LDL, may slow the progression of

As was pointed out above, the P/S (poliunsaturated/saturated) ratio is not suitable measure of the atherogenicity or thrombogenicity of a diet or foods. Currently they are expressed as

*Index of atherogenicity:* C12:0 + (4 x C14:0) + C16:0/ ω-3 + ω-6 + MUFA

*Index of thrombogenicity:* C14:0 + C16:0 +C18:0/(0,5 x C18:1) + (0,5 other MUFA) + 0,5(ω-6) + 3(ω-3) + (ω-3/ ω-6). The AI of the meat in this trial was particularly interesting, rather lower than the data reported by Ulbricht and Southgate (1991) for raw minced beef and than those reported by Badiani *et al.* (2002) for cooked beef (0.72 and 0.77, respectively); our data were similar to

FB SB Significance

SFA 365.1 39.1 330.6 26.9 Ns MUFA 244.3 39.5 271.7 29.4 Ns PUFA 186.6 20.5 201.9 15.7 Ns -6 174.2 19.9 188.3 12.9 Ns -3 12.39 1.5 13.57 0.8 Ns AI 0.54 0.04 0.49 0.07 Ns TI 1.46 0.12 1.20 0.09 Ns

*FB: faba bean; SB: soybean meal solvent extract;AI: atherogenic index; TI thrombogenic index.*

Table 7. Fatty acid profile of Longissimus thoracis muscle (mg 100g-1 of edible part).

Warren *et al.* (2003) for steers fed corn silage and concentrates (8.9).

1. diets high in C18:0, stearic acid, do not raise serum cholesterol;

those of Poli *et al.* (1996) on Chianina young bulls (AI: 0.58).

Ulbricht and Southgate (1991) reported that:

potential of palmitic acid.

atherosclerosis.

*ns: not significant.*

follows:

to those reported for Italian meat breeds (Poli *et al.* 1996; Cifuni *et al.,* 2004) and lower than those from other breeds (Migdal *et al.*, 2004).

Regarding the fatty acids profile in both groups and in each analysed tissues (intramuscular, perirenal and subcutaneous adipose tissues) palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids were the most widely represented fatty acids. In particular, in intramuscular fat, the sum of oleic, stearic and palmitic acids represents over 50% of total fatty acids, according to the observations of Cifuni *et al.* (2004) and Migdal *et al.* (2004).

Comparing the data of groups faba and soybean, the only significant (P<0.01) difference was for stearic acid being higher for bulls fed faba bean than for those fed soybean meal solvent extract. However this result did not significantly affect the SFA concentration, or AI and TI indexes (table 7).

Protein source did not affected the fatty acids composition of the analysed adipose tissues. This is probably due to the very low concentration of phospholipids in subcutaneous and perirenal fat tissue.

**C**omparing the fatty acid composition of the three adipose tissues the following differences were noted:

The LT fatty acid profile was similar to those reported by Raes *et al.* (2003) for *Longissimus lumborum* of Belgian Blue and Limousin beef (SFA: 338 and 506 mg 100g-1 edible portion; MUFA: 323 and 554 mg 100g-1 edible portion; PUFA: 195 and 195 mg 100g-1 edible portion; in Belgian Blue and Limousin bulls, respectively) and different from values found in Irish and Argentine beef. The latter showed significantly higher total intramuscular fatty acid content compared to the former, probably due to genetic selection.

This observation confirms that of Carnovale and Nicoli (2000) who concluded that Italian meat showed a favourable intramuscular fatty acid composition with high PUFA content. Muscle with a high percentage of unsaturated fatty acids (UFA) generally scored higher in

to those reported for Italian meat breeds (Poli *et al.* 1996; Cifuni *et al.,* 2004) and lower than

Regarding the fatty acids profile in both groups and in each analysed tissues (intramuscular, perirenal and subcutaneous adipose tissues) palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids were the most widely represented fatty acids. In particular, in intramuscular fat, the sum of oleic, stearic and palmitic acids represents over 50% of total fatty acids,

Comparing the data of groups faba and soybean, the only significant (P<0.01) difference was for stearic acid being higher for bulls fed faba bean than for those fed soybean meal solvent extract. However this result did not significantly affect the SFA concentration, or AI and TI

Protein source did not affected the fatty acids composition of the analysed adipose tissues. This is probably due to the very low concentration of phospholipids in subcutaneous and

**C**omparing the fatty acid composition of the three adipose tissues the following differences

The LT fatty acid profile was similar to those reported by Raes *et al.* (2003) for *Longissimus lumborum* of Belgian Blue and Limousin beef (SFA: 338 and 506 mg 100g-1 edible portion; MUFA: 323 and 554 mg 100g-1 edible portion; PUFA: 195 and 195 mg 100g-1 edible portion; in Belgian Blue and Limousin bulls, respectively) and different from values found in Irish and Argentine beef. The latter showed significantly higher total intramuscular fatty acid

This observation confirms that of Carnovale and Nicoli (2000) who concluded that Italian meat showed a favourable intramuscular fatty acid composition with high PUFA content. Muscle with a high percentage of unsaturated fatty acids (UFA) generally scored higher in

influenced by several factors such as adipocite size, nutrient supply, etc.;

according to the observations of Cifuni *et al.* (2004) and Migdal *et al.* (2004).

and an increased PUFA content (Scollan *et al.*, 2006);

tissues as well as to the high PUFA concentration of LT.

content compared to the former, probably due to genetic selection.

those from other breeds (Migdal *et al.*, 2004).

indexes (table 7).

perirenal fat tissue.

were noted:

tissues;

taste panel evaluation (Westerling and Hedrick, 1979) and food with high UFA, especially PUFA, is good for human health (Rusman *et al.*, 2003). The ω-6/ω-3 ratio was higher than the value (less than 3) reported by Scollan *et al.* (2006) but lower than that registered by Warren *et al.* (2003) for steers fed corn silage and concentrates (8.9).

It has to be underlined that the fatty acid profile o the food arouse high interest in human medicine due to their influence on the functionality of the cardio-circulatory apparatus. A number of epidemiological researches put in evidence that diets with high content of saturated fatty acids (SFA) were associated with high levels of serum cholesterol (especially of low density lipoprotein, LDL) which appear important in atheroma. Successively, Ulbricht and Southgate (1991) reported that:

PUFA are considered protective factors: ω-6 fatty acids show mainly anti-atherogenic activity while ω-3 fatty acids have anti-thrombogenic activity. More recently, high prominence is attributed to the role developed by the MUFA, and particularly by the oleic acid that, reducing the oxidation of the cholesterol LDL, may slow the progression of atherosclerosis.

As was pointed out above, the P/S (poliunsaturated/saturated) ratio is not suitable measure of the atherogenicity or thrombogenicity of a diet or foods. Currently they are expressed as follows:

$$\text{Index of otherwise:}\\\text{c.C12:}\\0 + (4 \ge \text{C14:} 0) + \text{C16:} 0\\\text{/ } \text{a-S} + \text{a-6} + \text{MUFA}$$

*Index of thrombogenicity:* C14:0 + C16:0 +C18:0/(0,5 x C18:1) + (0,5 other MUFA) + 0,5(ω-6) + 3(ω-3) + (ω-3/ ω-6).

The AI of the meat in this trial was particularly interesting, rather lower than the data reported by Ulbricht and Southgate (1991) for raw minced beef and than those reported by Badiani *et al.* (2002) for cooked beef (0.72 and 0.77, respectively); our data were similar to those of Poli *et al.* (1996) on Chianina young bulls (AI: 0.58).

*FB: faba bean; SB: soybean meal solvent extract;AI: atherogenic index; TI thrombogenic index. ns: not significant.*

Table 7. Fatty acid profile of Longissimus thoracis muscle (mg 100g-1 of edible part).

Protein Sources in Ruminant Nutrition 209

In addition, our *in vivo* studies concerning the *infra vitam* and *post mortem* performances of

From these results, it can be concluded that the use of grain legumes as a protein source in ruminant diets could be used as alternative to soybean meal. However, mainly in the intensive livestock system the soybean meal solvent extract represents the protein source par excellence for the high protein content (in particular undegradable fraction), the absence of anti-nutritional factors and the extremely favourable biological value of its proteins. Not insignificant, then is the economic assessment, soybean is, especially in the areas where the ruminants breeding has been particularly developed, the protein source with the

By contrast, it has to be underlined that biotech herbicide tolerant soybean continued to be the principal biotech crop in 2010, occupying 73.3 million hectares or 50% of global biotech area. Farm animals are currently fed soybean and soybean meal developed from genetic transformation. Europe is strongly dependent upon the American continent for its protein

Thus, the possible risk connected to genetically modified organisms use in animal breeding has led to the reconsideration of animal production processes with special reference to the use of alternative protein sources (e.g. faba beans, dried peas, lupine seeds, chickpeas) able to replace soybean. These legumes have agronomic importance because they improve soil fertility and reduce nitrogenous dressing, with positive effects on environmental pollution. Moreover, they need a limited initial investment for their modest requirements of chemical

A.S.P.A. (1991). Metodologie relative alla macellazione degli animali di interesse zootecnico ed alla valutazione e dissezione della loro carcasse. Ed. ISMEA, Roma. Akpinar, N., Akpinar, M.A. & Türkoglu, S. (2001). Total lipid content and fatty acid composition of the seeds of some Vicia L. species. *Food Chem.*, 74, pp. 449–453. Arieli, A., Bruckental, I., Kedar. & I., Sklan, D. (1995). *In sacco* disappearance Of starch nitrogen and fat in processed grains. *Anim. Feed sci. Techn*., 51, pp. 287-295. Bach Knudsen, K.E. (1997). Carbohydrate and lignin contents of plant materials used in

Badgley, C., Moghtader, J., Quintero, E., Zakem, E., Chappell, M.J., Avilés-Vázquez, K.,

Samulon, A. & Perfecto, I., (2007). Organic agriculture and the global food supply.

animal feeding. *Anim. Feed Sci. Technol.*, 67, pp. 319–338.

*Renew. Agric. Food Sys.,* 22, pp. 86–108.

young bulls fed either faba bean or soybean showed:

lower for the group receiving faba bean.

quality/price ratio more favourable.

requirements amounting up to 90 to 95%.

and energetic inputs and their short culture cycle.

**6. References**

biological efficiency of growth) and feed conversion indexes;

thrombogenic indexes, nor the cholesterol content were influenced;

However, the index of thrombogenicity in this trial was higher than the findings of the above-cited authors (1.27 and 1.30 for Ulbricht and Southgate, 1991 and Poli *et al.*, 1996, respectively); only the TI (1.77) reported by Badiani *et al.* (2002) was similar to our data.

These results show that the faba bean could be used as an alternative protein source to soybean meal solvent extract as it did not affect the growth rate (body weight, daily weight gain and biological efficiency of growth) or the feed conversion indexes during the whole experimental period, and offers decided agronomical, economical and healthy advantages.

Nevertheless, in the first period after weaning the faba bean reduced the growth rate, probably due to the higher concentrations of NPN and anti-nutritional factors. It might be useful in this period to use this protein source associated with other richer in rumen undegradable.

Our results contribute to show that both protein sources (soybean meal and faba bean) could be utilised in the diet for young Marchigiana bulls ensuring the high quality of the meat obtained by this breed.

Although the meat of group fed faba bean had significantly higher concentrations of stearic acid compared to the level found in soybean group, neither the atherogenic and thrombogenic indexes, nor the cholesterol content were influenced.

As regards the effect of protein source on organolepitc characteristics, our results on hydroxyproline content and water holding capacity were conflicting. While the meat of the group fed soybean meal solvent extract showed a potential low tenderness (higher level of hydroxiproline) the water holding capacity measured by compression was lower for the group receiving faba bean.

From our results it is also possible to formulate a favourable assessment of the nutritional characteristics of the of meat Marchigiana young bulls. Indeed, the cholesterol values were very close to those indicated by the PGI of the "Vitellone Bianco dell'Appennino Centrale" and lower than those found in other breeds. Moreover, the fatty acids profile of LT confirms that the meat of the Italian breed specialised in meat production has higher unsaturated fatty acids concentration and lower saturated fatty acids levels, which in turn ensures medium-low atherogenic and thrombogenic indexes.


Agricultural waste + Microorganisms → Biogas + Digestate (1)

In the fermentation process, which also works in the absence of oxygen, the production of valuable products such as alcohol, organic acids, and a mixture of gases due to microorganisms' action is observed. It is believed that the fermentation of agro-waste is difficult and time-consuming due to the presence of long-chain polymeric molecules and requires acid or enzymatic hydrolysis before fermentation to produce valuable endproducts. For instance, bio-butanol production can be achieved with the help of a bacteria called *Clostridium* spp., coupled with sugar production from various types of agro-waste. The process comprises two main steps, i.e., acidogenesis and solventogenesis, which is

referred to as acetone, butanol, and ethanol (ABE) fermentation. Hence, it is a promising technology, but it is costlier and time-consuming.


**1.2 Water as a solvent in the synthesis of heterocycles**

Water plays a dominant role as a solvent for the development of numerous heterocycles by replacing the use of hazardous organic solvents. Despite its overwhelming advantages as reaction solvent for organic synthesis, poor solubility of many organic compounds in water limits its use under standard conditions. However, when used in high temperature and pressure conditions, it undergoes a change in its properties and can act as a potent solvent for good number of organic reactions [9]. Owing to tremendous potential of water as a medium for production of heterocyclic moieties, in this chapter, we have tried to compile the 'in water' synthesis of fivemembered heterocycles.