*Article* **Antimicrobial Polyamide-Alginate Casing Incorporated with Nisin and** ε**-Polylysine Nanoparticles Combined with Plant Extract for Inactivation of Selected Bacteria in Nitrite-Free Frankfurter-Type Sausage**

**Kazem Alirezalu 1,\*, Milad Yaghoubi 2, Leila Poorsharif 2, Shadi Aminnia 3, Halil Ibrahim Kahve 4, Mirian Pateiro 5, José M. Lorenzo 5,6 and Paulo E. S. Munekata 5,\***

**Abstract:** The effects of combining a polyamide-alginate casing incorporated with nisin (100 ppm and 200 ppm) and ε-polylysine (500 ppm and 1000 ppm) nanoparticles and a mixed plant extract as ingredient in sausage formulation (500 ppm; composed of olive leaves (OLE), green tea (GTE) and stinging nettle extracts (SNE) in equal rates) were studied to improve the shelf life and safety of frankfurter-type sausage. The film characteristics and microbiological properties of sausage samples were evaluated. Sausage samples were packaged in polyethylene bags (vacuum condition) and analysed during 45 days of storage at 4 ◦C. Control sausages were also treated with 120 ppm sodium nitrite. Polyamide-alginate films containing 100 ppm nisin and 500 ε-PL nanoparticles had the highest ultimate tensile strength compared to other films. However, 100 ppm nisin and 500 ε-PL nanoparticles decreased water vapour permeability of films. The results also revealed that nisin nanoparticles had significantly (*p* < 0.05) low inhibitory effects against *Escherichia coli*, *Staphylococcus aureus*, molds and yeasts and total viable counts compared to control and ε-PL nanoparticles. Furthermore, 1000 ppm ε-PL nanoparticles displayed the highest antimicrobial activity. Based on the obtained results, the films containing ε-PL nanoparticle could be considered as a promising packaging for frankfurter-type sausages.

**Keywords:** polyamide-alginate film; nanoparticle; nisin; ε-polylysine; frankfurter; plant extract; active packaging

#### **1. Introduction**

Frankfurter-type sausages are a kind of emulsified meat products, which because of their high techno-functional components (bioavailable vitamins (B), essential amino acids, fatty acids, zinc, and heme-iron), ready to eat (RTE) product status, flavour acceptance and low cost, are widely consumed [1–3]. However, contamination in meat products like sausages, especially by foodborne bacteria, is the main concern of meat producers [4]. Moreover, frankfurter-type sausages with high fat content are sensitive to oxidation which leads to a reduction in quality (flavour and texture) during storage [5,6], hence researchers

**Citation:** Alirezalu, K.; Yaghoubi, M.; Poorsharif, L.; Aminnia, S.; Kahve, H.I.; Pateiro, M.; Lorenzo, J.M.; Munekata, P.E.S. Antimicrobial Polyamide-Alginate Casing Incorporated with Nisin and ε-Polylysine Nanoparticles Combined with Plant Extract for Inactivation of Selected Bacteria in Nitrite-Free Frankfurter-Type Sausage. *Foods* **2021**, *10*, 1003. https://doi.org/10.3390/foods 10051003

Academic Editor: Salam A. Ibrahim

Received: 24 March 2021 Accepted: 1 May 2021 Published: 4 May 2021

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**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/).

endeavour to reduce contamination in sausages using natural antimicrobials and antioxidants [7–9].

Increasing the safety of RTE products like sausages could be achieved using antimicrobial packaging [10–14]. Using natural antimicrobial compounds in packaging structures displays higher efficacy in comparison to direct use in the food matrix as an ingredient. The higher efficacy of active antimicrobial packaging may be attributed to the exposure of the food surface (where the risk of contamination is high) to antimicrobial compounds [15–17].

Polysaccharides like alginate due to their special colloidal properties such as filmforming, thickening, emulsion stabilizing agent, and gel producing are widely used as a biopolymer film or coating compounds [18]. For instance, sodium alginate has received great consideration due to promising properties in combination with calcium ions as delivery systems for active compounds [19–23]. In this regard, the U.S. Food and Drugs Administration (FDA) has indicated that alginate polymer has the Generally Recognized As Safe (GRAS) status for food use [24]. Furthermore, Surendhiran et al. [25] revealed that phlorotannin encapsulated in a alginate/poly(ethylene oxide) composite film inactivated *Salmonella* spp. and increased the shelf life of chicken meat.

Nisin is a nontoxic and stable bacteriocin produced from *Lactococcus lactis* with authorized use in food [26]. Nisin has strong antimicrobial properties against several spoilage bacteria in meat and meat products such as *Clostridia* and *Bacilli* spores [27,28]. In this regard, Churklam et al. [29] evaluated effects of carvacrol in combination with nisin on sliced Bologna sausage and they noticed that nisin and carvacrol inhibited microbial growth compared to control samples. Furthermore, other authors have reported similar results for nisin in ready-to-eat Yao meat products [30], pork loin [31], frankfurter-type sausage [6] and fresh sausage [32].

ε-Polylysine (ε-PL) is one of the most well-known natural components with high antimicrobial properties against a wide spectrum of bacteria like Gram-positive, Gramnegative bacteria (*Clostridium perfringens, Staphylococcus aureus* and *E. coli*), and yeast and molds [33]. ε-PL has high thermo-stability and is widely utilized in meat industry products like chilled beef [34], and frankfurter type sausage [35] as preservative. Furthermore, antimicrobial efficacy of ε-PL could be increased in combination with plant extracts [35]. Natural plant extracts like olive leaves (OLE), green tea (GTE) and stinging nettle (SNE) extracts are a good source of phenolic compounds with strong antimicrobial and antioxidant properties [36–38]. In OLE, the major polyphenols are oleuropein, oleuropein, hydroxytyrosol, tyrosol, luteolin-7-*O*-glucoside, apigenin-7-*O*-glucoside, *p*-coumaric acid, ferulic acid [39]. In the case of GTE the main polyphenols are catechin and its derivatives are epicatechin gallate, epigallocatechin gallate, and epigallocatechin [40]. The phenolic composition of SNE is comprised of flavonoids (kaempferol, rutin, isorhamnetin, and quercetin,) and phenolic acids (*p*-coumaric acid and ferulic acid) [41], making these plant materials rich sources of bioactive and nutrients compounds with potential ability as nitrite substitutes in frankfurter-type sausages [36].

The antimicrobial effect of combined ε-polylysine and nisin with natural antioixdants in frankfurter-type sausage has been reported [6]. To the best of our knowledge, there are no previous studies on the effect of active packaging containing ε-polylysine nanoparticles (ε-PLN) and nisin nanoparticles (NN) on the quality properties and stability of frankfurtertype sausage during storage. The aim of present study was thus to evaluate the effect of active polyamide-alginate films containing nisin and ε-PL nanoparticles in combination with plant extracts on quality and shelf life of frankfurter-type sausages.

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

#### *2.1. Materials*

All chemical components and microbial media were of analytical grade (purity >99%) and purchased from Merck (Darmstadt, Germany). Food-grade nisin (Nisaplin, 5000 IU/mL, and ε-polylysine powder (5000 IU/mL,) were purchased from Danisco (Copenhagen, Denmark) and FoodChemand (Shanghai, China), respectively. The high

molecular mass chitosan powder (molecular weight 3.1 × 105 g mol−1; 95% deacetylation degree) was also purchased from Sigma-Aldrich (Saint Louis, MO, USA). The polyamide film was acquired from Besharat Company (Tabriz, Iran).

#### *2.2. Preparation of Plant Extract*

The olive leaves (OLE), green tea (GTE) and stinging nettle (SNE) extracts were obtained following the procedure described by Ebrahimzadeh et al. [42] with some modifications. Plant leaves firstly dried in an oven (40 ◦C) for 48 h and sifted through sieves with 14-inch mesh. Then, 50 g of dried powder and 500 mL of ethanol solution (95%) in Erlenmeyer flask were mixed by magnetic stirrer at room temperature for 48 h. The ethanol in mixture was evaporated at 40 ◦C in a rotary evaporator after filtering the mixture through a Whatman No 1 filter paper. Equal amounts of GTE, SNE and OLE were prepared for further use in frankfurter-sausage samples.

#### *2.3. Preparation of ε-Polylysine Nanoparticle (ε-PLN) and Nisin Nanoparticle (NN)*

The stock solution of nisin and ε-PL were prepared according to method described by Alirezalu et al. [35] as follows: 2 g from each of these compounds were separately solubilized in 2% glacial acetic acid solution (200 mL) at 60 ◦C and filtered through 0.45 μm membrane filter (for sterilization) (Minisart NML, Sartorius, New York, NY, USA). The nanoparticles of nisin and ε-PL were produced according to the method described by Das et al. [43], and Bernela et al. [44] with some modifications. Calcium chloride and ε-PL solution (1:20, *v*/*v*) were mixed together. Then, this solution was mixed with 58.75 mL of sodium alginate (0.63 mg/mL), 12.5 mL of chitosan solution, and 6.25 mL of Pluronic F-68 (1 mg/mL). The final mixture was shaken slowly for 3.5 h (at room temperature). The nanoparticles of ε-PL were obtained after centrifugation (15,000× *g*) (at 4 ◦C for 0.5 h) and freeze-drying process. The similar technique was utilized for preparation of nisin nanoparticles.

#### *2.4. Preparation of Active Antimicrobial Film*

The polyamide-alginate films were produced according to the procedures described in [24,45]. Mechanical stirring was used to dissolve 3 g of sodium alginate in 200 mL of sterile deionised water for 30 min (70 ◦C). After that, for improving films characteristics (increase flexibility and decrease brittleness), glycerol (0.44 g/g of alginate) as a plasticizer was added. Mechanical stirring was utilized for dissolving glucono δ-lactone (5.4 g/g calcium carbonate) with calcium carbonate (0.03 g/g alginate) in 50 mL of distilled water. Then, for calcium alginate films production, the solution was dispersed at 150 mL sodium alginate solution. The ε-PLN (500 and 1000 ppm) and NN (100 and 200 ppm) were added to the mixture and homogenized for 3 min (13,500 rpm at 25 ◦C) and for integrate absorption of water and gelation of alginate, the attained solution was mixed slowly for 12 h by using mechanical stirring. After that, Petri dishes (10 cm in diagonal) were filled with 10 mL of the solution and dried in an oven (12 h at 45 ◦C). The Petri dishes were held in desiccators before peeling the films. Finally, polyamide and calcium alginate films were attached together to polyamide-alginate films.

#### *2.5. Mechanical Properties*

Mechanical properties of calcium alginate films including tensile strength (TS) and elongation at break (%E) were analysed following the ASTM procedure D882-91 [46]. The films were cut into 6 × 0.5 cm and were conditioned by saturated solution of calcium nitrite (RH = 55%) inside a desiccator for 24 h at room temperature. Initial grip separation with 50 mm and cross-head speed with 2 mm/min was used at this study with five replicates for mechanical analysis from each film samples.

#### *2.6. Water Vapor Permeability (WVP)*

The ASTM procedure E96-95 was utilized for gravimetrically evaluation of calcium alginate film WVP [46]. The calcium alginate films were sealed onto cups (2 cm diagonal and 10 cm height) with 3 g calcium sulfate and before hold inside desiccator containing saturated solution of potassium sulfate (RH = 97%) the cups were weighted. Finally, to attain a 97% RH gradient on the films the desiccator was placed inside the oven (25 ◦C). The cups were weighted twice a day (12 h to 12 h) for six days and the results were analysed by Fick and Henry's laws as follows:

$$WVP(\text{g mm}/\text{m}2 \text{ day kPa}) = \frac{\text{The rate of water vapor transmission} \times \text{film thickness (mm)}}{\text{differential vapor pressure of water through the film}} \tag{1}$$

#### *2.7. Preparation of Frankfurters*

Each repetition of frankfurter processing was carried out with beef from different animals. The same ingredients and formulation were used in the three batches during three successive days (3 treatments × 4 time periods × 3 repetitions × 3 runs). A local meat processing factory was utilized for sausage production. Frankfurter-type sausage formulation (g/kg) was comprised of 0.4 sodium ascorbate, 15 salt, 81.5 starch and other dry materials, 120 soybean oil, 3.5 polyphosphate sodium, 20 seasoning, 210 ice/water, 0.5 mixed plant extract, and 550 of beef meat. Beef meat was cut into cubes with 3 mm size and homogenized with half of the ice/mixed extract (500 ppm), salt (NaCl) and sodium polyphosphate in a cutter (EX3000 RS, Kilia, Schönkirchen, Germany) at 10 ◦C for 12 min. After that, other ingredients including seasoning, starch, and sodium ascorbate were added slowly into the mix and homogenized for 1 min. Finally, half of ice/mixed extract (final mixed extract concentration of 500 ppm) and microbial suspensions (103 CFU/g), along with remaining components were added and mixed for about 120 s. The sausages were stuffed mechanically (VF50, Handtmann, Biberach, Germany) into antimicrobial polyamide-alginate films before steam cooking (1.5 h at 80–85 ◦C).

Sausages were quickly chilled with a cold-water shower, packaged in vacuumed condition in polyethylene bags and stored at 4 ◦C. The following five treatments were prepared: control with polyamide-alginate films without nanomaterials; samples stuffed in the polyamide-alginate films incorporated with 100 and 200 ppm NN, and samples stuffed in the polyamide-alginate films incorporated with 500 and 1000 ppm ε-PLN. Film characteristics and microbial counts were analysed at 0, 15, 30, and 45 days of refrigerated (4 ± 1 ◦C) storage. A schematic illustration to show the overall workflow is presented in Figure 1.

**Figure 1.** Schematic diagram for frankfurter-type sausage production.

#### *2.8. Microbiological Properties*

Microbiological properties of sausage packaged in polyamide alginate films were analysed as follows: 225 mL of 0.1% (*w*/*v*) peptone water and 25 g of samples were homogenized using sterile lab-blender (Paddle Lab Blender, Neutec, Farmingdale, NY, USA) for 3 min. 0.1% of sterile peptone water was also used for serial dilution production. Brilliant Green agar (BGA, Merck, Darmstadt, Germany), Plate Count agar (PCA, Merck) and Sulfite Polymyxin Sulfadizine (SPS) agar (Merck) were used for enumeration of *E. coli*, total viable count, and *Clostridium perfringens*, respectively, by pour-plate technique. Incubation time and temperature for *E. coli*, total viable count, and *Clostridium perfringens* were 24–47 h at 37 ◦C, 48–72 h at 30 ◦C and 24 h at 37 ◦C, respectively. *Staphylococcus aureus* and yeast and molds were enumerated on Baird Parker agar (BPA, Merck) and Dichloran Rose-Bengal Chloramphenicol (DRBC) agar (Merck) following incubation for 48 h at 30 ◦C and 5 days at 25 ◦C, respectively. The microbiological results were reported as Log10 CFU/g of sausage samples.

#### *2.9. Statistical Analysis*

The experimental data resulted from 3 treatments × 4 time periods × 3 repetitions × 3 runs were analysed using the statistical software SAS (v.9, SAS Institute Inc., Cary, NC, USA). Normal distribution and variance homogeneity had been previously tested (Shapiro-Wilk). Random block design was utilized for evaluation of microbiological data, considering a mixed linear model, including replication as a random effect, and different treatments and storage period as fixed impacts. One-way ANOVA was also utilized for mechanical properties and WVP, and Tukey's test for means comparison (statistical significance at *p* < 0.05 value) and results were expressed as mean values ± standard error in all figures and tables.

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

#### *3.1. Mechanical Properties*

The mechanical properties of calcium alginate films including elongation at break (E%) and tensile strength (TS) are shown at Table 1. The results showed that tensile strength of calcium alginate films ranged between 63–67 MPa. Added NN and ε-PLN affected significantly (*p* < 0.05) the tensile strength of films. Pranoto et al. [47] showed that tensile strength of calcium alginate films could decrease by adding garlic oil which may be caused by its hydrophobic properties. In our experiment, tensile strength in calcium alginate films incorporated with 500 ppm ε-PLN was higher than other films (Table 1). Conversely, ε-PLN (500 ppm ε-PLN and 1000 ppm ε-PLN) could significantly (*p* < 0.05) decrease the tensile strength of the films. This fact could be due to the high ε-PLN content, which can reduce tensile strength of the films. Benavides et al. [48] also reported similar results.

σ: Ultimate tensile strength (CV: 0.54); E: Elongation at break (CV: 1.71). a–c In each row with different letters differ significantly, (*p* < 0.05).

The results also showed that elongation at break (E%) in control films were higher than other films. Incorporating nanoparticles decreased the flexibility of the films, therefore, elongation at break were also decreased in the films. Moreover, link between alginate, calcium and chitosan decreased the flexibility and E% of the films. The results of this study are in agreement with those reported by Guiga et al. [49] in polyamide films. The authors indicated that added nisin in polyethylene and polyamide films could decrease E% of the films from 271% to 130%. The appearance of the active films used are presented in Figure 2.

**Figure 2.** Calcium alginate films and polyamide-alginate film incorporated with NN and ε-PLN. (**A**) Calcium alginate film, (**B**) Calcium alginate film + 100 ppm NN, (**C**) Calcium alginate film + 200 ppm NN, (**D**) Calcium alginate film + 500 ppm ε-PLN, (**E**) Calcium alginate film + 1000 ppm ε-PLN, (**F**) Polyamide-alginate film.

#### *3.2. Water Vapor Permeability (WVP)*

The effects of nanoparticles in WVP of calcium alginate films are shown in Figure 3. According to Barzegaran et al. [50], calcium alginate films have lower water vapour permeability (6.16 × 10–7 g/m.s.Pa) in comparison to other polymers. Therefore, for production of films with antimicrobial activities with low water vapour permeability, calcium alginate films were produced. The results showed that adding 100 ppm of NN in calcium alginate films decreased WVP of the films, whereas incorporating 200 ppm of NN increased the WVP of the films.

**Figure 3.** Water vapour permeability of calcium alginate films incorporated with nisin and ε-PL nanoparticles. a–d Mean values among films not followed by a common letter differ significantly (*p* < 0.05).

Sodium alginate films can be considered as an edible casing because of its hydrophilic properties (low resistant against moisture) and mechanical stability [51]. Therefore, favouring the gel consistency in films (by adding calcium) can improve water vapour permeability of the films [52].

WVP of the alginate films were decreased by adding 500 ppm ε-PLN. The interaction between amines (chitosan) and carboxyl components (alginate) may be the main reason for the lower WVP in films. Intermolecular gaps and porous microstructure of films matrix significantly affect the permeability of the films. Turhan and ¸Sahbaz [53] showed that

plasticizers can increase water vapour permeability of the films by increasing intermolecular gaps. As shown in Figure 3, WVP of films incorporated with 1000 ppm ε-PLN and 200 ppm NN were significantly increased whereas the WVP of films with 500 ppm ε-PLN and 100 ppm NN were decreased. These results may be caused by increasing and decreasing intermolecular gaps, respectively.

#### *3.3. Microbiological Properties*

Total viable count in all sausage samples (*p* < 0.05) increased during storage. The polyamide-alginate films incorporated with 1000 ppm ε-PLN and 200 ppm NN showed (*p* < 0.05) higher inhibitory effects against TVC. Furthermore, polyamide-alginate films incorporated with ε-PLN presented higher antimicrobial effects compared to polyamidealginate films with NN. The antimicrobial effects of ε-PL against wide spectrum of microorganisms (Gram-positive, Gram-negative, and fungus) compared to nisin may be the main reason that explains the higher antimicrobial properties of ε-PLN.

TVC of frankfurter sausages packaged in polyamide-alginate films with 500 ppm ε-PLN and 100 ppm NN ranged between 5.54 and 5.87 Log CFU/g at the end of storage period (Figure 4). It is worth mentioning that the borderline for microbiological acceptability in meat products (especially due to odour changes) is around 6 Log CFU/g [54,55]. Conversely, TVC in sausage samples packaged in films with 1000 ppm ε-PLN and 200 ppm NN reached 4.22 Log CFU/g and 4.53 Log CFU/g, respectively. Therefore, the results showed that polyamide-alginate films incorporated with 500 and 1000 ppm ε-PLN could (*p* < 0.05) can increase the shelf life of the frankfurter-type sausages (Figure 4). In this regard, Feng et al. [56] indicated that ε-PL with rosemary extract could significantly decrease TVC and improve sensory properties of chicken breast muscle. Additionally, Alirezalu et al. [35] also indicated that ε-PLN displayed significantly higher inhibitory activity against TVC in comparison ε-PL (free form) in frankfurter-type sausage.

**Figure 4.** Total viable count of sausage samples packaged in polyamide-alginate films during refrigerated storage. A–D Mean values among treatments not followed by a common letter differ significantly (*p* < 0.05). a–c Mean values during storage not followed by a common letter differ significantly (*p* < 0.05).

Conversely, de Barros et al. [57] evaluated effects of natural casing incorporated with nisin in vacuum packaged sausage for the control of spoilage microorganisms, and reported an inhibitory effects of nisin against TVC. Similar results supporting the antimicrobial activity of nisin were also reported by Neetoo and Mahomoodally [58] on cold smoked salmon (by using cellulose-based films and coatings incorporated with nisin and potassium sorbate), and Ercolini et al. [59] on beef burgers coated in nisin and packaged in LDPE films.

Regarding the antimicrobial effect of films against *Clostridium perfringens*, counts between 2.43 and 2.86 Log CFU/g in all sausage samples were obtained at the first day of storage (Figure 5). During storage, significant reductions were observed among treatments and at the of storage the treatments control, polyamide-alginate films with 500 and 1000 ppm ε-PLN had lower values than those sausages packaged with polyamide-algine films with 100 and 200 NN. However, significant differences between sausages packaged in polyamide-alginate films containing 1000 ppm ε-PLN and control group after 45 days of refrigerated storage.

**Figure 5.** *Clostridium perfringens* of sausage samples packaged in polyamide-alginate films during refrigerated storage. A–D Mean values among treatments not followed by a common letter differ significantly (*p* < 0.05). a–c Mean values during storage not followed by a common letter differ significantly (*p* < 0.05).

Meira et al. [60] evaluated antimicrobial effects of polypropylene/montmorillonite nanocomposites containing different concentration of nisin as antimicrobial active packaging. The authors showed that nisin inhibited the growth of *Clostridium perfringens*, which was stronger in samples with higher concentrations of nisin. In addition, Cé et al. [61] also reported similar results against *Clostridium perfringens* in chitosan films containing nisin. It is important to comment that *Clostridium perfringens* is an anaerobic bacterium that mostly growth in inner sections of the sausage and the antimicrobial were in films that are in contact with the external surface of the samples. Therefore, our results did not show high inhibitory effects against *Clostridium perfringens* in sausage samples.

*Staphylococcus aureus* in meat and meat products is one of the most important bacteria because of its enterotoxin production [28]. Polyamide-alginate films incorporated with NN and ε-PLN had significant (*p* < 0.05) effects on *Staphylococcus aureus* count (Figure 6). During the refrigerated storage, *Staphylococcus aureus* significantly (*p* < 0.05) decreased in all sausage samples. Our results revealed that *Staphylococcus aureus* counts in samples packaged in polyamide-alginate films incorporated with 500 ppm and 1000 ppm ε-PLN and control sausages were significantly (*p* < 0.05) lower than obtained in other sausages. Elmani [62] evaluated the antimicrobial effects of lysozyme, chitosan, and nisin on Cig kofte (a traditional Turkish raw meatball) and showed that the inhibitory effect of nisin against *Staphylococcus aureus* was higher than chitosan and lysozyme. Our findings agree with data

reported by Millette et al. [63] who reported alginate films containing 1000 IU/mL of nisin could decrease 2 Log CFU/cm2 of *Staphylococcus aureus* in beef meat after 7 days of storage.

**Figure 6.** *Staphylococcus aureus* of sausage samples packaged in polyamide-alginate films during refrigerated storage. A–C Mean values among treatments not followed by a common letter differ significantly (*p* < 0.05). a–d Mean values during storage not followed by a common letter differ significantly (*p* < 0.05).

ε-PLN significantly (*p* < 0.05) affected *E. coli* in sausage samples (Figure 7). *E. coli* counts continuously decreased in all packaged sausages during storage except for samples packaged with NN (*p* > 0.05) due to lower antimicrobial effects of nisin against Gramnegative bacteria.

**Figure 7.** *E. coli* of sausage samples packaged in polyamide-alginate films during refrigerated storage. A–D Mean values among treatments not followed by a common letter differ significantly (*p* < 0.05). a–d Mean values during storage not followed by a common letter differ significantly (*p* < 0.05).

The presence of chitosan in nisin nanoparticles' structure leads to low inhibitory effects of polyamide-alginate films containing nisin against *E. coli*. Our outcomes agree with those reported by Cé et al. [61] who observed that higher concentrations of chitosan had weaker inhibitory effects against *E. coli*. Furthermore, Elmali [62] evaluated the antimicrobial effects of lysozyme, chitosan, and nisin on Cig kofte and reported there were any inhibitory effects against *E. coli* in samples treated with nisin after 72 h of storage. Polyamide-alginate films containing ε-PLN decreased (*p* < 0.05) *E. coli* in sausage samples during the storage time.

Our results showed that *E. coli* count in sausages packaged in films containing 1000 ppm ε-PLN reached 0 Log CFU/g after 45 days of storage. The high antimicrobial effects of ε-PLN against *E. coli* were also reported by Sun et al. [64] who evaluated the antimicrobial effects of nano-crystalline cellulose films containing ε-PL on fish meat.

At day 1, sausage samples packaged in polyamide-alginate films with NN and ε-PLN presented (*p* < 0.05) lower content of molds and yeasts in comparison to control group (Figure 8). During storage, molds and yeasts increased in all sausage samples. At the end of storage time, sausage samples packaged in films containing 100 ppm NN showed the highest counts of molds and yeasts. Our findings agree with data reported by Guerra et al. [65] who evaluated the antimicrobial effects of cellophane containing nisin and reported that bioactive cellophane packaging could be used for controlling microbial growth in chopped meat. Packaged sausage samples in polyamide-alginate containing ε-PLN (500 and 1000 ppm) decreased (*p* < 0.05) the rate on molds and yeasts growth during refrigerated storage. Furthermore, in control samples (with 120 ppm sodium nitrite) molds and yeasts counts until day 30 were within the standard range.

**Figure 8.** Molds and yeasts of sausage samples packaged in polyamide-alginate films during refrigerated storage. A–D Mean values among treatments not followed by a common letter differ significantly (*p* < 0.05). a–d Mean values during storage not followed by a common letter differ significantly (*p* < 0.05).

Alirezalu et al. [6] also reported similar results in frankfurter sausages. These authors evaluated the antimicrobial effects of nisin, ε-PL and chitosan and reported a similar inhibitory effect against molds and yeasts in meat products. Moreover, catechins in GTE as a phenolic compound not only can inhibit activity of intracellular enzymes and synthesis of fatty acid and protein but also can damage membrane compounds of molds and yeasts [66].

#### **4. Conclusions**

Our outcomes showed that polyamide-alginate casing incorporated with ε-PLN and NN with mixed plant extract (same rates of green tea, stinging nettle, and olive leaves extracts) could be potentially used for increasing frankfurter-type sausage shelf life. Sausages with 1000 ppm ε-PLN had significantly higher inhibitory effects against molds and yeasts, *E. coli*, *Staphylococcus aureus*, and total viable counts. Therefore, polyamide-alginate film incorporated with ε-PLN and NN with mixed plant extract could be used usefully for improving frankfurter type sausage quality and shelf life.

**Author Contributions:** Conceptualization, K.A., M.Y. and H.I.K.; writing—original draft preparation, K.A. and L.P.; writing—review and editing, K.A., M.Y., S.A., H.I.K., M.P., J.M.L., and P.E.S.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** Thanks to GAIN (Axencia Galega de Innovación) for supporting this review (grant number IN607A2019/01). José M. Lorenzo, Paulo E. S. Munekata and Mirian Pateiro are members of the HealthyMeat network, funded by CYTED (ref. 119RT0568).

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

#### **References**


**6. Conclusion**

In this chapter, we summarize our recent efforts in realizing reconfigurable FPC antenna basing on the reconfigurable MS. PIN diodes are added on the MS to realize the reflection phase control. Using the flexible phase control capability of the MS, it is easy to tune the frequency and radiation pattern of the FPC antenna, so as to realize reconfigurable FP antenna. The reconfigurable FPC antenna is a good way to improve the performance of antenna. This method can make the FPC antenna more widely used in the field of wireless communication systems.

### **Abbreviations**

*Advanced Radio Frequency Antennas for Modern Communication and Medical Systems*

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[4] Kim D, Jeongho J, Choi J. A

broadband Fabry-Perot cavity antenna designed using an improved resonance prediction method. Microwave and Optical Technology Letters. 2011;**53**(5):

[5] Wang N, Liu Q, Wu C, Talbi L, Zeng Q, Xu J. Wideband Fabry-Perot

[6] Konstantinidis K, Feresidis AP, Hall PS. Multilayer partially reflective surfaces for broadband Fabry-Perot cavity antennas. IEEE Transactions on Antennas and Propagation. 2014;**62**(7):

[7] Kim D. Noval dual-band Fabry-Pérot cavity antenna with low frequency separation ratio. Microwave and Optical

Technology Letters. 2009;**51**(8):

a single layer superstrate. IEEE

[8] Meng F, Sharma SK. A dual-band high-gain resonant cavity antenna with

resonator antenna with two complementary FSS layers. IEEE Transactions on Antennas and Propagation. 2014;**62**(5):2463-2471

transmission models to design wideband and dual-band Fabry-Perot cavity antennas. In: 2013 International Symposium on Electromagnetic Theory; 2013. pp. 1084-1087


**4. Methods to improve EMI performance of power converters**

#### **4.1 With** *RC* **snubber**

In the previous section, it has been shown that the ringing in the current (and voltage) waveforms degrades the EMI performance of the circuit. Therefore, damping out the ringing will improve the performance. A simple method to achieve the same is the use of an *RC* snubber. Considering the case of a buck converter, the snubber is connected across the freewheeling diode (snubber is to be connected between switching node and ground), as shown in **Figure 8**. The values of the snubber elements *Rs* and *Cs* are computed as follows:

*Cs*≈3 times the diode junction capacitance. Since the junction capacitance is a varying quantity (with junction voltage), the maximum value corresponding to zero bias value, i.e., *CJ*0 is considered for calculation. Therefore, *Cs*≈3 480 *pF*. This results in a value of *1.44 nF,* and so a value of *2 nF* is selected.

*Power Converters Electromagnetic Emissions with Methods to Measure, Compare and Reduce… DOI: http://dx.doi.org/10.5772/intechopen.99711*

*Rs*≈2*ω*0*Lp* where, *ω*0 is the angular frequency corresponding to the ringing oscillations to be damped and *Lp* is the parasitic inductance, *1 nH* at present.

The ringing oscillations in the current waveform shown in **Figure 7(a)** have a frequency of ≈500 *MHz*. With these values, *Rs* is computed to be *6.28 Ω,* and therefore, the value of *10 Ω* is selected.

With the snubber elements included in the buck converter circuit as shown in **Figure 8**, the input current is computed and is shown in **Figure 9(a)**. Also shown in the Figure is the current without snubber (shown earlier in **Figure 7(a)**) for comparison.

Clearly, the snubber results in damping out the ringing at the rising edge of the input current waveform. The same inference can also be drawn from the frequency domain characteristics shown in **Figure 9(b)**. The snubber has clearly damped out the resonant peak due to the high-frequency ringing. With snubber included in the circuit, the frequency spectrum plot clearly shows a reduction in the spectral amplitude of the high-frequency components, and hence an improvement in the emission performance.

It is also important to check the power loss in the snubber resistor and its impact on the efficiency of the circuit. The power loss is given by [5], *fswCfV*2 where, *fsw* is the switching frequency (100 kHz), *V*is the peak voltage which appears across the diode terminals (approximately equal to the input voltage *Vdc*).

The power loss is calculated and found to be *28.8 mW*. The output power is ð Þ 0*:*4�12 2 5 ¼ 4*:*6 *W*. Therefore, the power loss in the snubber is 28*:*2�10�3 4*:*6 � 100 ¼ 0*:*625% of the power delivered at the output and can be neglected for all practical purposes.

**Figure 8.** *Buck converter with input side* RC *snubber.*

#### **Figure 9.**

*Input currents in buck converters with and without snubber. (a) Temporal variation of input current and (b) comparison of frequency domain characteristics.*

Although the snubber improves the EMI performance of the converter, designing the snubber elements can prove to be difficult since it is difficult to estimate the parasitic inductance accurately and switch capacitance and the maximum voltage at the switching node (due to overshoot). In addition, the snubber damps out or attenuates the high-frequency oscillations, which take place at (or around) a certain frequency. If all high-frequency components (above a certain cutoff or threshold frequency) could be attenuated, further improvement in the EMI performance can be obtained. This can obviously be achieved by using a low pass filter. This is discussed in the following.

### **4.2 With damped** *LC* **filter**

One popular method to reduce emissions from power converter and improve their EMI performance is using filters. Considering the example of the buck converter shown in **Figure 6**, a damped *LC* filter is added on the input side, as shown in **Figure 10**.

The filter elements are the inductance *L f* , capacitance *Cf* and the damping resistor *Rf* . The value of the damping resistor is selected from the condition *Rf* << *R=D*2 [6]. Selecting *Rf* ¼ 1 *Ω* easily satisfies the criteria. The values of *L f* and *Cf* are selected to be *100 μH* and *100 pF,* respectively. With these values, the resonant frequency is ≈5 *MHz*. This is sufficiently away from the switching frequency of *100 kHz* and therefore does not slow down the converter's response. However, it provides sufficient attenuation to the high-frequency components and, therefore, is expected to improve EMI performance. To validate the same, the buck converter with the input side filter is simulated, and the input current *Isw* is obtained. This is shown in **Figure 11 (a)**, and the input current without the filter is shown in **Figure 7(a)** for a practical converter. The frequency spectrum of the currents with and without filters is shown in **Figure 11(b)**.

In the time domain current waveform, the effect of the damped *LC* filter in reducing the oscillation is clearly noticeable. The amplitude of the overshoot is also observed to have reduced. The attenuation provided to the high-frequency components can be seen from the frequency response. The reduction in the amplitude at some of the high-frequency components is around *20–30 dB.* This is a significant improvement in the performance of the converter operation from the EMI/EMC point of view. Also, it is worthwhile to note that the damped *LC* filter attenuates both the resonant peaks present in the frequency characteristics. This can be compared with the method employing snubber elements (**Figure 9(b)**), where only one peak has been attenuated.

However, it is important to mention here that the *LC* filter changes the converter dynamics, often leading to a significant degradation in the transient response. Moreover, for converters operated under closed-loop control, the addition of the filter may even make the control system unstable [7]. Therefore, the

**Figure 10.** *Buck converter with input side damped LC filter.*

*Power Converters Electromagnetic Emissions with Methods to Measure, Compare and Reduce… DOI: http://dx.doi.org/10.5772/intechopen.99711*

#### **Figure 11.**

*Input currents in buck converters with and without input side filter. (a) Temporal variation of input current and (b) comparison of frequency domain characteristics.*

design of the filter is not a straightforward task, and the converter performance, closed-loop response, and EMI specifications must all be considered in satisfying all the requirements.

So far, the mechanism behind emissions from power converters, along with some of the important factors responsible for degrading the EMI performance, has been discussed. In addition, some popular methods to improve the EMI performance have also been discussed. In the following sections, the measurement of the emissions and analysis of the same through standard receivers will be briefly discussed.

#### **5. Measurements of electromagnetic emissions**

Measurements of emissions are performed to test for EMC compliance of any product. Therefore, the measurements must be carried out so that the results are easy to correlate across different measurement sites or EMC labs. To understand this, assume that the noise current injected by a certain product into the power supply needs to be measured for compliance. This could be conveniently carried out by using a current probe. However, the noise current injected into the supply depends on the equivalent impedance measured between the supply's terminals. This impedance varies over frequency and will be different at different places, time of the day, seasons, etc. As a result, the noise current will also vary, and therefore the measurement results cannot be correlated.

In order to eliminate the above uncertainties, a Line Impedance Stabilization Network (LISN) is employed for the measurement of Conducted Emissions (150 kHz- 30 MHz) [8]. The block diagram of the measurement setup is shown in **Figure 12**.

The LISN is connected between the supply lines and the product, usually referred to as Device under Test (DuT) or Equipment under Test (EuT). LISN has the following two purposes [8]:

Therefore, with **Figure 12**, only the noise current from the DuT flows through the LISN to the supply terminals. Since the impedance is fixed, a linearly related equivalent noise voltage drop is produced at the LISN, which is then fed to a measuring receiver (EMI receiver). The receiver's measured noise is then analyzed, which will be briefly discussed in the following section.

In the case of radiated emissions, the emissions or noise is coupled directly through the air. Hence an antenna is used to capture the emissions, which are then fed to the measuring receiver for analysis. The block diagram is shown in **Figure 13**.

In the measurement of radiated emissions, typically, over the frequency range of 30–200 MHz, the biconical antenna is used, from 200 MHz – 1 GHz, a log-periodic antenna is employed, and beyond 1 GHz, horn type antennas are used. Discussion on antennas is beyond the scope of the present chapter. However, more detailed discussions are available in the literature [8].

The emission or noise measured by the antenna is sometimes passed through a pre-amplifier stage before feeding to the EMI receiver. The noise emitted by the devices or equipment must be within limits set by the standards. In the United States, the Federal Communications Commission (FCC) limits are required to be adhered to whereas, in most of the European countries, the limits specified by the International Special Committee on Radio Interference (CISPR) are followed. In order to test whether the device or equipment emits the noise, lies within the limit, the measured noise (by LISN or Antenna) is analyzed by a measuring receiver or EMI receiver. The standards also stipulate the analysis procedure to be followed by

**Figure 12.** *Block diagram of conducted emission measurement setup.*

**Figure 13.** *Block diagram of radiated emission measurement setup.*

*Power Converters Electromagnetic Emissions with Methods to Measure, Compare and Reduce… DOI: http://dx.doi.org/10.5772/intechopen.99711*

such a receiver. The receiver, according to CISPR 16–1-1 standards, is briefly discussed in the following.

### **5.1 EMI receiver according to CISPR 16–1-1**

As specified by the CISPR 16–1-1 standard, a measuring EMI receiver must be employed to obtain the noise produced by the DuT. The receiver is coupled to different devices (LISN for CE or different antennas for RE) depending upon the type of emission to be tested. The input impedance of the receiver must be 50 Ω or as close as possible. Otherwise, impedance mismatch might lead to standing waves which will introduce error in the results. The receiver can be operated in different modes, such as Peak, Quasi-Peak, Average, or RMS. The modes are selected based on the type of signal to be measured [9].

In the following, the peak detector mode is briefly discussed since it can be used for different types of input signals and indicates the worst-case scenario. In other words, if the emission of equipment is above the stipulated limit in the peak detector mode, it will fail all the other modes as well. Discussion and modeling of the receiver in quasi-peak, average and RMS modes can be found in literature [10, 11].

The receiver frequency range is divided into a number of different bands. Each band has its range as well as bandwidth for the Intermediate Filter (IF). **Table 1** specifies the important requirements for the receiver in peak detector mode.

**Table 1.**

*Requirements of CISPR 16–1-1 EMI receiver in peak detector mode.*

**Figure 14.** *EMI receiver output for the input current to the buck converter.*

The input time-domain signal to the EMI receiver is typically transformed to the frequency domain through the FFT algorithm. Thereafter, the windowing operation is performed with the IF filter, determined by the band specified in **Table 1**. An extremely short time constant for charging and a very long time for discharging is employed for the peak detector mode. For the other modes, the time constants are specified in the standard. The output of the peak detector stage is obtained across the frequency range of the EMI receiver to produce the emission result for the DuT [6].

As an example, consider the input currents to the buck converter shown in **Figures 7(a)** and **11(a)**, i.e., without and with the damped input filter, respectively. The noise currents are analyzed with the CISPR 16–1-1 EMI receiver (numerically implemented). The results are presented in **Figure 14**, up to 1 GHz.

The improvement in the emission due to the buck converter with the damped *LC* filter is clearly observed in the EMI receiver output. The output of the EMI receiver can be compared with the limits set by the standards to test if the equipment is compliant with the EMC standards or not. Since the standardized procedure is followed, the results can be correlated across different sites.

### **6. Conclusion**

In this chapter, the emission from power electronic converters has been discussed. Beginning with a discussion on the origin of the emission, which was attributed to the rapid switching transients taking place during the operation of the switches, the effect of the device characteristics as well as circuit parasitic elements were analyzed. At each step, through simulations and computations, the effects were demonstrated to degrade the converter's EMI performance compared to an ideal buck converter performance. Thereafter, a couple of methods employed to improve the EMI performance were discussed and demonstrated.

The importance of stipulating a standard procedure for measuring and analyzing the emissions was briefly discussed. The different coupling devices used in measuring emissions over different frequency ranges were introduced along with the measuring EMI receiver according to CISPR 16–1-1 standard. The results of the buck converter currents were analyzed with a numerically modeled EMI receiver as per the standard, and it was shown that the input filter dramatically improves the EMI performance of the power converter. Finally, since the switching transient is common to all power electronic converters, the discussions hold good for power converters in general.


**5. Virus infection of spermatozoa**

Virus capsids morphologically identical to capsids of *Herpesviridae* family were detected in the nucleus and cytoplasmic droplet of infected spermatozoa by TEM (**Figure 8a**, **b**) (for the review see [99]). The structures shown in **Figure 8** (**8a**, **b**) are capsids of herpes simplex virus

**Figure 8.** (a) Longitudinal section of normal sperm; (b) section through the middle piece of the tail of sperm. VC, HSV capsids; M, mitochondria; A, acrosome; CH, chromatin; and C, centriole. The hexagonal structure of some capsids is visible. Phase-contrast microscopy (c) and IF (d) of infected sperm. HSV antigen (arrows) and DAPI stain for DNA (blue); (e) FISH with probes to DNA of HSV. Localization of HSV DNA in sperm heads (arrows).

(HSV), we proved this by immunofluorescence (IF) using the monoclonal antibodies to HSV1 and HSV2 (**Figure 8c**, **d**) and *in situ* hybridization (ISH) with biotinylated probes for HSV (**Figure 8e**). Infection was observed in both the total sperm fraction and the isolated fraction of motile spermatozoa. The persistence of the HSV genome in motile, morphologically normal sperms indicates that the virus may be transmitted vertically to the offspring via natural fertilization or various ARTs, including IVF or ICSI.

Herpetic infection of spermatozoa was significantly more common in infertile men and men whose spouses had a history of spontaneous miscarriage or ART failure as compared with fertile men. Specific antiherpetic treatment of men diagnosed with HSV infection of spermatozoa results in a substantial, almost fivefold increase in the rates of blastocyst formation after ICSI and clinical pregnancy after ART [100].


**5. Conclusions**

Overall, construction companies implement sustainable construction strategies focused on introducing their construction projects to the market. Despite companies provide timely responses to market pressure, this approach is usually incompatible with long-term organizational changes. Although companies adapt their processes to meet customers' requirements, they lack a transversal strategy involving all of the processes within the organization. This gap is evidenced in management processes that prioritize customer-oriented strategies such as energy-efficient facilities and architectural design strategies instead of other management processes that address waste and materials recycling, acquisitions and supply chains. Although these strategies are visible to the market, they only approach the finished product, not a deep organizational change.

Nevertheless, this study showed that although construction firms need to respond to the sustainable demands of society, they implement more and deeper sustainable construction strategies when regulations are stricter, such as the case for construction companies working in the mining industry. There is also evidence that company size directly affects sustainability. Large companies, as indicated by the number of employees, are more likely to develop and implement strategies to address new social, environmental and economic requirements. Likewise, the greatest effect on sustainability is observed in companies with a higher turnover.

This research showed that the participating companies did not pursue strategies that encourage profound organizational change toward corporate sustainability. The function of sustainability management is not a permanent role in the organizational structure, as evidenced by the lack of a permanent sustainability role such as a Director of Sustainability. The lack of a role and organizational structure related to sustainability is a significant barrier for establishing a corporate sustainable strategy and the dissemination, follow-up, and results of sustainable strategies. This strategy should be conceived from the senior management and extended downwards to the entire organization. Thus, the strategy's fragmentation indicates a gap in the dissemination of the policy and a defective communication of the long-term sustainable vision.

As evidenced by this study, only 32% of construction companies declare that they implement sustainable strategies. However, 71% of the respondents believe these strategies could be applied and 88% indicate that these strategies are important for business sustainability. From this perspective, companies recognize sustainable strategies' positive impact on society and on the environment, along with their inherent economic benefits for the firm. Nevertheless, their efforts are focused on the market's demands. The lack of a long-term vision may affect the transformation of sustainable strategy into a competitive advantage. Therefore, if companies do not proceed opportunely, the delay might represent a disadvantage when competitors have experienced transformation based on a sustainable business model.

**Acknowledgments:** The authors thank the Chilean Construction Chamber for providing the database of the construction companies registered in their records, and the Vice Chancellor's research (VRI) scholarship of the Pontificia Universidad Católica de Chile. The authors also express their gratitude for the support of the Center for Sustainable Urban Development (CEDEUS), research grant CONICYT/FONDAP 15110020.

**Author Contributions:** Carlos Giannoni, Luis Fernando Alarcón and Sergio Vera, conceived, designed, and wrote together this paper.

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


**3. Refractive index profile of GRIN optical fibre using the multilayer model**

The GRIN optical fibre sample can be considered as a phase object. When it is implemented in the path of one of the interfering beams of a Mach-Zehnder interferometer, the produced fringes suffer shifts in the GRIN optical fibre region. These shifts represent the optical path difference between the light beam traversing the fibre and that traversing the surrounding medium.

In GRIN optical fibre, the core is surrounded by a homogeneous cladding of refractive index *ncl*. In the multilayer model, the core and cladding of the graded index optical fibre are assumed to consist of a large number of thin layers. Each layer has an annular cross section and is considered to have a thickness *a* and refractive index *nj* where the layers are numbered by *j =1,...,N* with *N = R/a* . *R* is the core radius. If this optical fibre is immersed in a liquid of refractive index *nL*, there will be a refraction of the incident beam at the boundary between the liquid and the cladding. Hamza et al. obtained a recurrence relation which predicts the optical path of the refracted beam through *Q* layers of the fibre. The GRIN fibre is illuminated by a collimated beam, with a ray crossing the centre of the core dening the optical axis (Hamza et al., 1995). An arbitrary beam transverses the fibre at a distance *dQ* from the optical axis and leaves the fibre at a distance *xQ*. Assume a coordinate system whose origin is at the fibre centre, then the corresponding fringe shift is defined as *ZQ* and the optical path difference *δQ* is given by the recurrence formula

$$\begin{split} \frac{\lambda Z\_{\mathcal{Q}}}{h} &= \frac{\lambda}{2\pi} \, \Delta \phi\_{\mathcal{Q}} = \sum\_{j=1}^{\mathcal{Q}} 2n\_{j} \Big( \sqrt{\left(\mathcal{R} \cdot (j \cdot 1) a\right)^{2} \cdot d\_{\mathcal{Q}}^{2} n\_{L}^{2} \left/ \ n\_{j}^{2}} \cdot \sqrt{\left(\mathcal{R} \cdot j a\right)^{2} \cdot d\_{\mathcal{Q}}^{2} n\_{L}^{2} \left/ \ n\_{j}^{2}} \right) \Big) \\ &+ 2n\_{\mathcal{Q}} \Big( \sqrt{\left(\mathcal{R} \cdot (\mathcal{Q} \cdot 1) a\right)^{2} \cdot d\_{\mathcal{Q}}^{2} n\_{L}^{2} \left/ \ n\_{Q}^{2}} \right) \cdot n\_{L} \Big( \sqrt{\mathcal{R}^{2} \cdot d\_{\mathcal{Q}}^{2}} + \sqrt{\mathcal{R}^{2} \cdot x\_{Q}^{2}} \Big) \end{split} \tag{10}$$

with *Q* running from 1 to *N* , *h* is the interfringe spacing and *ΔφQ* is the interference phase difference.

This recurrence relation also describes the shape of the fringes which is produced as a result of the refraction of the incident beam through the *Q* layers of the fibre. Without loss of generality, we can assume that the incident light beam passes through the middle of the *Q*th layer. Thus the value of *dQ* in Eq. (10) can be obtained from the following relation

$$d\_Q = \frac{\eta [R - (Q - 0.5)a]}{n\_L} \tag{11}$$

Eq. (10) can now be used to get the refractive index profile for GRIN optical fibres (*N*→∞). Hamza et al. have shown that inhomogeneous refraction must be considered in the measurement and calculation of optical fibre parameters, so that the accuracy of the measured parameters of GRIN optical fibre is increased (Hamza et al., 1994, 1995).

### **4. Characterization of TE modes in the GRIN optical waveguides**

The study of the GRIN optical waveguides using the wave equation to achieve the allowed propagating modes is familiar from basic quantum mechanics. The analogy between the particle in box and optical waveguide problem is very strong: both situations describe waves, which are confined between two reflecting boundaries. In both cases, the waves partially tunnel into the surrounding potential barrier before turning around. Only certain allowed energies (in the case of the particle), or propagation coefficients *β* (in the case of the optical waveguide) create a standing wave in one-dimensional system.

### **4.1 Symmetric optical waveguide**

The squared value of the refractive index distribution at the core of the GRIN optical waveguide is shown in Fig. 2. At the centre of the core the refractive index has a maximum value *no* and then decreases to reach *nclad* at *x=±R* (*R* is the radius of the core). The squared value of the refractive index distribution is given by,

$$m^2(\mathbf{x}) = \begin{cases} m\_{\text{dual}}^2 & -R > \mathbf{x} \text{, } R < \mathbf{x} \\\\ n\_o - \Delta n \left(\frac{\mathbf{x}}{R}\right)^2 & -R < \mathbf{x} > R \end{cases} \tag{12}$$

where *∆n = no - nclad*.

74 Advanced Holography – Metrology and Imaging

The GRIN optical fibre sample can be considered as a phase object. When it is implemented in the path of one of the interfering beams of a Mach-Zehnder interferometer, the produced fringes suffer shifts in the GRIN optical fibre region. These shifts represent the optical path difference between the light beam traversing the fibre and that traversing the surrounding

In GRIN optical fibre, the core is surrounded by a homogeneous cladding of refractive index *ncl*. In the multilayer model, the core and cladding of the graded index optical fibre are assumed to consist of a large number of thin layers. Each layer has an annular cross section and is considered to have a thickness *a* and refractive index *nj* where the layers are numbered by *j =1,...,N* with *N = R/a* . *R* is the core radius. If this optical fibre is immersed in a liquid of refractive index *nL*, there will be a refraction of the incident beam at the boundary between the liquid and the cladding. Hamza et al. obtained a recurrence relation which predicts the optical path of the refracted beam through *Q* layers of the fibre. The GRIN fibre is illuminated by a collimated beam, with a ray crossing the centre of the core dening the optical axis (Hamza et al., 1995). An arbitrary beam transverses the fibre at a distance *dQ* from the optical axis and leaves the fibre at a distance *xQ*. Assume a coordinate system whose origin is at the fibre centre, then the corresponding fringe shift is defined as *ZQ* and the optical path difference *δQ* is given by the recurrence

*Q j Q L j Q L j*

*n R j a dn n R ja dn n*

2 22 2 2 2 2 2

2 2 22 2 22 2

(11)

, (10)

*Q QL Q L Q Q*

with *Q* running from 1 to *N* , *h* is the interfringe spacing and *ΔφQ* is the interference phase

This recurrence relation also describes the shape of the fringes which is produced as a result of the refraction of the incident beam through the *Q* layers of the fibre. Without loss of generality, we can assume that the incident light beam passes through the middle of the *Q*th

[ ( 0.5) ]

*L nR Q a d n*

Eq. (10) can now be used to get the refractive index profile for GRIN optical fibres (*N*→∞). Hamza et al. have shown that inhomogeneous refraction must be considered in the measurement and calculation of optical fibre parameters, so that the accuracy of the

The study of the GRIN optical waveguides using the wave equation to achieve the allowed propagating modes is familiar from basic quantum mechanics. The analogy between the particle in box and optical waveguide problem is very strong: both situations describe waves, which are confined between two reflecting boundaries. In both cases, the waves

*n R Q a dn n n R d R x*

layer. Thus the value of *dQ* in Eq. (10) can be obtained from the following relation

measured parameters of GRIN optical fibre is increased (Hamza et al., 1994, 1995).

**4. Characterization of TE modes in the GRIN optical waveguides**

*Q*

2 ( 1) / / 2

1

*Q*

*j*

1

2 ( 1) /

**3. Refractive index profile of GRIN optical fibre using the multilayer model**

medium.

formula

difference.

*Q*

*Z*

*h*

The guided wave problem requires only the knowledge of the square of the refractive index distribution.

Fig. 2. The core of the GRIN optical fibre is confined between cladding media, arranged such that the squared refractive index *n2(x)* is larger than that of the surrounding media 2 *nclad* .

In the TE case, the E field is polarized along the *y-* axis and propagating along the *z-* axis. The TE modes of the GRIN optical waveguide are obtained as the solutions of the following equations (Conwell, 1973; Marcuse, 1973),

$$\frac{d^2 E\_y}{dx^2} + \left(k\_o^2 \, n^2(\mathbf{x}) - \beta^2\right) E\_y = 0 \qquad , \tag{13}$$

$$-\frac{i}{w\mu\_o}\frac{\partial E\_y}{\partial z} = H\_x$$

$$\epsilon\_r \tag{14}$$

$$\frac{\text{i}}{w\mu\_o} \frac{\partial E\_y}{\partial \mathbf{x}} = H\_z \tag{15}$$

Digital Holographic Interferometric Characterization of Optical Waveguides 77

On the other hand, to find the allowed TE modes and the propagation coefficients of the asymmetric GRIN optical waveguide, we assume that, the GRIN optical waveguide, as shown in Fig. 3, consists of two graded index zones; both have a Gaussian refractive index profile. The first zone is in the range *0 < x < b1*, and the second one is in the range *b2 < x < b4*. There is a very thin layer of constant refractive index *ns1* confined between the two zones in the range *b1 < x < b2*. The plane at *x=0* represents the interface between the surface of the first zone of the optical waveguide and the immersion liquid (of refractive index *nL*) and the plane at *x = b4* represents the interface between the second zone of the waveguide and the

The refractive index profile for each zone is fitted using a Gaussian function which could be used to determine the allowed TE modes for the optical waveguide sample. The first zone of the waveguide in the range *0 < x < b1* is represented by the Gaussian refractive index profile given by

> 2 1 1 1 ( ) , 0 *x b nx n n e s x b*

where *b* is the depth of the first zone (between *0* and *b1*), and *n1* is the difference between the refractive indices at *x = 0* and *x = b1*. The conditions for the wave propagation along the first zone of the waveguide are: (1) a zigzag of the beam path and (2) the total phase change must be a multiple of 2*π*. Using WKB approximation (Marcuse, 1973; Mathey, 1996), the

12 13

*o k n x dx*

where *β = koneff* is the propagation constant of the *m*th mode; *m* is an integer. *M* is the total number of modes, *ko = 2π/λ* is the wave-vector, *λ* is the wavelength, and *neff* is the effective

**Seconed zone**

*xc*

*b b3 4 b1 b2*

Fig. 3. The waveguide confined between liquid and substrate media, arranged such that the

refractive index *n(x)* is larger than that of the surrounding media (*nL* ) and (*ns*).

**Depth (***b***) m**

2 () 2 2 2 , 1

(22)

*m mM* (23)

*ns*

*x*

**Substrat region**

**4.2 Asymmetric optical waveguide**

substrate, which has refractive index *ns*.

**4.2.1 TE modes in the first zone of waveguide**

1

*b*

0

**0**

**Refractive index** *n(x)*

*nL*

lateral resonance condition for the first zone is given by

22 2

**First zone**

*ns1*

where

$$k\_o^2 = o^2 \,\varepsilon\_o \,\mu\_o \tag{16}$$

*ko*, *μo*, *ω* and *β* are the wave number, the permeability constant, the angular frequency and the allowed propagation coefficient along the *z*-axis, respectively.

Applying the continuity boundary conditions that connect the solutions at the interfaces *x = R* and *x = –R* and for *Δn<<no*, the solutions of the Eqs. (13)-(15) in the three regions (symmetric waveguide) are given in terms of Hermite polynomials (*Hq*) of degree *q*, as

$$E\_{y}(\mathbf{x}) = \begin{cases} A e^{-\frac{k\_{o}(\Delta\varepsilon)^{\frac{1}{2}}R}{2}} H\_{q}(R\sqrt{\frac{k\_{o}(\Delta\varepsilon)^{\frac{1}{2}}}{2R}}) e^{-\gamma(x-R)}, & \text{, } x > R \\\\ A e^{-\frac{k\_{o}(\Delta\varepsilon)^{\frac{1}{2}}x^{2}}{2R}} H\_{q}(\sqrt{\frac{k\_{o}(\Delta\varepsilon)^{\frac{1}{2}}}{2R}}), & -R < x < R \\\\ A e^{-\frac{k\_{o}(\Delta\varepsilon)^{\frac{1}{2}}R}{2}} H\_{q}(-R\sqrt{\frac{k\_{o}(\Delta\varepsilon)^{\frac{1}{2}}}{2R}}) e^{\gamma(x+R)}, & x < -R \end{cases} \tag{17}$$

where *q* is an integer that identifies the mode. *Hq* is the appropriate Hermite polynomial defined by

$$H\_q(\mathbf{x}) = (-1)^q \ e^{\mathbf{x}^2} \quad \frac{d^q}{d\mathbf{x}^q} \ e^{-\mathbf{x}^2} \tag{18}$$

The first three Hermite polynomials in *x* are

$$\begin{aligned} H\_0(\mathbf{x}) &= \mathbf{1}, \\ H\_1(\mathbf{x}) &= \mathbf{2} \,\mathbf{x}, \\ H\_2(\mathbf{x}) &= \mathbf{4} \,\mathbf{x}^2 - \mathbf{2} \end{aligned} \tag{19}$$

Also *γ* in Eq. (17) is the attenuation coefficient in the clad region and 2 *n n o* . The allowed propagation coefficient and the effective index for every mode are given respectively by

$$\beta = \sqrt{k\_o^2 \varepsilon\_o - \frac{k\_o \sqrt{\Delta \varepsilon}}{R} \left(1 + 2q\right)}\tag{20}$$

and

$$m\_{\rm eff} = \frac{\beta}{k\_o} \,. \tag{21}$$

where *εo* is the dielectric constant at the center of the symmetric optical waveguide, and it is related to the refractive index by 2 *o o n* .

### **4.2 Asymmetric optical waveguide**

76 Advanced Holography – Metrology and Imaging

*ko*, *μo*, *ω* and *β* are the wave number, the permeability constant, the angular frequency and

Applying the continuity boundary conditions that connect the solutions at the interfaces *x = R* and *x = –R* and for *Δn<<no*, the solutions of the Eqs. (13)-(15) in the three regions (symmetric waveguide) are given in terms of Hermite polynomials (*Hq*) of degree *q*, as

( ) 2 ( ) 2

*k Ae H R e x R R*

( ) 2 ( ) 2

where *q* is an integer that identifies the mode. *Hq* is the appropriate Hermite polynomial

*R*

*k Ae H R e x R R*

( ) 2

*k E x Ae H x RxR*

*q*

( ) ( 1)

*q q d Hx e e*

> 0 1

*H x Hx x Hx x*

( ) 1, () 2, () 4 2

2

Also *γ* in Eq. (17) is the attenuation coefficient in the clad region and 2 *n n o*

allowed propagation coefficient and the effective index for every mode are given

*n*

where *εo* is the dielectric constant at the center of the symmetric optical waveguide, and it is

*k k q R*

*o*

*k*

*q*

1

( ) ( ), 2

*o x R*

1

1

*o x R*

(18)

(20)

. (21)

(19)

. The

( ) ( ), 2

*q qx x*

*dx*

2

(16)

(17)

2 2 *o oo k*

the allowed propagation coefficient along the *z*-axis, respectively.

*o*

*o*

*o*

2 2

2 1 2 *o o o*

*eff*

related to the refractive index by 2

*o o n* .

The first three Hermite polynomials in *x* are

1 2

*k R*

1 2 2

2

*k R*

*k x R o y q*

1 2

( ) ( ) ( ), 2

where

defined by

and

respectively by

On the other hand, to find the allowed TE modes and the propagation coefficients of the asymmetric GRIN optical waveguide, we assume that, the GRIN optical waveguide, as shown in Fig. 3, consists of two graded index zones; both have a Gaussian refractive index profile. The first zone is in the range *0 < x < b1*, and the second one is in the range *b2 < x < b4*. There is a very thin layer of constant refractive index *ns1* confined between the two zones in the range *b1 < x < b2*. The plane at *x=0* represents the interface between the surface of the first zone of the optical waveguide and the immersion liquid (of refractive index *nL*) and the plane at *x = b4* represents the interface between the second zone of the waveguide and the substrate, which has refractive index *ns*.

### **4.2.1 TE modes in the first zone of waveguide**

The refractive index profile for each zone is fitted using a Gaussian function which could be used to determine the allowed TE modes for the optical waveguide sample. The first zone of the waveguide in the range *0 < x < b1* is represented by the Gaussian refractive index profile given by

$$m(\mathbf{x}) = n\_{s\_1} + \Delta n\_1 \ e^{-\left(\frac{\mathbf{x}}{b}\right)^2} \qquad \qquad \qquad 0 < \mathbf{x} < b\_1 \tag{22}$$

where *b* is the depth of the first zone (between *0* and *b1*), and *n1* is the difference between the refractive indices at *x = 0* and *x = b1*. The conditions for the wave propagation along the first zone of the waveguide are: (1) a zigzag of the beam path and (2) the total phase change must be a multiple of 2*π*. Using WKB approximation (Marcuse, 1973; Mathey, 1996), the lateral resonance condition for the first zone is given by

$$2\int\_{0}^{h\_1} \sqrt{k\_o^2 \ n^2(\mathbf{x}) - \beta^2} \, d\mathbf{x} \quad -2\Phi\_{1\not\!2} - 2\Phi\_{1\not\!3} = 2\,\text{m}\,\pi \,, \quad 1 \le m \le M\tag{23}$$

where *β = koneff* is the propagation constant of the *m*th mode; *m* is an integer. *M* is the total number of modes, *ko = 2π/λ* is the wave-vector, *λ* is the wavelength, and *neff* is the effective

Fig. 3. The waveguide confined between liquid and substrate media, arranged such that the refractive index *n(x)* is larger than that of the surrounding media (*nL* ) and (*ns*).

Digital Holographic Interferometric Characterization of Optical Waveguides 79

optical field. The position of MO2 is precisely adjusted. These two beams are recombined at

2D transition Stage

M1

BS2 MO1

CCD

MO2

*d*

Optical cell

The mirror M2 in the digital holographic set-up reflects the reference beam. This mirror can be tilted to control the position of the reference beam on the CCD camera. The reference beam reaches the CCD camera at a small incident angle with respect to the propagation direction of the object wave. Therefore, the off axis holographic configuration is used (Wahba & Kreis, 2009a, 2009b, 2009c; Yassien et al., 2010). In addition, the mirror M2 in the digital holographic setup is mounted on a piezoelectric transducer (PZT), which acts as the phase shifting tool. It is positioned in the reference arm and enables us to obtain four phase shifted holograms. The phase shifting steps start with 0o and the holograms are shifted mutually by *π*/2. These phase shifted holograms are recorded by an Allied Vision Marlin F145B2 CCD camera with pixel pitch 4.65 µm × 4.65 µm and pixel numbers 1392, and 1040 in the horizontal and vertical

Phase shifting digital holographic interferometry is used to investigate the optical parameters of GRIN optical waveguide samples. Fresnel off–axis holograms have been produced by using a Mach-Zehnder holographic arrangement; see Fig. 4. A piezoelectric transducer (PZT), acting as a phase-shifting tool, is applied in the reference arm and helps us to obtain four phase-shifted holograms. Fig. 5 represents the phase shifted digital holograms for a GRIN optical fibre sample. The GRIN optical fibre sample is immersed in a liquid of refractive index 1.46, which is greater than that of its cladding. The phase of the hologram (Fig. 5(a)) is assumed to be zero, whereas the phase shifts of the holograms (Fig.

the beam splitter *BS2*, which is identical to *BS1*.

Laser S

Fig. 4. Digital holographic interferometric set-up.

M2

PZT

L BS1

**6.1 Refractive index profile of GRIN optical waveguide**

5(b) to 5(d)) are *π*/2, *π*, and 3*π*/2; respectively.

directions, respectively.

**6. Results and discussion**

mode index for light that propagates along the optical waveguide. *1/2* and *1/3* are the phase changes at the film-liquid interface and at the first and second zones interface, respectively. Generally these last quantities are approximated to *1/2 π/2* and *1/3 π/4* (Mathey, 1996). The effective indices (*neff*) for the first zone are obtained numerically by substituting Eq. (22) into Eq. (23).

### **4.2.2 TE modes of second zone waveguide**

Referring to Fig. 3, we suppose that, without loss of generality, the second region of the waveguide in the range *b2< x < b4* is divided into two asymmetric parts which have an individually Gaussian refractive index profile. The first and the second parts within the ranges are *b2< x < b3* and *b3< x < b4*, respectively. The refractive index profile in these two parts of the second zone of the waveguide is given by

$$m(\mathbf{x}) = \begin{cases} m\_{s\_1} + \Delta m\_2 \ e^{-\left(\frac{\mathbf{x} - \mathbf{x}\_c}{b}\right)^2} & , b\_2 < \mathbf{x} < b\_3 \\\\ m\_{s\_2} + \Delta n\_3 \ e^{-\left(\frac{\mathbf{x} - \mathbf{x}\_c}{b}\right)^2} & , b\_3 < \mathbf{x} < b\_4 \end{cases} \tag{24}$$

where *b* is the irradiation depth for both, the first part between *b2* and *b3*, and the second part between *b3* and *b4. n2* is the difference between the refractive indices at the interface between the first and the second parts at *x = b3* and that at *x = b2*, whereas *n3* is the difference between the refractive indices at the interface between the first and the second parts at *x = b3* and that of the substrate at *x = b4*.

Similarly, by analogy with Eq. (23) we have for the first and second parts

$$2\int\_{b\_2}^{b\_3} \sqrt{k\_o^2 \ n^2(\mathbf{x}) - \beta^2} \, d\mathbf{x} \quad -2\Phi\_{1\not\equiv 1} = 2\,\mathrm{m}\,\pi \quad \mathbf{1} \le \mathbf{m} \le M \tag{25}$$

$$2\int\_{b\_3}^{b\_4} \sqrt{k\_o^2 \ m^2(\mathbf{x}) - \beta^2} \, d\mathbf{x} \quad -2\Phi\_{1\not\!3} = 2m\pi \ , \quad 1 \le m \le M \tag{26}$$

since *1/2 = 0* at *xc*.

The effective indices (*neff*) for the two parts of the second zone are obtained numerically by using Eq. (24) in Eqs. (25) and (26).

### **5. Digital holographic set-up**

The digital holographic setup is a Mach-Zehnder system, see Fig. 4. The optical waveguide sample is immersed in a liquid of refractive index *nl* near the cladding refractive index *nclad* of the GRIN optical fibre or the refractive index of the substrate of the waveguide sample. An Abbe refractometer with an accuracy of ±0.0001 is used to measure the refractive indices of the immersion liquids. The collimated laser beam crosses the sample and passes through the microscope objective MO1 with magnification 10x and N.A. 0.25. An identical microscope objective MO2 is installed in the reference arm to eliminate the curvature of the

phase changes at the film-liquid interface and at the first and second zones interface,

(Mathey, 1996). The effective indices (*neff*) for the first zone are obtained numerically by

Referring to Fig. 3, we suppose that, without loss of generality, the second region of the waveguide in the range *b2< x < b4* is divided into two asymmetric parts which have an individually Gaussian refractive index profile. The first and the second parts within the ranges are *b2< x < b3* and *b3< x < b4*, respectively. The refractive index profile in these two

2

2

, ( ) ,

*n ne b xb*

*c*

*n ne b xb*

*c*

*x x b*

> *x x b*

where *b* is the irradiation depth for both, the first part between *b2* and *b3*, and the second part between *b3* and *b4. n2* is the difference between the refractive indices at the interface between the first and the second parts at *x = b3* and that at *x = b2*, whereas *n3* is the difference between the refractive indices at the interface between the first and the second

2 2 3

3 3 4

,

>

*1/2* and

*1/2 π/2* and

*1/3 π/4*

(24)

*m mM* (25)

*m mM* (26)

*1/3* are the

mode index for light that propagates along the optical waveguide.

1

*s*

2

Similarly, by analogy with Eq. (23) we have for the first and second parts

1 3 2 () 2 2 , 1

1 3 2 () 2 2 , 1

*k n x dx*

*k n x dx*

The effective indices (*neff*) for the two parts of the second zone are obtained numerically by

The digital holographic setup is a Mach-Zehnder system, see Fig. 4. The optical waveguide sample is immersed in a liquid of refractive index *nl* near the cladding refractive index *nclad* of the GRIN optical fibre or the refractive index of the substrate of the waveguide sample. An Abbe refractometer with an accuracy of ±0.0001 is used to measure the refractive indices of the immersion liquids. The collimated laser beam crosses the sample and passes through the microscope objective MO1 with magnification 10x and N.A. 0.25. An identical microscope objective MO2 is installed in the reference arm to eliminate the curvature of the

22 2

22 2

*s*

respectively. Generally these last quantities are approximated to

substituting Eq. (22) into Eq. (23).

since

**4.2.2 TE modes of second zone waveguide**

parts of the second zone of the waveguide is given by

*n x*

parts at *x = b3* and that of the substrate at *x = b4*.

3

*b*

2

4

*b*

3

*b*

*b*

using Eq. (24) in Eqs. (25) and (26).

**5. Digital holographic set-up**

*1/2 = 0* at *xc*.

*o*

*o*

optical field. The position of MO2 is precisely adjusted. These two beams are recombined at the beam splitter *BS2*, which is identical to *BS1*.

Fig. 4. Digital holographic interferometric set-up.

The mirror M2 in the digital holographic set-up reflects the reference beam. This mirror can be tilted to control the position of the reference beam on the CCD camera. The reference beam reaches the CCD camera at a small incident angle with respect to the propagation direction of the object wave. Therefore, the off axis holographic configuration is used (Wahba & Kreis, 2009a, 2009b, 2009c; Yassien et al., 2010). In addition, the mirror M2 in the digital holographic setup is mounted on a piezoelectric transducer (PZT), which acts as the phase shifting tool. It is positioned in the reference arm and enables us to obtain four phase shifted holograms. The phase shifting steps start with 0o and the holograms are shifted mutually by *π*/2. These phase shifted holograms are recorded by an Allied Vision Marlin F145B2 CCD camera with pixel pitch 4.65 µm × 4.65 µm and pixel numbers 1392, and 1040 in the horizontal and vertical directions, respectively.