Introduction

Genipin (GN) is an iridoid derivative that is chiefly procured from Genipa americana and Gardenia jasminoides fruits. G. americana is native to Central, South America, and Brazil whereas G. jasminoides is found in numerous Asian countries including Japan, India, Pakistan, and China [1]. The fruits of both G. americana and G. jasminoides were exploited in traditional medicine [2, 3]. GN itself was recognized for its therapeutic attributes in variable health conditions, such as gastric cancer [4], oral squamous cell carcinoma [5], and rotavirus-caused diarrhea [6]. GN also demonstrated a beneficial impact on male mice with circadian rhythm abnormalities in terms of their reproductive health [7]. Furthermore, GN was regarded as a synergistic agent that could be co-administered with the antileishmanial medicine, sodium antimony gluconate [8].

GN is also acknowledged for its cross-linking capabilities. Cross-linking allows molecules to bind together, and establish 3D networks. These networks would meliorate the stability and structural integrity of the procured materials [1]. Meliorating the stability and structural integrity of natural polymers (biopolymers), such as chitosan and gelatin, would improve their mechanical attributes and would also retard their degradation rates, and this would encourage their utilization in food packaging, wound dressing, and tissue engineering applications [9,10,11,12,13,14]. Nonetheless, the biocomptability and safety of biopolymers are essential to allow their utilization in these applications[10, 11, 15]. Thus, bio-compatible cross-linkers, such as GN [16], which would meliorate the biopolymers attributes whilst retaining their bio-comptability [17, 18], would be preferred to the toxic cross-linkers, such as glutaraldehyde (GA) and epoxide [1]. Noteworthy, epoxides could be carcinogenic owing to their capacity to covalently bind nucleophilic residues in proteins and nucleic acids [19]. Moreover, the toxic residual aldehyde residues of GA could devitalize cells, induce calcification and also initiate immune responses [20]. On the other hand, GN is a natural and biocompatible cross-linker, which was stated to demonstrate 5000–1000-folds lower cyto-toxicity than did GA [16].

GN biocomptability has also enabled its incorporation in drug delivery vehicles where its cross-linking capabilities were exploited to meliorate the mechanical attributes and extend drug release from the biopolymer-based drug delivery vehicles [21, 22]. Biopolymer hydrolysates, such as the antioxidant casein phosphopeptides, which were intended for the treatment of the inflammatory bowel disease, were also cross-linked with GN in order to promote their stability in the GIT [23]. Noteworthy, GN cross-links the primary amine entities of polymers and proteins [24]. Such cross-linking capability has enabled it to activate altered supports, such as the amino-grafted silica and activated carbon [25, 26] and, also altered biopolymers, such as chitosan, gelatin, and amino-grafted cellulose [27,28,29], into covalent immobilizers, which were capable of binding the amino-containing enzymes via the sturdy covalent linkages.

Owing to the benefits of cross-linking biopolymers with GN, we attempted to provide a comprehensive review on GN as an efficient cross-linker for biopolymers. We started by reviewing GN botanical sources and how to extract it via safe and biocomptabile solvents. Afterward, GN structure and mode of action were discussed. Moreover, the GN cross-linked biopolymers and their applications in tissue engineering, wound dressings, drug delivery, and packaging were discussed. GN capability to activate biopolymers, such as chitosan and gelatin, and other supports into covalently reactive enzyme immobilizers was also discussed. Moreover, other important GN applications, such as exploiting it as a colorant for foods and textiles and incorporating it in altered biosensors, were discussed.

GN sources

Genipa americana L.

Genipa americana L. plant is native to Central, South America, and Brazil [1]. It belongs to family Rubiaceae [30], and it is a small plant that could reach moderate height in the range of 26.2 to 65.6 feet. It grows best at 18–28°C within slightly acidic soils [1]. Its fruit is termed genipap [3, 30]. Genipap is an edible, aromatic fruit [1] that is around 8–10 cm long and exhibits an elliptical morphology [3, 30]. Genipap is exploited in beverages production. Its flesh could also be exploited as a surrogate to commercial pectin in order to promote jelling of pectin-deficient fresh juices [30]. Genipap together with G. americana leaves and barks were exploited in traditional medicine as therapy for gastric and kidney conditions, prostate cancer, influenza and throat illness [2].

Genipa americana L. comprises 1–3% GN [30]. Various researchers have extracted GN from unripe (UR) genipap [24, 31]. On one occasion, 60.77 mg GN /g was acquired from the endocarp of the UR genipap endocarp [31]. On other occasions, GN was acquired from the UR genipap endocarp at 38 mg/g [32] and 3.4 mg/g. Such variations could be ascribed to the altered fruit varieties, and also to their varied handling and extraction techniques [31].

Geniposide was also shown to be present in genipap. Geniposide is the glycosylated form of GN, which could be hydrolyzed via β-glucosidase in order to attain GN [33]. Moreover, intestinal and liver β-D-glycosidases are also capable of releasing GN from geniposide [34]. Geniposide was present in the ripe (R) genipap endocarp (89.48mg/g) [31] and also in its UR mesocarp (117.99 and 20.7 mg/g) [31, 32]. Noteworthy, geniposide constitutes > 70% of UR genipap iridoid constituents [33]. Genipap also comprises geniposidic acid, genipin-1-β-gentiobioside, gardenoside, 6′'-O-p-coumaroyl1-β-gentiobioside geniposidic acid, 6′-O-feruloyl-geniposidic acid, and 6′′-O-p-coumaroylgenipin-gentiobioside [31].

Gardenia jasminoides J. Ellis

Gardenia jasminoides J.Ellis (gardenia) is a Rubiaceae family evergreen shrub whose height ranges from 2 to 6 feet. It is found in numerous Asian countries including Japan, India, Vietnam, Pakistan, and China [1, 3, 30]. It is also cultivated as a house plant in subtropical and warm temperate regions owing to its evergreen leaves and aromatic flowers [34]. Gardenia fruits extracts were adopted in traditional Chinese medicine as an efficacious oral remedy for inflammation and hepatic disorders [3]. Moreover, its dried fruits were used to cure acute conjunctivitis and hematuria, dysentery caused abdominal pain, and hepatic pain caused by cirrhosis [30]. Gardenia fruit extracts have also shown fine therapeutic traits versus central nervous system diseases, such as cerebral stroke and depression.These fruits were also exploited as functional food supplements within China and East Asia [3]. Geniposide is the chief bioactive iridoid of the ripe gardenia fruits. GN is also presented by gardenia; nonetheless, at 0.17% only [30].

GN extraction

In this section, GN extraction via safe and biocompatible solvents, such as water, ethanol, natural deep eutectic solvents (NADESs) [24, 35], and milk [36] was discussed. Exploiting these solvents instead of the toxic organic solvents, such as acetone, methanol, and formic acid [24], would render the extraction process more eco-friendly. Moreover, the procured extracts might be regarded as ready to use extracts, and this would eliminate the need for the expensive purification steps that would be necessary if toxic solvents were employed. For instance, the NADESs extracts were regarded by some workers as ready-to-use owing to the low-toxic attributes of NADESs which could enable their direct incorporation within pharmaceutical and food products [24]. Moreover, GN ethanolic solution was formerly exploited during the fabrication of scaffolds, which were intended for tissue engineering [9]. Thus, the GN ethanolic extract might also be regarded as ready-to-use.

Enzymes were also involved in GN extraction. For instance, β-glucosidases are capable of hydrolyzing geniposide into GN [1]. Thus, they could be exploited to hydrolyze the geniposides, which constitute the chief bioactive iridoid in the ripe gardenia fruits [30]. This would subsequently enlarge the GN yield from gardenia fruits, which originally comprise only 0.17% GN [30]. Cellulases were also reported to hydrolyze geniposide into GN. Moreover, they could also break-up plant cells [1], and this would further boost the extraction process. Thus, the two above-mentioned enzymes were also discussed in this section.

Water and ethanol extraction

Water and ethanol are solvents that are generally-recognized-as-safe (GRAS) [24]. Thus, they were exploited in GN extraction. For instance, ethanol extraction recovered 47 mg GN/g from UR genipap after applying 2 MPa pressure at 50 °C [32]. On another occasion, ethanol and water efficiencies for GN extraction from peeled UR genipap were compared. Water was more efficient as its GN extraction reached 92.2 mg/g after applying 0.1 MPa pressure at 40 °C (Table 1). The low pressure utilized herein (low-pressure-extraction) would be recommended owing to the simple operation of the equipment, which would contribute to the low cost of the attained product. The GN extraction process was further meliorated via subjecting the UR genipap fruit powder to a mechanical pressing procedure prior to its aqueous extraction. Such pressing procedure reduced the extraction time from 22.2 to 5.84 min as it probably improved mass transfer rate [35].

Table 1 Altered GN and geniposide extraction protocols and their yield
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The superiority of water as a solvent for GN extraction was further confirmed by Náthia-Neves et al. [33]. Such superiority was regarded to the larger polarity of water. Both water and ethanol were exploited to extract GN and geniposide from the UR genipap, which was semi-defatted via supercritical-fluid extraction with CO2. Semi-defatting the UR genipap and removing a large proportion of their non-polar components provided a matrix rich in polar moieties. Thus, the UR genipap polar moieties, such as GN, became more accessible for extraction via water or ethanol. The extraction process was further meliorated via implementing ultrasound techniques. The ultrasound-triggered acoustic cavitation would break-up the genipap cell walls, and this would promote the diffusion of the desired moieties to the solvent [33]. Furthermore, ultrasound techniques could create proficient processes with low energy requirements and short operation times [36]. At low ultrasound power (150 W), the amount of extracted GN was raised from 101.0 to 121.7 mg/g extract upon prolonging the process time from 1 to 7 min. Nonetheless, at high power (450 W), the amount of extracted GN was reduced from 118.80 to 108.58 mg/g extract after akin process prolongation. Such reduction was regarded to GN degradation owing to the excessive cavitation at such incremented power. Noteworthy, ethanol was the superior solvent for the geniposide ultrasound-assisted extraction from the semi-defatted UR genipap, and 312 mg geniposide/ g extract was recorded at 150 W after 7 min. The geniposide affinity for ethanol was regarded as the cause of such superior extraction [33].

Extraction via natural-deep-eutectic-solvents

Natural-deep-eutectic-solvents (NADESs) were utilized to extract GN and geniposide from genipap and gardenia, respectively. NADESs are mixtures comprising a minimum of one hydrogen-bond acceptor (HBA) and one hydrogen-bond donor (HBD), which are joined together, chiefly, via hydrogen-bonds. The HBA and HBD are bio-renewable moieties or primary metabolites. Thus, the NADESs are biocompatible and exhibit low-toxicity. Moreover, NADESs exhibit superior solvation capability. Hence, they are auspicious surrogates to the toxic and non-biodegradable solvents, such as acetone and methanol. However, NADESs exhibit incremented viscosity [24, 37] and low volatility. Such low volatility would impede the separation amidst the NADESs and their extracted moieties. Nonetheless, owing to the low-toxic attributes of NADESs, they might be directly merged within pharmaceutical and food products, and accordingly, NADESs extracts were regarded by some workers as ready-to-use [24]. As for the incremented viscosity of the NADESs, which could lower the mass transfer and impede the extraction process, it could be surmounted via mixing the NADESs with water [24, 37].

Noteworthy, NASESs utilization was formerly coupled with the application of ultrasound techniques. Ultrasound techniques are eco-friendly as they lessen the amount of required solvent, the process duration and also the required energy [24, 37]. On one occasion, high-intensity ultrasound (HIUS) was applied at 21.4 W acoustic power in conjugation with the NASESs system, 1 betaine: 3 lactic acid, in presence of 40% water (solvent/feed ratio 19). Such HIUS application raised the GN yield from ≈ < 2 mg/g to ≈11 mg/g powdered unripe genipap. The HIUS induced micro-turbulence which promoted mass transfer and raised the contact area amidst the genipap powder and the NASESs, and this eventually raised the amount of extracted GN [24]. Efficient geniposide extraction from gardenia fruits was also achieved when 600 W utrasonic power was coupled with the NASES system: 1 choline chloride: 2 propylene glycol, in presence of 25% water. Such extraction acquired 57.99 mg geniposide/g [37].

Extraction with milk

Milk was also exploited in GN extraction. However, it was not regarded solely as a solvent. Its copious primary amine moieties cross-linked the extracted GN, and this created a natural innovative blue colorant. Thus, milk was the GN solvent, the GN interaction medium and also the carrier for the concocted blue complexes. The milk-mediated GN extraction from the UR peeled genipap was assisted via low-frequency ultrasound processing. The ultrasound processing promoted the GN extraction, and the content of free GN, which was estimated after 2h of cold storing (7 °C) the milk extracts, was raised upon implementing the ultrasound processing and also upon raising the adopted power from 100 to 400 W. However, the recorded free GN gradually cross-linked the milk proteins, and this caused its amount to be reduced to only 20% after 24 h of cold storing. Such gradual cross-linking induced a gradual intensification of the milk extracts blue color (Fig. 1) [36].

Fig. 1
figure 1

Alterations observed in the color of the milk, which was processed at different ultrasound nominal power, during its cold storage (7 ± 2 °C) [36]

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β-Glucosidase assisted extraction

β-Glucosidases hydrolyze geniposide into GN. Variable microbial strains were utilized to acquire the needed β-glucosidases, such as Penicillium nigricans, Trichoderma reesei [38], T. harzianum CGMCC 2979 [39], T. viride, Aspergillus niger [40] and Bacillus altitudinis JYY-02 [41]. Recombinant E. coli was also utilized for Aspergillus niger β-glucosidase heterologous expression [38]. Moreover, lactic acid bacteria (LAB) were utilized to acquire β-glucosidase for geniposide hydrolysis. LAB are GRAS and exhibit probiotic qualities. Thus, they would be favored for GN production, specially if this GN was intended to prepare the blue food colorant. The whole cells of LAB strains Lactobacillus plantarum S3 and KCTC3104 bio-converted 93.4% and 100% of geniposide into GN (Table 2) within 12 h at pH 7 [38]. On some occasions, the cells of the β-glucosidase producing micro-organisms were immobilized [38, 40]. For instance, the T. reesei cells were immobilized within calcium alginate beads. These immobilized cells were proficient as they bio-converted 89% of geniposide to GN within 34 h whereas their free compeers bio-converted only 24% after 72 h [40].

Table 2 Examples of the β-glucosidase mediated geniposide to GN bio-conversions
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Noteworthy, lots of the research performed on β-glucosidase mediated geniposide hydrolysis adopted purified geniposides [38, 40, 41]. Nonetheless, some research exploited the crude gardenia fruits as their geniposide source. On one occasion, 95% of gardenia geniposides were bio-converted into GN after 108-h incubation with P. nigricans. A loftier bio-conversion of 97.8% was accomplished after a 48-h fermentation process with T. harzianum CGMCC 2979. The medium exploited during such fermentation comprised 80g/l gardenia dried fruit powder dispersed in buffer (pH 6.1). Afterward, the acquired GN was purified with 62.3% recovery [39].

Cellulase-assisted extraction

Cellulase would perform two tasks in GN extraction. Initially, it would degrade the plant cell walls, and liberate the intra-cellular components. This would promote extraction of geniposides from their plant-rich material. Such geniposides would then be acted upon by cellulase in order to liberate GN. The cellulase-mediated extraction successfully recovered GN from gardenia fruits and Eucommia ulmoides barks, which mainly comprised geniposide [42, 43]. Processing the gardenia fruits powder with Aspergillus niger cellulase at a 10 mg/ml concentration for 24 h at pH 4 and 50 °C recovered 35 mg GN/g. The amount of recovered GN was then raised to 58.83 mg/g after adopting the two-phase system during extraction. The two phases were the aqueous phase, which would be favored by the cellulase enzyme and the extracted geniposides, and the ethyl acetate phase, which would be favored by GN. The partition of the liberated GN away from the aqueous phase would partially purify GN, It would also prohibit GN acid hydrolysis at the adopted pH 4 (20% of GN decomposed after 24 h at pH 4). Moreover, GN would be placed away from the proteinaceous cellulase, and this would prevent the cross-linking amidst GN and cellulase. Thus, the amount of recovered free GN was raised to 58.83 mg/g. Noteworthy, ethyl acetate is amidst the least toxic organic solvents [43].

The cellulase-mediated extraction and in-situ hydrolysis of geniposide to GN were also performed in the presence of ionic liquids (ILs), which are green solvents. ILs comprise large organic cations together with organic or inorganic anions. ILs offer numerous profitable traits, such as their stability; chemically and thermally, their fine ability to extract a vast range of organic moieties, and their miscibility with water as well as organic solvents. In order to efficiently extract geniposide and GN from the E. ulmoides barks, the efficiencies of variable ILs were initially compared. The IL; 1-hexyl-3-methylimidazolium chloride; was selected. Utilizing this IL at 0.5 mol/L concentration, 303 K temperature, and pH 5 for 120 min in conjugation with 20mg/ml cellulase allowed the recovery of 0.76 mg/g GN [42].

GN structure and mechanism of action

GN structure is presented in Fig. 2 [44]. Polymeric forms of GN were also reported. It was argued that at the elevated pH of 13.6, the abundant OH− entities would attack GN. This would trigger GN ring opening and would elaborate aldehyde residues (Fig. 3). The elaborated aldehyde residues would then mediate GN polymerization via aldol condensation. The polymerized GN could comprise 7–88 GN monomers and its molecular weights could reach up to 20,000 Da (monomeric GN m.w is 226 Da). Such escalated molecular weight could be the reason of the viscous traits conferred to GN solution at pH 13.6. Noteworthy, the pH 13.6 GN solution demonstrated a brownish color in contrast with its transparent morphology within the 1.2–9 pH range, which might also be regarded to its polymerized forms [45].

Fig. 2
figure 2

GN structure recreated as per Butler et al. [44]

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Fig. 3
figure 3

Proposed mechanism of GN polymerization at pH 13.6 which was recreated as per Mi et al. [45]

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The polymerized GN was debated to interact with the primary amine residues via its terminal aldehyde residues. Schiff’s base would be established betwixt these aldehyde residues and the amine residues [45]. Thus, long GN cross-linking bridges would be established with the primary amine providing moieties, e.g., chitosan (CHS). These long cross-links would increment the distance amidst the cross-linked CHS and would lower its cross-linking density [45].

As regards the monomeric GN (Fig. 2), two mechanisms were considered for its interaction with the primary amine presenting moieties, such as amino acids, proteins, and CHS [44, 46]. The first, faster and immediate interaction comprised the amine nucleophilic attack on GN C3. This attack would induce GN ring opening, the merger amine presenting moiety (as a secondary amine) with GN, and would also elaborate an aldehyde residue (Fig. 4). The secondary amine would then attack the elaborated aldehyde and a hetero-cyclic GN derivative would be established. The second, slower interaction comprised a SN2 nucleophilic substitution in which GN ester would be substituted by a secondary amide, and methanol would be procured (Fig. 4) [44]. Noteworthy, this substitution reaction requires acid catalysis [44, 45].

Fig. 4
figure 4

GN interaction with the primary amino residues [44]

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One of the crucial applications of the GN-amine interactions is the attainment of the blue pigment. Nonetheless, both the GN and the GN-amino acid complex exhibit their uttermost absorption at 240 nm [46]. That is, they are colorless. Moreover, monitoring the GN-glycine interactions (at pH 6) via high-performance liquid chromatography-diode array detection-mass-specrometry revealed that the GN-glycine monomeric complex was procured at 15 min. Nonetheless, the GN-glycine mixture became visually blue after 25 min [46]. This further confirmed that the GN-amino monomeric complex was not responsible for the blue coloration. In order to procure the GN-derived blue pigment further interactions were undergone. The GN-amine hetero-cyclic complexes are subjected to oxygen free radical-triggered polymerization, and this polymerization produce the blue-colored pigments. That is why the blue color of the GN cross-linked CS was most intense in proximity of the air-exposed surface [44]. Such polymerization could reach up to the tetramer level (Fig. 5) and could involve the five or the six-membered GN rings [45]. Noteworthy, such GN polymerization and blue pigment formation could only occur following the GN-amino cross-linking. If no cross-linking occurred, as in case of mixing GN with acetyl-glucosamine, no blue pigments would be attained. It should also be noted that the polymerized GN-amino complex is the one mediated via ring opening (Fig. 4) rather than the one mediated via ester substitution [44].

Fig. 5
figure 5

Cross-linked GN-amino complexes at acidic and neutral circumstances [45]

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Noteworthy, evidence of GN interaction with secondary and tertiary amines was formerly presented. The methanolic mixtures of GN and benzylamine (primary amine), N-methylbenzylamine (secondary amine), or N,N-dimethylbenzylamine (tertiary amine) were monitored for 48–72 h at room temperature while placing the mixtures in open containers in order to provide oxygen. The GN blue pigment was observed only with the primary amine; benzylamine [47], which is consistent with the above-mentioned reaction mechanisms. As for the secondary and tertiary amines, a reddish golden color was elaborated which indicated the interactions amidst these amines and GN. Such interactions were further confirmed via 1H-NMR [47].

Noteworthy, despite that the above-mentioned reaction mechanisms of the monomeric and polymeric GN are the ones that are chiefly considered when inspecting GN reactions [9, 45, 48], the GN-mediated physical interactions could not be discarded. GN has the capability of establishing H-bonds, and this could influence the procured GN complexes. On one occasion, glucose oxidase cross-linked enzyme aggregates (CLEA) were procured via GN cross-linking. These GN-CLEA were then immobilized onto native carbon felt via the H-bonds, which were established amidst the GN hydroxyl residues and the residues presented by the carbon felt [16]. H-bonds were also debated to be established amidst GN and gelatin (GL) when GN was exploited to cross-link the GL films, which were impregnated with cinnamon oil-loaded pickering emulsion. The cross-linking amidst GN and GL incremented their inter-molecular interactions, and promoted the construction of H-bonds [13].

GN structure also presents hydrophobic traits. These hydrophobic traits were argued to help convert the primary amine-containing immobilizers, which would be activated via GN, into hetero-functional immobilizers. Such hetero-functional immobilizers would be capable of binding enzymes via covalent and hydrophobic forces, owing to GN, and also via anion exchange owing to the protonated amine residues of the immobilizer main matrix [49] (Fig. 6). On another occasion, when GN was exploited to cross-link the CHS graphene oxide (GO) sponges that were intended to function as adsorbents for polystyrene microplastics, triclosan, and diclofenac, a new band was elaborated in the FT-IR spectrum of the GN-CHS-GO at 2917 cm−1 [50]. This newly elaborated band could indicate the contribution of hydrophobic interactions in the interactions amidst GN and the CHS-GO as it was formerly mentioned that changes in the CH-stretching region (3100–2800 cm−1) were associated with alterations in hydrophobic interactions [51]. It was also observed that GN cross-linking incremented the adsorption capacity of the GN-CHS-GO sponges. Nonetheless, the loftiest increment was recorded in case of the polystyrene microplastics, which were the most hydrophobic among the three tested adsorbates [50]. Promoting the adsorption of these hydrophobic moieties might also be regarded to GN hydrophobic traits. It should also be noted that GN cross-linking was frequently argued to increment the hydrophobicity of its cross-linked materials owing to the established covalent linkages [12] and also to the replacement of the hydrophilic entities with the hydrophobic ester residues [13].

Fig. 6
figure 6

Schematic diagram showing the hetero-functional traits of the GN activated primary amine containing immobilizer (recreated as per the postulate of Tacias-Pascacio et al. [49])

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As regards the GN-mediated electrostatic interactions, GN was argued to get de-protonated at highly alkaline pHs [45]. If such de-protonation occurred in an already formed GN complex, electrostatic repulsions would be induced amidst the negatively charged GN and any anionic moieties present within the GN cross-linked complex, such as the anionic biopolymer alginate. Repulsions could influence the cross-linked matrix traits as they could trigger its expansion and increment its water up-take [52].

Influence of pH on GN interactions

Neves et al. [46] inspected the influence of pH and amino acid type on the development of the GN-derived blue pigment, and they deduced that pH was the more influential factor. The pH governs the protonation state of the available primary amino residues, and this is crucial for the interaction with GN as the NH3+ residues could not mediate the nucleophilic attack on GN C3 (Fig. 4) [46], and thus, could not initiate the GN cross-linking reactions (Fig. 5). The inability of NH3+ residues to cross-link GN caused the acidic amino acids, aspartic and glutamic acid, to fail in forming blue color with GN at their native acidic pH (pH 3). However, upon raising their pH to 6.7, the needed NH2 residues were provided, and the GN blue color was attained (Fig. 7). Glycine also offered altered reactivities at altered pHs. At pH equal to its pKa (pH 6) its reactivity was lower than at pH 6.7 (Fig. 7). This lower reactivity was also evidenced by the rates of blue color creation at 590 nm, which were 0.5 and 21.0 absorbance/min at pH 6 and 6.7, respectively. The loftier NH2 abundance and the lower NH3+/NH2 ratio presented at the pH 6.7 were credited for such higher reaction rate. The pH also modified lysine and arginine interactions with GN. The rates of blue color creation at 590 nm were 39 and 17 absorbance/min, respectively, at pH 6.7 whereas these rates elevated to 57 and 23 absorbance/min, respectively, at the native basic pHs (pHs 9 and10, respectively) of lysine and arginine. This was regarded to the presence of more NH2 residues at the higher pHs 9 and 10. Nonetheless, when lysine was exploited to cross-link genipap extract rather than pure GN, the uttermost blueness-index (BI) was recorded amidst pH 6–7. Raising the pH to 9 reduced the BI. The genipap extract comprised proteins and amino-acids which could influence the blue color creation. Glycine also recorded its uttermost BI at pH 6–7 with the genipap extract. Accordingly, the pH 6–7 was recommended for the attainment of the GN blue pigments [46]. Similarly, pH 7 (accomplished via distilled water) was recommended for the attainment of the GN blue pigments following GN-egg protein interactions at 60 °C as no blue color was acquired at the acidic pHs, whereas mauve and dark red colors were acquired at pHs 9 and 12, respectively. Such altered colors were regarded to GN saponification at alkaline pHs and the consequent generation of genipinic acid which interacted with the proteins creating pigments other than blue [53].

Fig. 7
figure 7

The blue colors procured after 40 min at 50 °C via the interaction of GN with altered amino acids in a aqueous solutions, or in b PBS (pH 6.7) buffered solutions [46]

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The near neutral pH 7.4 was also favored by Mi et al. [45], who inspected the GN-CHS cross-linking at altered pHs, as the loftiest GN-CHS cross-linking (96%) was attained at such a pH. The superior cross-linking at pH 7.4 improved the GN-CHS stability, and reduced its swelling and enzymatic degradation as compared to the GN-CHS gels cross-linked at pHs 5, 9, and 13.6 [45]. The NH2 residues presented by CHS (pKa 6.3 [54]) at pH 7.4 would be more than its presented NH3+ residues. Thus, its nucleophilic attack on GN (Fig. 4) would be more efficient and the cross-linking interactions would be promoted. On the other hand, lesser NH2 residues would be presented by CHS at pH 5, and this reduced the cross-linking to 39.9%, and also elevated the GN-CHS swelling and degradation rates. As regards the GN-CHS cross-linked at pH 13.6, it exhibited 1.4% cross-linking only. Such low cross-linking was regarded to GN ring opening polymerization at pH 13.6 (Fig. 3). The polymerized GN would constitute long cross-links amidst the CHS chains. These long cross-links would increment the distance amidst the cross-linked CS and would lower the cross-linking density. Moreover, the solid-state 13C-NMR provided evidence on the reduced GN-CHS cross-linking reactivity at strongly alkaline pHs [45].

On another occasion, the pHs 4, 7.4, 8.8, and 10.5 were inspected during the porcine small intestinal submucosa (PSIM) cross-linking via GN. The PSIM is chiefly collagen (90%). Thus, it could interact with GN via the amino residues of lysine, hydroxylysine, and arginine (0.25%). The authors anticipated that such interaction involved GN ring opening and the formation of the heterocylic GN-amino complex (Fig. 4), which would then get polymerized. The cross-linking reactions were performed with 0.25% GN for 24 h at 37 °C. The most diminished cross-linking (33.31%) was recorded at pH 4 owing to the reduced NH2/NH3 + molar ratio. On the other hand, sizeable cross-linkings of 83.32, 94.18, and 90.77% were acquired at pHs 7.4, 8.8, and 10.5, respectively. These sizeable cross-linkings were coupled with comparable reduced swelling percent (48.57, 43.34, and 45.52%, respectively) and collagenase degradation degrees (3.20, 3.25, and 3.10%, respectively). Nonetheless, the tensile strengths of the pH 8.8 and pH 10.5 GN cross-linked PSIM (11.73 MPa and 12.61 MPa, respectively) were significantly elevated as compared to the pH 7.4 cross-linked specimen (8.01 MPa) despite the elevated cross-linking of the three specimens (> 80%). The inconsistent alterations amidst the cross-linking degree and the tensile strength were observed for altered specimens during this study, and it was regarded to PSIM anisotropy [48]. Based on the aforementioned findings, if the GN-PSIM scaffold was intended for the regeneration of a tissue with tensile strength ≈12 MPa, the alkaline cross-linking would be favored.

Influence of amino acid type on GN interactions

It was stated that amino acids proficiency in the procurement of the GN blue pigments depended on the quantity of primary amino residues that they presented and also on the simplicity of their construction. Thus, when lysine, arginine, glycine, aspartic acid and glutamic acid were compared, the fastest color development (< 5min) was achieved by lysine, which offered 2 primary amino residues. Lysine superior performance was evident at both its native alkaline pH 9 and at pH 6.7 where the loftiest blue color creation rates (at 590 nm) of 57.0 and 39.0 absorption/min were recorded, respectively. Moreover, lysine totally consumed GN after just 30 min of interaction in either of the aforementioned pHs at 60 °C. The next loftiest blue color creation rate, after those recorded for lysine, amounted to 23.0 absorption/min and was recorded for arginine when it was utilized at its native alkaline pH 10 [46]. That is, arginine was less reactive than lysine although both amino acids offered 2 primary amino residues. Arginine pKa (12.48) was loftier than that of lysine (pKa = 10.53). Thus, arginine would offer more NH3+ than would arginine at pHs 9, 10 & 6.7, and this would reduce its reactivity as compared to lysine [46]. Moreover, arginine molecular weight (174.20 g/mol) was loftier than that of lysine (146.19 g/mol) which might have negatively influenced its interaction and reduced its reactivity. Noteworthy, glycine was also profoundly reactive, and it offered a blue color creation rate of 21.0 absorption/ min when it was utilized at pH 6.7. This rate was loftier than that offered by di-primary amino presenting arginine at pH 6.7 (17.0 absorption/min). Thus, glycine exhibited escalated reactivity although it comprised only a single primary amino residue. Such escalated reactivity could be regarded to glycine simple construction and small size which could have lowered its steric effects [46].

Influence of temperature on GN interactions

Elevating the temperature would elevate the energy of GN and the available amino residues. Thus, the collision frequency and the quantity of colliding moieties with enough energy to overcome the barrier would rise [48]. Subsequently, GN reaction rate and cross-linking density would rise upon raising the temperature. For instance, when 0.25% GN was exploited to cross-link the porcine small-intestinal submucosa for 24 h at pH 7.4, raising the temperature from 4 to 25, 37, and 50 °C, raised the cross-linking degree from 41.39 to 55.28%, 83.32, and 91.16%, respectively [48].

On another occasion, when GN-egg protein interactions were exploited to attain blue pigments. The pigment creation rate elevated upon raising the temperature. At 40 and 50 °C, blue pigments were created after 96 h whereas they were created after 72 h at 60 °C. At 70 °C, the pigments were created after 48 h, but a sediment was also observed. Thus, 60 °C was selected for the GN-egg proteins interactions [53].

Influence of GN concentration

The exploited GN concentration could influence altered aspects of the procured GN-amino complexes, such as their cross-linking degree, their water uptake, and their degradation degree [9, 12, 48]. Processing an amine containing matrix with increasing GN concentrations would lead to the cross-linking of increasing amounts of amine moieties, and this would reduce the matrix content of free amine moieties, and would increment its cross-linking degree [12]. For instance, the cross-linking degree of the GN cross-linked CHS films, which were loaded with the astaxanthin, was raised from 22.73 to 45.45% upon raising the GN concentration from 0.5 to 1% (w/w of CHS), respectively [12]. The cross-linking degree of the GN cross-linked porcine small-intestinal submucosa (PSIM), which is mainly constituted of COL (90%), was also raised from 61.54 to 74.47% and then to 81.41% upon raising the GN concentration from 0.03% to 0.06% and then to 0.12% (w/w), respectively. Nonetheless, further raising the GN concentration to 0.5% insignificantly raised the cross-linking degree to 89.33%. Moreover, raising the GN concentration from 0.5 to 1% didn’t increment the GN-PSIM cross-linking degree indicating that 0.5% GN completely consumed the amine moieties in the PSIM [48].

It should also be noted that raising the cross-linking degree of a matrix, as a consequence of incrementing the exploited GN concentration, would cause the matrix to exhibit a denser micro-configuration, and this would lower its free volume. Thus, its water up-take and swelling would be diminished [48]. Moreover, in such a dense micro-environmnet, the penetration of moieties, such as the degrading enzymes, would be sterically hindered. Thus, the matrix degradation would also be diminished upon incrementing the exploited GN concentration. On one occasion, when the GN concentration was raised from 0 to 0.06% and then to 0.12%, the swelling of the GN cross-linked PSIM was reduced from 112.72 to 82.77% and then to 57.96%, respectively. The degradation degree of the GN-PSIM scaffolds were also reduced from 7.48 to 4.69% and then to 3.77% upon raising the GN concentration from 0, to 0.03%, and then to 0.06% [48]. Noteworthy, the exploited GN concentration would also influence other aspects of the procured GN-amino complexes, such as their mechanical traits and the barrier traits [12, 13, 27]. Nonetheless, these influences will be discussed in details in the up-coming sections (Sect. "Bio-plastics and packaging supplies" & "Enzymes immobilization").

Table 3 unveiled that GN was generally utilized at reduced concentrations (≤ 1%) [17, 18, 27, 48, 55,56,57,58,59]. On one occasion, the concentration of GN, which was exploited to cross-link the gelatin (GL)-polycaprolactone (PCL) scaffolds, was raised up-to 2.5%. Nonetheless, raising the GN concentration from 1% to 2.5% induced a modest increment in the GN-GL-PCL scaffolds dry elastic modulus (from 22.0 ± 0.76 kPa to 23.4 ± 0.96 kPa). Another modest increment was recorded in the scaffolds cross-linking degree following such a raise in GN concentration. Moreover, it was stated that both the 1% and the 2.5% GN cross-linked GL-PCL scaffolds kept > 92% of their inceptive mass following 8 weeks in PBS [9]. These results and the acquired modest increments wouldn’t justify the 2.5 fold raise in the exploited GN concentration. Thus, ≤ 1% GN concentrations would be favored. Noteworthy, the cytotoxic cross-linker, glutaraldehyde (GA) [16], was formerly utilized at concentrations reaching up-to 25% [60, 61]. Utilizing GN at the ≤ 1% reduced concentrations would compensate for its escalated cost as it was reported that the cost of GN exceeded that of GA [25]. It is worth mentioning that when both GA and GN were exploited to cross-link and activate CHS microspheres into covalent immobilizers, GA was exploited at 5% concentration whereas GN was exploited at only 0.5% concentration [55].

Table 3 The GN concentrations and cross-linking conditions, which were adopted during the fabrication of altered GN cross-linked materials
Full size table

GN application conditions

Table 3 summarized the conditions adopted during some GN cross-linking procedures which involved altered primary amine providing matrices, such as the polysaccharide, chitosan (CHS) [17], and the proteins, gelatin (GL) [56] collagen (chief component of the porcine small-intestinal submucosa) [48], and soy protein isolate [59]. It could be observed that GN was sometimes applied to solid or already gelled specimens, such as the collagen rich porcine small-intestinal submucosa (PSIM) [48] and the CHS micro-spheres [55], respectively. Moreover, GN was also applied to liquid solutions and suspensions [11, 18, 22, 56]. In such a situation, the GN introduced cross-links might induce the gelation of these liquids, as was the case with the CHS-polyvinyl alcohol solution blend which was converted into hydrogel via GN cross-linking [17]. However, the GN introduced cross-links might only contribute to the gelation processes of these liquids. In such a case the gelation processes would be mainly mediated via another technique, such as the sodium hydroxide induced CHS gelation [18] or the thermo-reversible gelation traits of GL, which would provide GL hydrogels upon cooling [11] (Fig. 8).

Fig. 8
figure 8

Proposed interactions amidst GN, GL, and amino-grafted microfibrillated cellulose (AMFC) [11]

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Table 3 also revealed that, mostly, there was a correlation between the temperature adopted during the GN cross-linking procedure and the duration of such procedure. When escalated temperatures, such as 60 °C and 70 °C were adopted, the cross-linking was allowed to proceed for only 1 or 2 h, respectively [27, 59]. On the other hand, when lower temperatures were adopted (2–8 °C, 4 °C, room temperature or even 37 °C), the cross-linking duration was extended to 8 h [17] and also to 24 h [9, 18, 22, 48]. This could ascribed to the fact that the GN reaction rate would rise upon raising the temperature [48]. Noteworthy, extending the 60 °C incubation of the casted GN-GL solution to 6 h, was mainly intended to dry these solutions [13]. Drying the GN cross-linked CHS-polyvinyl alcohol and CHS-glycerol solutions was also probably the reason for incubating these specimens for 2 days at room temperature and at 35 °C, respectively [12, 58].

GN applications

GN as a colorant

Colorants are critical in altered industries, such as textile and food industries. Nonetheless, there are growing demands for natural colorants as synthetic ones were shown to be carcinogenic and allergenic [35]. The GN blue colorants are natural colorants. Moreover, they present finer thermal, light, and pH stabilities than other commercially attainable natural blue colorants [31]. Thus, they hold significant potential as colorants. Gardenia GN has been utilized as food colorant for many years in East Asia, such as in Japan and Korea [35]. Moreover, GN colorant has been regarded as a “fruit juice” color additive in the United States (US) (Title 21 CFR, Code of Federal Regulations) [35]. Several US, European, and Canadian patents, which regarded GN as a food colorant, were also recently published, such as CA2718604C, which was published on 15 September 2010, and US9376569, which was published on 28 June 2016 [3].

Noteworthy, GN was also exploited in the dyeing of altered fabrics, such as wool, cotton, and silk [53]. Recently, exploited was GN in silk dyeing. Nonetheless, GN was regarded as a cross-linker rather than a dye. The dye was the blue phycocyanin (PC). PC is procured from cyanobacteria, and it presents antioxidant traits. Thus, it could be considered as a sustainable, bio-active dye. Nonetheless, PC comprises a polypeptide chain, and this escalates its molecular weight (220 kDa). Such escalated molecular weight would impede its diffusion into the silk fibres and would lower its up-take. Moreover, bio-dyes exhibit reduced fastness as compared to synthetic dyes. These hurdles were subdued via GN. GN cross-linked the amino entities of PC with those of the silk fibres and immobilized PC onto silk. Utilizing GN at pH 5.8 raised the amount of the silk adsorbed PC from 23.5 mg/g to 74.4 mg/g. The GN-PC silk dyeing was also optimally performed at 70°C rather than the conventional 95°C silk dyeing temperature, which deteriorates silk strength and causes its surface abrasions. Moreover, the GN-PC dyed silk presented escalated fastness versus washing and rubbing (> 4). Immobilizing PC onto the silk fibres via GN also enabled these fibres to achieve escalated ultraviolet protection factor (up to 39) owing to the developed dark shade and the PC UV adsorptive capability. These fibres also presented fine antioxidant attributes. Thus, the application of these GN-PC dyed silk fibres could be extended to pharmaceutical applications [62].

Tissue engineering

An ideal tissue engineering scaffold should be cyto-comptabile [63] and bio-degradable. The scaffolds gradual bio-degradation would permit their gradual substitution with the endogenous cells, and this would lead to the construction of the new tissues [18]. Nonetheless, if the bio-degradation rate was much incremented, the tissue regeneration process would be disrupted [18] as the scaffolds would degrade prior to the construction of the new tissues. Noteworthy, the natural bio-comptabile biopolymers, such as gelatin, chitosan, and collagen, are known for their escalated degradation rates [14, 18, 48]. Thus, these biopolymers were cross-linked with GN in order to retard their degradation whilst retaining their bio-comptability [9, 17, 18, 48] (Table 4). GN cross-linking was also exploited to meliorate and tune other aspects of tissue engineering scaffolds, such as porosity, mechanical attributes, and swelling [9, 17, 18]. Porosity is a critical attribute as sufficient porosity should be demonstrated by the scaffold in order to facilitate the translocation and proliferation of cells. It would also facilitate the translocation of nutrients and metabolites and waste discharge. Nonetheless, if the porosity was much incremented the mechanical attributes of the scaffolds would be impaired [18]. Such mechanical attributes are critical as the scaffold should keep its mechanical stability during the inceptive in-vitro seeding step and also after its in-vivo implantation. Moreover, the scaffolds mechanical attributes should simulate those of the native tissue [9]. A scaffold should also be capable of swelling in order to convey nutrients to the regenerating tissues. The swelled scaffold would also confer larger space for cell adhesion, and would create a finer micro-environment for proliferation [18].

Table 4 Key aspects influenced by GN-cross-linking of altered tissue regeneration scaffolds and membranes
Full size table

GN cross-linked gelatin scaffolds and membranes

Gelatin (GL) is a naturally procured. Moreover, it is commercially acquirable at low cost and it is bio-compatible and bio-degradable. Hence, it was widely investigated in biomedical studies. Nonetheless, GL presents impaired mechanical attributes and escalated degradation rate [14]. Thus, GL was frequently cross-linked with GN in order to subdue these obstacles. On one occasion, the freeze dried scaffolds, which were constituted from a uniform gelatin (GL)-polycaprolactone (PCL) nanofibers dispersion, were cross-linked with GN. The GN cross-linking didn’t significantly impact the, overall, markedly porous construction of the scaffolds as the porosity was only slightly reduced from 98.8% (0% GN) to 98.2% (2.5% GN). On the other hand, the GN cross-linking retarded the 3D-scaffolds degradation rate where > 92% of the GN-GL-PCL scaffolds (0.5–2.5% GN) inceptive masses were kept following 8 weeks in PBS. On the other hand, only 41% mass was kept by the GL-PCL scaffolds. The GN cross-linking also promoted the scaffolds mechanical attributes and progressively raised their dry Young’s modulus from 10.6 kPa (0% GN) to 23.4 kPa (2.5% GN). The scaffolds wet Young’s modulus was also raised from 6.5 kPa (0.5% GN) to 6.7 kPa (1.0% GN), and then to 7.8 (2.5% GN). Noteworthy, such stiffness values were proximate to those of variable organs (kidneys (5–10 kPa), intestines (20–30 kPa), and cardiac muscles (10–15 kPa). Taking into account that a scaffold should exhibit mechanical attributes that mimics its targeted tissue [9], it could be implied that the procured GN-GL-PCL scaffolds could be adequate for the regeneration of altered tissues. In order to further confirm the adequacy of the GN-GL-PCL scaffolds for tissue-regeneration, the scaffolds were seeded with human dermal fibro-blasts (HDF), it was shown that these cells proliferated, spread and adhered to the scaffolds. Thus, the scaffolds cyto-comptability was ensured [9].

GN cross-linked GL membranes were also fabricated and inspected as barrier membranes in dental guided bone and tissue regeneration [56]. The barrier membranes are required to guarantee that the space needed for bone regeneration is not contaminated with undesired cells and is available for bone growth [64]. These membranes should be bio-compatible and should also keep their structural stability at least throughout the early healing stage. The inspected GN cross-linked barrier membranes comprised both GL and hyaluronic acid (HA). GN was postulated to cross-link the amino containing GL. On the other hand, HA was blended in via the action of GL. The lysine NH3+ residues of GL cross-linked the HA carboxyl residues and created stable amides (Fig. 9). The GN-GL-HA membranes were also impregnated with the antimicrobial, hinokitiol, in order to impede any microbial contamination. These membranes were proven to be bio-compatible and antibacterial, and they didn't trigger cytotoxic influences [56].

Fig. 9
figure 9

Proposed interactions amidst GN, GL, and hyaluronic acid [56]

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On another occasion, GN-HA cryogels were formulated. It was postulated that HA reactive hydroxyl entities established glycosidic linkages with GN. The GN-HA cryogels exhibited a lamellar porous construction and their pores size was adequate for cell culture. Hence, the GN-HA cryogels were regarded as a possible non-cytotoxic tissue-engineering scaffold. They could also be adopted in drug delivery and wound healing [63].

GN cross-linked chitosan based scaffolds

Chitosan (CHS) was inspected for tissue regeneration applications owing to its bio-comptability and bio-degradability. Moreover, its structure is comparable to that of the extracellular matrix [17]. On one occasion, CHS was blended with the bio-compatible polyvinyl alcohol (PVA), and this blend was cross-linked with either GN or glutaraldehyde (GA) in order to acquire cross-linked CHS-PVA scaffolds for liver tissue regeneration. Both the GA and GN cross-linked specimens presented little weight losses during the inspection of their bio-degradability in PBS for 7 days whereas the uncross-linked CHS-PVA specimen lost 100% of its weight within 3 days. The uncross-linked CHS-PVA specimen was also shown to be unstable and to dissolve quickly in water during the swelling study. As regards to the GA and GN cross-linked scaffolds, GN provided a somewhat loftier swelling ratio than that provided by the GA cross-linked specimen. However, the swelling difference was small; thus, it could be implied that both specimens demonstrated comparable incremented cross-linking density [17]. Nonetheless, the GN cross-linking provided more bio-compatible scaffolds that permitted loftier cell viability for the human hepatoblastoma cell line (HepG2) than did the GA cross-linked scaffolds [17]. Thus, the GN cross-linked scaffolds hold potential in liver tissue regeneration.

Both GN and GA were also exploited to cross-link the de-cellularized nerve extracellular matrix-CHS (NECM-CHS) scaffolds which were intended for nerve reparation. NECM chiefly comprises chondroitin sulfate, collagen, fibronectin, and laminin, and it is procured from natural nerves. Thus, its physicochemical attributes would be analogous to the autologous nerves, and this would lessen immunological rejection. The GN and GA cross-linking served chiefly to retard the degradation of the scaffolds in order to last during neural tissue in-vivo regeneration. Both the GA and GN cross-linked scaffolds demonstrated significantly reduced dissolution in presence of collagenase and lysozyme as compared to the uncross-linked scaffolds where 30.46%, 27.36%, and 56.12% dissolution degrees were recorded, respectively, following the immersion in collagenase and lysozyme supplemented solutions for 14 days. Moreover, after the in-vivo subcutaneous implantation of the scaffolds, the GN and GA cross-linked scaffolds were persistent after 4 weeks whereas their uncross-linked compeers fully degraded. It was also observed that GN demonstrated loftier histo-comptability than GA. Following 1 week of implantation, copious inflammatory cells were evident amidst the GA-NECM-CHS scaffold whereas only a small quantity of scattered inflammatory cells were evident amidst the GN-NECM-CHS scaffold. Furthermore, the GN-NECM-CHS scaffolds were given the 0 cytotoxic grading whereas the GA-NECM-CHS scaffolds were given the 2 grading. Another merit of the GN-NECM-CHS scaffolds as compared to the GA-NECM-CHS scaffolds was their significantly loftier porosity which reached 89.07%. Pores in scaffolds are critical for cell migration and growth and also for conveying nutrients and metabolites. Noteworthy, the GN-NECM-CHS scaffolds porosity rate was also loftier than the uncross-linked NECM-CHS scaffolds (74.48%). The porous configuration of the GN-NECM-CHS scaffolds was regarded to the GN induced gelation, which occurred prior to the solid–liquid phase separation of the scaffolds [18].

GN cross-linked collagen based scaffolds and membranes

Collagen (COL) is amidst the most significant and prevalent extracellular matrix proteins in mammals. Thus, it was much inspected in tissue engineering [65]. GN was also frequently exploited to cross-link COL in order to meliorate its attributes, such as its impaired mechanical attributes and its rapid degradation, and also its contraction [57, 65]. On one occasion, GN was exploited to avert COL contraction in the 3D gingival tissue equivalents, which were constituted of COL gel impregnated with gingival fibro-blasts and seeded, on top, with gingival keratinocytes. The results indicated that 50 μM GN significantly lowered the 3D gingival tissue equivalents contraction whilst presenting commensurate cell proliferation capability to that presented by their uncross-linked analogue. Nonetheless, it was reported that 150 μM GN exhibited cytotoxic traits with respect to skin fibro-blasts [65]. Thus, GN concentration should be carefully selected.

Noteworthy, the COL in Wharton's Jelly (WJ) also enabled its GN cross-linking. WJ matrix is procured from the umbilical cord and it comprises mesenchymal stromal cells embedded in an extracellular matrix (ECM). Such ECM comprises growth factors and pro-healing macro-molecules, such as hyaluronic acid and COL. Thus, WJ could be exploited in regenerative medicine. Nonetheless, perinatal tissues are unstable and are active for only a brief duration after postpartum. Such hurdle was subdued by the homogeneous de-cellularization of WJ. However, other hurdles averted the exploitation of acellular WJ in bone regeneration, such as its impaired mechanical attributes and its quick in-vivo resorption. WJ was completely resorbed during 3 weeks [57] which would be insufficient for bone regeneration. Thus, WJ films were cross-linked with GN in order to meliorate their mechanical attributes and slow their degradation. GN cross-linking significantly raised the films Young’s modulus indicating the attainment of stiffer films. Furthermore, it rendered the COL containing WJ films much resilient to collagenase mediated degradation. The uncross-linked WJ films lost 100% mass in the collagenase supplemented solution whereas the WP films, which were cross-linked with 0.05 mg GN/mg dry WJ, lost < 40% mass. The degradation was further reduced by raising the GN concentration till 0.2 mg GN/mg dry WJ. The GN cross-linked WJ films were regarded as auspicious bio-materials if GN concentration was carefully selected. The reduced GN concentration (0.05 mg GN/mg dry WJ) maintained adequate films hydrophilicity and porosity (> 85%) which would promote cellular adhesion and proliferation. On the other hand, more hydrophobic and less porous films were acquired with the loftier GN concentrations (0.1 and 0.2 mg GN /mg dry WJ). Furthermore, calcified nodules, which reflect intense immunological response to implants, were evident following 8 weeks of implanting the WJ films, which were cross-linked with 0.2 mg GN/mg dry WJ [57].

GN cross-linked scaffolds were also procured after GN had cross-linked the porcine small-intestinal submucosa (PSIM). PSIM is a de-cellularized extracellular-matrix which is mainly constituted of COL (90%). PSIM scaffolds exhibits reduced immunogenicity, fine bio-compatibility, and are approved by FDA for altered reconstructions[48]. The PSIM scaffolds were cross-linked with GN so as to retard their degradation in order to extend their existence during tissue regeneration. Cross-linking the PSIM scaffolds at pH 7.4 and 37 °C for 24 h lowered their collagenase mediated degradation degrees from 7.48% (0% GN) to ≈3.5% (0.06–1% GN). Such lowered degradation was regarded to the emended collagenase cleavage points and also to the steric hindrance imposed on collagenase penetration by the closely packed cross-linked GN-PSIM matrix. GN cross-linking also lowered the swelling percent of the PSIM scaffolds from 112.72% (0% GN) to 57.96% (for 0.12% GN concentration at pH 7.4 & 37 °C for 24 h) [48]. That is the GN-PSIM were still capable of swelling and up-taking water which is crucial for conveying nutrients to the regenerating tissues. Moreover, GN cross-linking raised the scaffolds tensile strength (from 6.26 MPa (0% GN) to 7.55 MPa (0.12% GN) and 9.95 MPa (0.5% GN)) whilst keeping their cyto-comptability [48].

Wound dressings

Altered GN cross-linked hydrogels were fabricated and inspected as wound dressings. On one occasion, a potential wound dressing was fabricated after utilizing GN to create covalent cross-links betwixt the amino entities of gelatin (GL) and those of the amino-grafted microfibrillated cellulose (AMFC). GL and AMFC were also postulated to interact together via the construction of the hydrogen bonds which would stabilize the triple helix configuration of GL (Fig. 8). Noteworthy, the merger of AMFC with GL, in absence of GN, didn’t significantly modify the hydrogel compressive stress (at 80% strain). On the other hand, when AMFC (5%; with respect to GL) was merged with GL in presence of 1.0% GN (with respect to GL), the compressive stress was raised from 0.04 to 0.75 MPa. The GN established covalent cross-links raised the hydrogel mechanical attributes. These cross-links also kept the structural integrity of the hydrogel during its swelling which amounted to 32.9% after 24 h immersion in PBS at 37 °C. On the other hand, the AMFC-GL films, without GN, dissolved after 5 h of immersion in PBS at 37 °C [11]. Noteworthy, wound dressings should be capable of swelling in order to uptake the wound exudate. Nonetheless, they should keep their wholeness after swelling in order to avert secondary wound damage, and this was the case with the GN-AMFC-GL films. The GN-AMFC-GL films cyto-comptability was also proven via in-vitro analysis. As regards to the in-vivo analysis, the percent of remaining wound-area in case of the 2% GN-5% AMFC-GL films was significantly lower than the control after 2 days [11].

On another occasion, a wound dressing was fabricated from the GN cross-linked chitosan (CHS)-collagen (COL) hydrogel [10]. Both CHS and COL comprise primary amine entities. Moreover, CHS could encourage wound healing as its constituting units, N-acetyl glucosamine, stimulate hemostasis and speed-up tissue regeneration. CHS also exerts antimicrobial traits via its cationic entities which could induce cytoplasmic matter seepage and cell death following their interactions with the bacterial cell-wall. As regards to COL, it presents fine homoeostatic traits in addition to its bio-degradability and reduced immunogenicity. The GN-CHS-COL was further impregnated with cefotaxime sodium loaded silver nanoparticles in order to boost its antimicrobial attributes, and was tested as a wound dressing. This dressing achieved > 98% wound closure following 2 weeks [10].

GN-chitosan (CHS)-polyvinyl alcohol (PVA) films were also inspected as wound dressings. GN mediated CHS cross-linking raised the films tensile strength from 18.53 MPa (0% GN) to 27.99 MPa (0.5% GN). The GN-CHS-PVA were also capable of up-taking water and retaining their wholeness, and this indicated their suitability as wound dressings as they could up-take the wound exudate and retain appropriate moisture amidst the wound to fasten heeling. Curcumin was also integrated within the GN-CHS-PVA in order to encourage wound healing. The curcumin loaded GN-CHS-PVA films were bio-compatible. These films also mediated quicker healing in the wounds induced on albino Wistar rats, which further verified their suitability as wound dressings [58].

Noteworthy, Zheng et al. [66] incorporated GN in a dual cross-linked wound dressing material. The initial cross-linking was the physical cross-linking amidst a self-assembling peptide, which comprised a biologically effective peptide (Gly-Phe-Phe-Tyr-Gly-Arg-Gly-Asp) fused to indomethacin (IDM). The physical-cross-linking of this peptide, which was termed IDM-1, was allowed to proceed in presence of CHS and GN which would get chemically cross-linked to each other. CHS was exploited owing to its hemostatic and antimicrobial traits. On the other hand, the utilized peptide would be hydrolyzed via the proteases in the wound area and would release the anti-inflammatory, IDM, and the Arg-Gly-Asp tripeptide, which could promote wound healing via interacting with integrin receptors [66]. The GN-CHS cross-linking meliorated the strength of the acquired hydrogels, and this was reflected by the rise in the hydrogel storage modulus (G’) from 69 Pa (plain IDM-1) to 4630 Pa (IDM-1-GN-CHS). Moreover, IDM-1 degradation was significantly lowered following the incorporation of GN and CHS. Around ≈18% of IDM-1 in the IDM-1-GN-CHS hydrogel was degraded following 24 h processing with 30U/ml proteinase-K whereas ≈ 67% IDM-1 in the plain IDM-1 hydrogel was degraded following akin processing. The authors regarded such lowered degradation to the covalent GN-CHS cross-links which wouldn’t be influenced by the proteinase. Nonetheless, the IDM-1 also presented primary amine entities [66]. Thus, it might also be covalently cross-linked with GN and this could have contributed to its much lowered degradation rate which would prolong the lifetime of the wound-dressing and would also provide a more extended release of the incorporated active entities. Noteworthy, the proposed IDM-1-GN-CHS hydrogel was proven to be bio-compatible. Moreover, its in-vivo topical administration speeded up wound closure and accomplished full recovery after 15 days [66].

Drug delivery vehicles

GN cross-linked nano and micro-particles

Curcumin (CR) is a hydrophobic phenolic bio-compound which presents antioxidant and anti-inflammatory traits. However, CR solubility is impaired, and this lowers its absorption and its bio-availability. Moreover, CR is temperature, light, O2, and pH sensitive [67, 68]. If CR was encapsulated, its absorption and bio-availability would be meliorated. Furthermore, the capsule material would shield it from the destabilizing agents, and this would meliorate its stability. Accordingly, CR was nano-encapsulated within GN cross-linked human serum albumin (HSA) nano-particles which were then grafted with tannic acid (TA). The procured CR-GN-HSA-TA and the CR-GN-HSA nano-particles were stable for 7 days whereas the size of the CR-HSA nano-particles, which were devoid of GN, was ≈4 folds incremented after 7 days. Moreover, the GN devoid CR-HSA nano-particles presented a 0.303 polydispersity index (PDI) on day 3 whereas the CR-GN-HSA nano-particles PDI amounted to only 0.190. These findings reflected the stabilizing effect conferred by the cross-linking action of GN. Such GN cross-linking also escalated CR encapsulation efficiency. Moreover, encapsulating CR regulated its release, and 60% cumulative release was observed after placing the CR-GN-HSA-TA within dialysis bags whereas all the free CR was released within 10 h. Noteworthy, the CR-GN-HSA-TA nano-particles were inspected as vehicle for CR oral delivery in order to treat ulcerative colitis. In this particular aspect, the TA averted the particles gastric disruption, and promoted their adhesion to the colon lesions via its anionic traits. Thus, the oral uptake of the CR-GN-HSA-TA significantly relieved colitis symptoms during the in-vivo mice study [67].

Theaflavin-3,3′-digallate (TF-3) is another phenolic bio-active moiety. TF-3 is procured from black tea and it is a poly-hydroxylated hydrophilic poly-phenol. Theaflavins were shown to inhibit altered cancer cells. Nonetheless, their in-vitro efficiencies might not be displayed in-vivo owing to their low intestinal permeability. Chitosan (CHS) is an intestinal permeation promoter that is capable of promoting the intestinal permeation of hydrophilic moieties via opening the narrow junctions betwixt the epithelial cells of the intestines in a temporary and reversible manner. Thus, CHS nano-particles could be exploited to encapsulate theaflavins and promote their intestinal permeation [69]. However, ionic complexation is commonly exploited for the construction of CHS complexes [70]. Such ionic CHS complexes would be adversely influenced by the pH alterations that would be encountered in the GIT. Accordingly, the electro-statically associated CHS-casein phosphopeptides (CHS-CP) nano-particles were cross-linked with GN and were inspected as a potential delivery vehicle for TF-3 [69]. The GN cross-linking provided the nano-particles with stable covalent cross-links and promoted their pH stability. Such promoted pH stability was proved via comparing the size of the CHS-CP nano-particles, which were placed for 2 h at pH 2 (to mimic the gastric incubation), with their inceptive size (at pH 6). The size of the uncross-linked CHS-CP nano-particles was much enlarged owing to the induced acid disruption. On the other hand, the size of the GN cross-linked CHS-CP nano-particles was much less affected by the acid incubation. For instance, the size of the GN cross-linked CHS-CP particles, which were formulated with 0.5 or 1 mg/ml GN and were allowed to cross-link for 8 or 16 h, remained more or less the same following the acid incubation [69]. Nonetheless, the above-mentioned 0.5 mg/ml GN cross-linked CHS-CP particles were more favored owing to their loftier cyto-comptability where they achieved > 80% cell viability in-vitro. The TF-3 loaded GN cross-linked CHS-CP particles also significantly promoted the epithelial permeation of the TF-3 in-vitro [69].

Casein phosphopeptides (CP) were also individually cross-linked with GN. The antioxidant attributes of CP`were intended for the treatment of the inflammatory bowel disease. Nonetheless, these peptides are prone to hydrolysis and enzymatic disruption. Thus, they were stabilized via GN cross-linking. The procured GN-CP nano-particles were stable in simulated gastric fluid at 37°C, and no significant morphological modifications (via transmittance-electron-microscope) were observed even after 72 h [23]. Moreover, the 600 nm absorption intensity (GN blue color was reported to present maximum absorption at 578–603 nm [71]) of GN-CP wasn’t remarkedly lowered after the 72 h gastric simulated incubation. GN stabilized the GN-CP nano-particles versus the acidic and enzymatic disruption of the gastric environment [23], and this would promote their oral administration. The GN-CP nano-particles were also stable in simulated intestinal fluid, but their size was slightly incremented after 72 h owing to their aggregation. Nonetheless, their desired antioxidant attributes weren’t altered after 24 h of the intestinal simulated incubation as compared to the GN-CP nano-particles, which were kept in water. The GN-CP cyto-comptability was also evident and > 80% cells viability was recorded for the GN-CP 100μg/ml concentration. Moreover, the fluorescently-labeled GN-CP were shown to preferentially aggregate at the site of inflammation in colon (Fig. 10). The inflamed colon has abundant charged proteins and aggregated inflammatory cells, and this could promote the preferential aggregation of the negatively charged GN-CP nano-particles. The GN-CP were also tested in-vivo in mice with DSS induced colitis, and they were shown to encourage crypt regeneration and lower the inflammatory cells infiltration. Thus, the GN-CP could be regarded as auspicious oral therapy for inflammatory bowel disease [23].

Fig. 10
figure 10

a The preparation of GN cross-linked casein phosphopeptide nano-particles (GCPP NPs as abbreviated by Ma et al. [23]), and b their preferential aggregation in the colon inflamed site in order to scavenge reactive oxygen species (ROS) and treat the inflammatory bowel disease [23]

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On another occasion, GN was exploited to cross-link the bovine serum albumin nano-particles, which were loaded with the anticancer drugs, neratinib and silibinin. These nano-particles were intended for treating triple negative breast cancer via intravenous mode [72]. Noteworthy, GN cross-linked micro-particles were also fabricated and inspected for drug delivery. For instance, GN cross-linked the bio-engineered mussel-adhesive-protein (MAP) in order to fabricate uniform micro-particles which were also impregnated with magnetic nano-particles (iron oxide). These micro-particles were regarded as magnetic microbots. Microbots are small mechanical robotics which could be controlled and guided toward a specific area by means of outside cues or environmental modifications. The proposed magnetic GN-MAP micro-particles were intended to get localized in the esophagus via an external magnetic field (Fig. 11). The bio-engineered MAP would then cause these particles to adhere to the esophagus despite the highly dynamic nature of the esophagus which would normally clear the drugs out as MAP presents outstanding underwater adhesive capabilities.Thus, the drug loaded magnetic GN-MAP micro-particles could be exploited in the targeted therapy of esophageal cancer [73].

Fig. 11
figure 11

Diagrammatic illustration of targeted medications delivery via the magnetic GN-MAP micro-particles [73]

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GN cross-linked hydrogels

GN cross-linked CHS and GL hydrogels were occasionally fabricated as delivery vehicles for drugs and bio-active moieties. For instance, the bio-active curcumin (CR) was nano-encapsulated within chito-oligosaccharides (COS)/poly-ɣ-glutamic acid (PGA) nano-particles, and the procured CR-COS-PGA nano-particles were impregnated within GN cross-linked GL films. Such impregnation raised the stability of the nano-particles by 174%, and it also significantly retarded CR in-vitro release. Moreover, the GN-GL incorporated CR-COS-PGA nano-particles presented 1.17 folds loftier anti-oxidant traits than their free analogues. Both porcine GL and GN were shown to present anti-oxidant traits which were potentiated by their combined presence (synergistic). Accordingly, the GN-GL films impregnated with the CR-COS-PGA nano-particles could formulate an efficacious CR delivery system which could be utilized in food industry [68].

On another occasion, GN cross-linked CHS hydrogels were inspected for the oral delivery of the non-steroidal anti-inflammatory drug, diclofenac sodium. Only 4.9% and 6.1% of the incorporated diclofenac were discharged following 1h and 2h incubation, respectively, in simulated-gastric-fluid whereas 80.5% and 88.2% were discharged following 1 h and 2 h incubation, respectively, in simulated-intestinal-fluid [22]. The GN covalent cross-links stabilized CS at the gastric acidic pH and lowered the diclofenac release. Thus, the GN cross-linked CHS hydrogels enterically protected diclofenac while permitting its discharge within the highly absorptive intestines, and this would make suitable for diclofenac oral administration [22].

Muco-adhesive buccal patches are auspicious vehicles for oral drug-delivery. GN was exploited to cross-link CHS in a buccal patch that comprised 4 electrospun layers, 3 electro-spayed antibiotic loaded nano-carrier layers, and a poly(caprolactone) layer. The electrospun layers comprised two muco-adhesive polymers, poly-vinyl alcohol (PVA) and CHS, and the analgesic, ibuprofen. GN cross-linked CHS in these electrospun layers in order to modulate the swelling of the patches and to increment their mechanical attributes. Swelling is critical for muco-adhesion as the inceptive muco-adhesion step comprises the wetting, spreading, and swelling of the patches in order to establish intimate contact with the mucosa. Nonetheless, if the swelling was aggravated, the patches surfaces would become slippery and their muco-adhesion would be lowered [74]. Thus, the patches should swell but not to an aggravated extent. GN (0.05 g) modulated the swelling index of the 80 PVA/20 CHS formulated patches and lowered it from > 300% to 208%. Such lowering was coupled with a rise in the muco-adhesion strength from 21.37 to 39.11 g. Further incrementing the GN content to 0.1 and 0.3 g, further reduced the patches swelling. Nonetheless, such reductions were coupled with reductions in muco-adhesion. Thus, the 0.05 g GN containing patches were the optimal with respect to their swelling. The tensile strength of these patches was also 2.04 folds loftier than their uncross-linked analogues. Moreover, they allowed > 90% cells viability in-vitro. It was also shown that the 0.05 g GN cross-linked patches released 19% of their loaded ibuprofen after their incubation in simulated saliva solution for 2 h (burst release). Such burst release was lower than that recorded for the uncross-linked patches (39%) [74]. Lowering the ibuprofen burst release in such a manner would still enable a quick analgesic effect. Nonetheless, it would avert the dangerous influences of releasing excess ibuprofen. The ibuprofen cumulative 48 h release was also significantly retarded for the 0.05 g GN cross-linked patches as compared to their uncross-linked analogues. Such retarded drug release was ascribed to the GN introduced cross-links, which rigidified the gel construction and restricted its chains motility [74].

GN was also incorporated within trans-dermal patches. Trans-dermal drug delivery vehicles discharge medications into systemic circulation. Thus, liver metabolism could be avoided [21]. This would be beneficial in case of drugs that might induce liver injury, such as paracetamol. Moreover, the trans-dermal administration of paracetamol would avert the incidence of the side effects, which are coupled with its oral administration, such as diminished appetite and stomach upset. Accordingly CHS-glycerol-phosphate di-sodium salt (GPD) thermo-responsive hydrogel, which was deposited onto silk, was inspected as a trans-dermal patch for paracetamol delivery. Nonetheless, the aforementioned CHS hydrogel presented wide pores (≈50 μm). These wide pores wouldn’t restrain the small sized paracetamol moieties, and thus, wouldn’t allow for paracetamol controlled release, which would lower the frequency of paracetamol administration. Thus, GN was exploited to cross-link the CHS based hydrogel in order to narrow its pores and also to meliorate its mechanical attributes. Utilizing GN at 0.01 wt% (of the CHS/GPD weight) narrowed the hydrogel pores to ≈5 μm owing to the GN introduced cross-links which created a denser hydrogel construction. The compressive strength (at 50% strain) of the hydrogel was also incremented from ≈1 kPa to 10 kPa following GN cross-linking. Moreover, paracetamol sustained release duration was extended from 6 to 12 h upon incrementing GN content from 0 to 0.025 wt%. The narrower pores impeded the fast diffusion of paracetamol and prolonged its release duration. Nonetheless, when GN content was incremented to 0.04 wt%, paracetamol release duration was lowered to 3h. It was argued that the excessive GN cross-linking blocked the majority of the hydrogel minute pores and lowered its trapping capability, and thus, paracetamol was instantly discharged [21]. Thus, the GN content shouldn’t exceed 0.025 wt% in order to achieve paracetamol sustained release.

GN in sensors and analysis

GN based sensors were prepared in order to mark food spoilage via the detection of biogenic amines (BA). BA are created during food spoilage via amino acids oxidative microbial hydrodecarboxylation. The BA are the reason of the rotten meat smell. Nonetheless, human health could be much harmed at BA concentrations lesser than the smell threshold [47]. Thus, BA sensors are important. BA present primary amino residues; hence; in presence of oxygen, they can interact with GN creating visible blue pigments. The proposed BA sensor comprised 2% alginate beads in which GN was entrapped at a 5 mg/ml concentration. These beads were placed in the vicinity of the volatile BA, putrescine (0.1 mmol) (Fig. 12), at room temperature. Within 24 h, the beads morphology was altered from colorless to blue indicating the interaction amidst the entrapped GN and putrescine vapor. Nonetheless, when the entrapped GN and putrescine were placed at 4 °C, no color alteration was observed [47], and this confirmed the impact of temperature on the GN-amine interactions.

Fig. 12
figure 12

GN loaded beads after being exposed to putrescine vapors for 24 h at room temperature [47]

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The impact of temperature on the GN-amine interactions probably caused the selection of escalated temperatures (80°C and 100°C) during the GN based colorimetric analysis of altered amines. Such escalated temperatures enabled the colorimetric analysis to be accomplished in ≤ 1h [71, 75]. On one occasion, the amino residues in water-soluble chitin-derivatives were quantitatively inspected via GN. Chitin, the second most plentiful natural polysaccharide, comprises N-acetyl-D-glucosamine units. These units could be de-actylated into D-glucosamine, and this would lead to the procurement of CHS. Thus, estimating the quantity of the glucosamine free amino residues could distinguish chitin from CHS. The proposed analysis comprised mixing 500 µl of the water soluble specimens (chitin-oligosaccharides, CHS-oligosaccharides, glucosamine) with 500µl of a GN solution (1 mg/ml; 4 days old). Such mixtures were then put for 30 min in a 100°C water-bath, cooled in an ice-bath (20 min), and measured (after 5 min) at 589 nm. The acquired blue color was very stable and the Beer-Lambert’s law was followed along a 50–300 mg/l D-glucosamine concentration range [75].

On another occasion, GN proficiency in the colorimetric detection and quantitation of amino-acids was evaluated in comparison to the commonly utilized, ninhydrin. Gardenia geniposide was extracted and was hydrolyzed to GN via β-glucosidase [71]. Afterwards, 0.1 mM ethanolic GN solutions were mixed with the 20 standard amino acids, which were dissolved in 100 mM phosphate buffer (pH 7). The GN-amino mixtures were placed for 1 h at 80°C. A blue color, with maximum absorption at 578–603 nm, was elaborated with all inspected amino acids. Nonetheless, a yellow color (440 nm) was elaborated with proline [71]. This different color indicated a different mode of interaction which could be regarded to the absence of primary amino residues in proline. It was also observed that, with the exception of histidine and aspartic acid, the molar absorptivities of the blue GN-amino complexes were loftier than those of the respective ninhydrin-amino reaction products. The largest differences were recorded in case of the GN-glutamic acid and the GN-asparagine whose molar absorptivities were 11.05 and 14.66 folds loftier, respectively, than their ninhydrin analogues. Afterwards, the influence of amino acids concentration was inspected for glycine, tyrosine, arginine and glutamic acid. The absorbance (590 nm) exhibited linear increments along 10 µM to 1 mM amino acid concentration range. It was also observed that the GN-amino blue color was stable for monthes on TLC plates [71].

GN was also exploited in the detection and degradation of chlorpyrifos (CPF), which is amongst the most frequently utilized organo-phosphorus pesticides [29]. GN was initially exploited to activate amino grafted cellulose nanofibres (CFs) in order to covalently bind acetylcholinesterase enzyme (AChE). Afterwards, the GN activated CFs were exploited as a fluorophore. GN interaction with the primary amine residues is known to create blue pigments. However, fluorescence emission was also observed. Such fluorescence was probably induced by the transitions within the sizeable heterocyclic conjugated system of the GN-amino complex. The fluorescence emission peak of the GN-aminated cellulose complex was recorded at 421 nm with a 320 nm excitation wavelength. Thus, the GN activated CFs would present fluorescence emission peak. Such peak wasn’t modified following AChE immobilization. Nonetheless, when the AChE substrate, acetylthiocholine iodide (ATCI), was introduced such peak was quenched (Fig. 13). AChE breaks ATCI into thiocholine and acetic acid. Thiocholine would bind to the nitrogen in the GN activated CFs via its sulfur entities, and this would quench the fluorescence. However, if the pesticide, CPF, was present, it would block the active site of AChE. Thus, ATCI breakage and thiocholine production would be diminished and the quenching of the GN activated CFs fluorescence would also be diminished. Accordingly, if the concentrations of the AChE loaded GN activated CFs (AChE@CFs) and ATCI were kept constant, the alteration in the fluorescence would be directly related to CPF concentration [29].

Fig. 13
figure 13

Detection of chlorpyrifos (CPF) via acetylcholinesterase, which was bound to GN activated amino grafted cellulose nanofibres (AChE@CFs) [29]

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Noteworthy, the CPF detection limit by the proposed AChE@CFs was 90 nM and the response was linear upto 80 μM CPF. Moreover, this system was selective for CPF in presence of heavy metals, amino acids, and also other pesticides. It should be noted that, the immobilized AChE would also degrade CPF into non-toxic metabolites. This degradation reached 89% following 36 h incubation with the AChE@CFs [29].

Bio-plastics and packaging supplies

Petroleum-based plastics are extensively exploited in packaging [76]. However, the disposal of these non-degradable plastics triggers environmental issues [15]. Moreover, exploiting these plastics could trigger food safety issues [13]. The extensive exploitation of these petroleum-based plastics could also contribute to resource depletion [15]. Thus, bio-based and biodegradable packaging supplies are currently widely inspected as surrogates to the petroleum-based packaging supplies in order to subdue their disadvantages [13, 15, 76]. The bio-based packaging supplies could be derived altered biopolymers, such as the polysaccharide, chitosan [12] and the proteins, soy protein and gelatin [59, 77]. Nonetheless, bio-based packaging supplies generally exhibit impaired mechanical and water barrier attributes [12, 13, 59, 77, 78]. The mechanical attributes of packaging supplies are responsible for keeping their integrity during the products processing, transporting, and commercial display [76]. Moreover, water is critical for food spoilage reactions [12]; thus, the packaging supplies should present fine water barrier attributes to minimize water content and prolong the shelf-life of the packaged goods. In order to alleviate the reduced mechanical and barrier attributes of the bio-based packaging supplies, these supplies were occasionally cross-linked with GN. Some examples of this were discussed in the following sections and were given in Table 5.

Table 5 Key aspects influenced by GN-cross-linking of altered packaging supplies and bio-plastics
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GN cross-linked chitosan based packaging supplies

Chitosan (CHS) is a biodegradable and safe polysaccharide. It is also cost-effective and available as it is acquired from the second most prolific natural polysaccharide, chitin [78, 79]. Furthermore, CHS presents antioxidant and antimicrobial attributes, and it could be simply prepared as films [12, 15, 78, 80]. Accordingly, CHS was much inspected for the food packaging applications [81]. Nonetheless, CHS presents reduced stability in acidic media, reduced moisture barrier attributes, and reduced mechanical strength which impedes its actual exploitation in food packaging [12, 78]. Thus, CHS films, which comprise abundant primary amine entities, were occasionally cross-linked with GN in order to meliorate their attributes.

On one occasion, GN was included within the core of the thymol loaded CHS-polyethylene oxide (PEO) nano-fibres in order to cross-link the CHS in the fibres shells. The films fabricated from the GN (0.1–1%) cross-linked nano-fibres were more resistant to water dissolution. Moreover, exploiting GN at 0.1–0.7% concentrations raised both the films tensile strength (TS) and elastic modulus (EM). On the other hand, the films elongation percent (E%) got progressively reduced as the GN concentration escalated from 0.1 to 1%. The covalent cross-links betwixt CHS and GN rigidified the films and lowered their mobility [81]; hence, their E% values were reduced. On another occasion, the CHS films, which were loaded with the antioxidant, astaxanthin, were cross-linked with GN at 0.5, 1 and 1.5% concentrations (w/w of CHS). Such cross-linking influenced the mechanical attributes of the CHS-astaxanthin films. The films TS values got progressively raised from 10.17 MPa (0% GN) till 17.82 MPa (1.5% GN). On the other hand, the films E% got progressively reduced from 65.69% (0% GN) till 11.87% (1.5% GN). It was argued that the GN introduced cross-links strengthened the CHS-astaxanthin films. Nonetheless, the created 3D network impeded the sliding of CHS molecular chains [12], and thereby, reduced the E% (Table 5). GN cross-linking also influenced the CHS-astaxanthin films water vapor transmittance rate (WVTR). The WVTR was initially reduced from 4.83 g/h m2 (*10–6) (0% GN) to 4.55 g/h m2 (*10–6) (0.5% GN) and then to 4.47 g/h m2 (*10–6) (1% GN). Nonetheless, further escalating the GN concentration to 1.5% significantly raised the films WVTR to 5.76 g/h m2 (*10–6). Such WVTR raise was regarded to the imbalanced hydrophilic/hydrophobic components ratio which caused the CHS matrix to be more accessible for water transfer. Noteworthy, the GN cross-linked CHS-astaxanthin films also presented loftier oxygen transfer rate than their uncross-linked analogue. For instance, the oxygen transfer rate of the 1.5% GN cross-linked films amounted to 41.13 cc−1 m−2 day−1 whereas that of the uncross-linked films was 38.44 cc−1 m−2 day−1. The GN-CHS covalent linkages escalated the films surface hydrophobicity and this promoted the adsorption of the un-polar O2 and raised the oxygen permeability [12].

Noteworthy, GN cross-linking would lower the transparency of the CHS films [12] owing to the developed bluish color. This lowered transparency would boost the films light protective traits [82]. Nonetheless, the bluish coloration could hamper the exploitation of the CHS films if transparent packaging films were required. Accordingly, it was attempted to decolorize the GN-CHS films via oxidative reactions [78]. These reactions were mediated via H2O2 which would yield HO• and HOO• radicals. These radicals would disrupt the GN-CHS films conjugated double bonds which granted the films their bluish-green appearance. However, in order to limit the chemical exposure of the GN-CHS films to H2O2, the multi-copper oxidase enzyme, laccase, was also involved in the oxidative decolorization of the GN-CHS films together with ABTS (2,2'-azinobis-(3-ethylbenzothiazoline6-sulphonic acid)). Noteworthy, ABTS functioned as a mediator. Mediators create stable radicals capable of oxidizing the moieties that couldn’t be directly oxidized via the exploited enzyme. Accordingly, the optimal GN-CHS decolorization process comprised processing the GN-CHS films for 24 h at 37°C with 1U/ml laccase in presence of 1 mM ABTS in pH 7 sodium phosphate buffer (0.1M). This step altered the color of the films from bluish-green to light brown. The light brown films were then processed for 30 min at 40°C with 5% (v/v) H2O2 at pH 11 in order to acquire the yellowish transparent appearance. It should be noted that the aforementioned decolorization protocol didn’t significantly modify the GN-CHS films acid stability or antioxidant attributes. Moreover, it raised the films TS from 26.59 to 63.3 MPa. Nonetheless, it lowered the films E% from 18.2 to 3.23%. Noteworthy, the raised TS of the decolorized samples was coupled with 33% reduction in their thickness [78]. This thickness reduction could contribute to the raised TS as during the reckoning of the TS the force applied to the film is divided by its inceptive cross-sectional area [83]. It was also argued that aldehyde residues were elaborated during the decolorization process. These aldehyde residues would create Schiff’s bases with the amine entities, and this could contribute to the stability of the polymer. As regards to the lowered E%, it was ascribed to the loss of glycerol residues following H2O2 interactions. Glycerol was the plasticizer in the GN-CHS films [78].

GN cross-linked gelatin based packaging supplies

Gelatin (GL), which is procured via collagen denaturation, is biodegradable, and it also presents outstanding film constituting capability. Thus, it could be exploited to formulate edible packaging films. Nonetheless, its impaired mechanical and barrier attributes and its water sensitivity [13, 77] should be first meliorated. This was formerly accomplished via GN cross-linking. On one occasion, cross-linking the GL films, which were impregnated with cinnamon oil (CO) loaded pickering emulsion, with increasing GN concentrations progressively raised their TS from 3.54 MPa (0% GN) to 20.22 MPa (5% GN with respect to the GL content). The E% of the GL-CO films was also progressively raised as the GN concentration escalated from 0 to 1 and then to 3% (with respect to the GL content). Nonetheless, further escalating the GN concentration to 5% lowered the E% to 233.19%, but this value was still loftier than that of the uncross-linked films (E% = 187.23%). These meliorated mechanical attributes were ascribed to the GN introduced cross-links which created a polymer network with dense construction. Such dense network would allow the loads from external forces to be quickly transferred amongst the molecular chains; thus, more force would be withstood. Furthermore, GN cross-linking averted the accumulation of the pickering particles, and created a uniformly distributed micro-structure, which would efficiently distribute load and avert stress concentration [13]. Thus, the mechanical attributes of the GL-CO films were meliorated. The films barrier attributes were also meliorated, and this was evidenced by the progressive reductions recorded in the films oxygen permeability and water vapor permeability (WVP) upon escalating the GN concentration. For instance, a 0.24 *10–8 g cm−1.s−1.Pa−1 WVP was recorded for the 5% GN cross-linked films whereas a significantly loftier 0.48*10–8 g cm−1.s−1.Pa−1 WVP was recorded for their uncross-linked compeers. The reduced WVP was ascribed to the cross-linked 3D network, which was created following GN cross-linking, as it presented a superior barrier to water. Moreover, the replacement of the hydrophilic entities with the hydrophobic ester residues [13] raised the films hydrophobicity and lowered their WVP. Noteworthy, GN cross-linking also potentiated the antimicrobial attributes of the GL-CO films. It was argued that the denser construction of the GN-GL-CO films prolonged the path for CO release. Thus, the loss of CO was lowered and it was released in a sustained manner [13].

Iahnke et al. [77] exploited the residual GL (R-GL), which remained after the fabrication of the soft GL capsules, in order to formulate packaging films. The R-GL was cross-linked with either GN or the cytotoxic glutaraldehyde (GA) at 0.15% (w/w) concentration. GN cross-linking raised the films TS from 2.72 MPa to 4.23 MPa. A comparable escalation in the films TS was also recorded following their GA cross-linking despite that GA achieved a loftier cross-linking degree (50%) than did GN (41%). Moreover, GN raised the films E% from 168 to 244% [77]. That is it boosted their deform-ability and play-ability whereas GA didn’t significantly modify the films E%. The escalation recorded in the E% of the GN-R-GL films was regarded to the escalated glycerol content (60 g/100 g GL). Such escalated plasticizer content caused its influence to be still evident even after GN cross-linking. It was also revealed that GN and GA significantly lowered the films WVP from 1.02 to 0.88 and 0.76 g mm/h‐1 m2 kPa, respectively. Thus, it was concluded that GN and GA cross-linking procured films with comparable attributes [77]. Nonetheless, the cyto-comptability of GN would promote its exploitation in food packaging.

Noteworthy, CHS-GL packaging films were formerly fabricated and cross-linked with GN (Fig. 14). The uncross-linked CHS-GL films presented fine TS (77.3 MPa). This TS was significantly escalated to 83.7 MPa following the cross-linking with 1% GN (w/w% of the polymers weight). The GN cross-linking also significantly lowered the films E% from 6.3% to 5.4%. Meanwhile, the films WVP wasn’t significantly modified by GN. The GN-CHS-GL films were also successfully impregnated with rosemary essential oil, which presents antibacterial, antioxidant, and detoxifying attributes, and quercetin, which presents antioxidant attributes. The impregnation of these two active entities further escalated the films TS to 86.7 MPa [15].

Fig. 14
figure 14

Schematic diagram showing the interactions amidst polymers within the quercetin and rosemary loaded GN cross-linked CHS-GL films [15]

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Altered GN cross-linked bio-plastics and packaging supplies

Non-toxic and bio-degradable bio-plastics could be derived from the proteins [84], which are involved in food industries, such as pea protein [84], which represents 20–25% of pea seeds [61], soy protein [59], which represents 40% of soybeans [60], and sodium caseinate [76]. Nonetheless, the protein derived bio-plastics present reduced mechanical and barrier attributes [84]. Thus, they could be cross-linked with GN in order to raise their mechanical and barrier attributes. On one occasion, pea protein isolate (PP) bio-plastics were cross-linked with GN. The cross-linking degrees of these GN-PP bio-plastics reached up 10.94%, when 0.25% (w/w) GN was exploited, and 22.35%, when 0.5% (w/w) GN was exploited. The GN-PP bio-plastics exhibited significantly reduced water-uptake as compared to the PP bio-plastics, and this was regarded to the GN derived aromatic residues, which raised the hydrophobic attributes of the GN-PP, and also to the GN introduced cross-links. It was also unveiled that the TS values of all the 0.25% and 0.5% GN processed PP bio-plastics were insignificantly reduced compared to the TS of the PP bio-plastics. Nonetheless, the 0.5% GN processed PP bio-plastics, whose blends were cured for 10 days, presented a significant reduction in their TS from 2.83 MPa to 2.00 MPa. As regards to the E% values (deform-ability), they were incremented following GN processing. This was true for all the 0.25% and 0.5% GN processed PP bio-plastics except the 0.5% GN processed PP bio-plastics whose blends were cured for 5 and 10 days where the E% values were lowered [84]. On another occasion, the GN cross-linking escalated both the TS and E% of the soy protein isolate-glycerol (SPI-G) films from 3.22 MPa and 22.53% (0% GN) to 3.28 MPa and 26.71% (0.1% GN (w/w of SPI)) and then to 4.16 MPa and 45.84% (1% GN). Further escalating the GN concentration from 2.5% to 10% (w/w of SPI) brought about insignificant alterations in the TS and reduced the E% of the GN-SPI-G films. It was argued that lower GN concentrations (≤ 1%) induced inter-molecular cross-linkings amongst the SPI chains whereas the escalated GN concentrations (2.5–10%) induced intra-molecular cross-linkings as the peripheral SPI amino entities were already involved in GN interactions. The TS would be influenced by the inter-molecular cross-linkings; thus; it escalated upon escalating GN concentration to 1%, and it wasn’t significantly influenced by further GN escalations. It was also argued that the lower GN concentrations (≤ 1%) would disrupt some of the SPI intrinsic interactions. Such disruption would cause the SPI networks to become more expansible and to present loftier E%. On the other hand, the inter and intra-molecular cross-linkings brought about by the loftier GN concentrations rigidified the SPI and lowered its E%. Noteworthy, the GN-SPI-G films, prepared with ≥ 2.5% GN, exhibited lower bio-degradation rates than the SPI-G films. Moreover, GN cross-linking was shown to lower the WVP of all the GN-SPI-G films. For instance, cross-linking the SPI-G films with 1% and 2.5% GN lowered their WVP from 2.41 to 1.88 and 1.72 *10–10 g m Pa−1 s−1 m−1, respectively [59]. Thus, GN cross-linking meliorated the SPI-G films mechanical and barrier attributes.

GN cross-linking also meliorated the mechanical and water barrier attributes of the keratin-polyvinyl alcohol-tris(hydroxymethyl)aminomethane films (K-PVA-Tris). Keratin is a biodegradable protein, which is bountiful in animal hair, hooves, and feathers. Taking into account that > 1 million tons of feathers, feather poles, and scraps are procured annually from animals’ slaughter, and that these materials are chiefly discarded as wastes [85], it would be clarified that exploiting keratin as a packaging material would be recommended so as to valorize these wastes and promote their application. GN meliorated the mechanical attributes of the K-PVA-Tris films as it gradually raised their TS from 9.58MPa (0% GN) to 11.04 MPa (0.5% GN). The films E% values were also raised from 10.83% (0% GN) to 50.5% with the 0.05% GN. Further escalating the GN concentration to 0.5% reduced the films E%. It was argued that GN, initially, lowered the structural defects of the K-PVA-Tris films. However, the cross-links introduced via the escalated GN concentrations tightened the 3D construction of the K-PVA-Tris films, and this impeded the molecular chains ability to slide and reduced the elongation at break. As regards to the K-PVA-Tris films barrier attributes, their WVP was reduced following GN cross-linking and the loftiest reduction was acquired at 0.5% GN (from 3.09*10−12g. cm−1.s−1.Pa−1 (0% GN) to 2.02*10−12g. cm−1.s−1.Pa−1). On the other hand, the GN cross-linking progressively escalated the oxygen permeability of the films from 11.78*10–5 cm3.m−2.d−1.Pa−1 (0% GN) to 3531.00*10–5 cm3.m−2.d−1.Pa−1 (0.5% GN). It was argued that the K-PVA-Tris surface hydrophobicity got incremented upon incrementing the GN concentrations. Such hydrophobicity promoted the adsorption of the un-polar O2 and raised the oxygen permeability [85].

Amongst the bio-materials that could be exploited as packaging supplies is the locust-bean milling dust (LBMD). LBMD is the waste dust procured following the milling and sieving of LB seeds. LBMD comprises copious protein (56 g/100 g) and polysaccharide (28 g/ 100 g) contents, and it is also capable of creating films. Nonetheless, these films mechanical resistance needs to be meliorated. Moreover, their hydrophilic attributes need to be lowered [82] in order to boost their water barrier traits. Thus, the proteinacious components (56 g/100g) of LBMD were exploited, and the LBMD films were cross-linked with GN (0.01, 0.03, and 0.05 g/ 100g). Escalating GN concentration progressively lowered the WVP of the films formulated with 5 g/ 100 ml LBMD. GN also progressively incremented the water contact angles of the LBMD top surfaces, and raised their hydrophobic attributes up to 0.03 g/ 100 g GN concentration. It was argued that the GN-protein cross-links rendered the OH entities unavailable for water interactions. Moreover, GN progressively raised the films elastic modulus and lowered their E% up to 0.03 g/ 100 g GN concentration. The GN-protein cross-links reinforced the films molecular construction [82]. Thus, the films became stiffer [83] and less flexible. Noteworthy, the blue color imparted to the LBMD films following GN cross-linking lowered the films transparency. This lowered transparency would boost the films light protective traits. Moreover, 0.03 g/100 g GN significantly raised the intrinsic antioxidant attributes of the LBMD films [82]. Thus, these films could be exploited for active food packaging.

Enzymes immobilization

Enzymes are adept at a variety of reactions. Moreover, enzymes are regarded as superior alternatives to chemical catalysts owing to their selectivity, which restricts the procurement of the undesired and environmentally vesicant byproducts. Enzymes also operate under mild circumstances, and this diminishes their energy needs. Hence, they have been taken into consideration for a variety of biotechnological applications, including the production of biodiesel and the food, pharmaceutical and textile industries. However, enzymes have some drawbacks that prevent their widespread industrial use. Enzymes demonstrate moderate stability at extreme pH and temperature. Additionally, the troublesome extraction of free enzymes from their reaction media raises their operation costs. Enzymes immobilization could provide a way around the aforementioned obstacles. Immobilizing enzymes and binding them to solid immobilizers would allow their straightforward detachment from their reaction mixture. As a result, the enzymes won't contaminate the intended final products, and the recovered enzymes will be re-exploited [86]. Immobilization could also boost monomeric and polymeric enzymes stability [87]. Amidst the known immobilization protocols, covalent binding is the sturdiest binding which averts enzymes detachment from their immobilizers [88], and hence, extends the lifespan of the proposed biocatalysts. GN cross-linking has provided a variety of immobilizers with the covalently functioning entities needed for enzymes covalent binding. Some examples were discussed in the following sections.

GN cross-linked chitosan and gelatin immobilizers

Chitosan (CHS) immobilizers were frequently reported owing to CHS abundance, biodegradability, and safety [79]. Moreover, CHS amino entities could be simply cross-linked via GA or GN [55] in order to covalently bind enzymes. Nonetheless, CHS micro-particles display vitiated mechanical attributes, and this would hamper their industrial exploitation [79]. GN cross-linking would meliorate CHS mechanical attributes. On one occasion, porous CHS beads were cross-linked with altered GN concentrations. The smallest GN concentration (0.1%) didn’t yield mechanically stable CHS beads as such beads were fragmented upon mixing them with the pH 4.5 β-galactosidase immobilization solution. Such fragmentation wasn’t observed at the loftier GN concentrations (0.15–0.25%). Moreover, the 0.15% GN-CHS beads, which were loaded with β-galactosidase at pHs 6, 7.5, and 9, kept 98.9%, 94.5%, and 93.3%, respectively, of their intactness after being re-used for 50 catalytic cycles at 40°C. The above-mentioned beads also kept 96, 81.7, and 89.1% of their inceptive β-galactosidase activity during their 50th catalytic cycle [27].

It should be noted that GN concentration would also influence the quantity and the activity of the bound enzyme entities. Incrementing GN concentration would provide more binding sites for the target enzyme. Thus, more enzyme residues would be loaded onto the immobilizer [27]. This would be initially coupled with increments in the observed immobilized activity. Accordingly, incrementing GN concentration from 0.2 to 0.6 g/L, during the activation of magnetic CHS beads, incremented the immobilized cutinase activity. However, further incrementing GN concentration to 1.2 g/L diminished the immobilized cutinase activity [89]. Such incremented GN concentration might have caused the binding of excessive enzyme residues, and this might have induced molecular crowdedness, and might have hindered the enzymes ability to perform conformational modifications required for their catalytic activity. Moreover, protein–protein interactions might have occurred between the bound enzyme residues [60], and diffusional restrictions might have also been imposed [27]. All of this would reduce the activity of the immobilized enzyme residues upon further incrementing GN concentration. On another occasion, incrementing GN concentration from 0.1 to 0.25%, during the activation of the porous CHS beads, incremented the β-galactosidase immobilization yield, which indicated that more β-galactosidase residues were bound. Nonetheless, the immobilization efficiency was concurrently gradually reduced [27] owing to the deactivation amongst the excessively bound β-galactosidase residues.

Noteworthy, it was attempted to co-immobilize cellobiose dehydrogenase and laccase onto GN, GA, and polyethylene-imine (PEI) activated CHS beads. These two enzymes were intended for the fabrication of the bioactive, lactobionic acid (LBA) from lactose. The GN-CHS beads presented the loftiest cellobiose dehydrogenase activity yield (100%). Nonetheless, no laccase activity was recovered by the GN-CHS beads despite that some laccase moieties were bound to these beads [55]. The GN covalent cross-linkings might have deactivated the bound laccase after distorting its construction. Noteworthy, the covalently active GA-CHS beads didn't also recover any laccase activity. Laccase immobilized activity was only evident with the PEI-CHS beads. Accordingly, the GN-CHS immobilized cellobiose dehydrogenase and the free laccase were exploited to prepare LBA from lactose in presence of ABTS (redox mediator), and they acquired 14.80 mM LBA. This acquired LBA was loftier than the 9.81 mM LBA acquired by the PEI-CHS beads which co-immobilized cellobiose dehydrogenase and laccase [55].

GN cross-linked gelatin (GL) immobilizers were also fabricated. On one occasion, GL was blended with the anionic bio-polymer, sodium alginate (SA), and this blend was formulated into beads via the SA calcium mediated ionotroptic gelation. The GL-SA beads were then grafted with GN in order to covalently bind β-galactosidase. It was observed than incrementing GN concentration from 0.05% to 0.15% incremented the immobilization yield to the value of 84%. Nonetheless, further incrementing GN concentration to 0.25% diminished the yield to 69.7% [28]. That is lesser β-galactosidase moieties got attached to the GN-GL-SA immobilizers despite incrementing the cross-linking GN. It was argued that incrementing GN concentration induced self-polymerization amidst the GN entities, and lowered the GN reactive residues which would be available to bind the enzyme. Moreover, incrementing GN concentration would lower the pore size of the immobilizer [28]. This would diminish the surface area accessible for the enzyme binding and would diminish the quantity of bound enzyme entities. Based on the aforementioned results, 0.15% GN was chosen for the grafting of the GN-GL-SA immobilizers. The β-galactosidase attached to these immobilizers demonstrated considerable operational and storage stability where it demonstrated 80% of its inceptive activity on its 175 storage day. Moreover, it demonstrated 90% activity during its 11th catalytic cycle [28].

Altered GN cross-linked immobilizers

Silica is a highly porous inorganic matrix. Such porosity raises silica surface area, and this consequently, promotes its exploitation in enzymes immobilization [25]. GN cross-linked silica immobilizers were fabricated. On one occasion, SiO2 micro-particles were initially processed with polyethylene-imine (750,000 Da) [90] in order to provide them with the amine entities [91] needed for interacting with GN. Afterwards, these particles and the enzyme, Alcalase (endo-peptidase) or Flavorzyme (exo-peptidase), were cross-linked together via either GN or the more toxic cross-linker; glutaraldehyde (GA). Noteworthy, due to the faster nature of the GA interactions, the GA cross-linking proceeded for up to 2 h whereas the GN cross-linking proceeded for up to 24 h. GA achieved considerable immobilization yields after 2h (> 35% for Alcalase, and > 40% for Flavorzyme) [90], which reflected the considerable enzymes quantities which got bound to the matrix. Nonetheless, the detected immobilized activities of both Alcalase and Flavorzyme were extremely reduced. GA triggered intra and intermolecular cross-linking within the immobilized enzymes, which led to their deactivation. On the other hand, GN cross-linking achieved more escalated immobilization yields after 24 h (> 80% for Alcalase, and < 70% for Flavorzyme). Moreover, considerable immobilized activities were detected for both Alcalase (3617.8 U/g) and Flavorzyme (605.9 U/g), and this reflected the superiority of GN cross-linking [90].

Activated carbon is porous and resilient. Moreover, its surface could be simply grafted with the functional entities that could establish covalent links with enzymes. On one occasion, activated carbon was processed with ethylenediamine in order to attain the primary amine entities which were later cross-linked with GN. The GN-activated carbon immobilizers were then exploited to covalently bind pepsin, and they were compared to the similarly processed GA-activated carbon immobilizers. It was observed that the specific surface area of activated carbon (1082 m2/g) was significantly diminished following its GA grafting (40 m2/g). Such specific area diminution was assigned to establishment of an ethylenediamine-GA coat around the activated carbon which blocked its pores. The specific area of the activated carbon was also diminished following its GN grafting, but to a lesser extent (221 m2/g) [26]. The loftier specific area of the GN-activated carbon would provide more space for the enzymes immobilization. Moreover, the GN-activated carbon immobilized pepsin demonstrated loftier capacity for casein hydrolysis than its GA-activated carbon analogue [26], and this proved the adequacy of GN to replace the toxic GA.

Cellulose, the most profuse biopolymer, is chemically stable, bio-compatible and bio-degradable. It also presents outstanding specific strength and it could be simply grafted owing to its copious OH entities. On one occasion, the cellulose nano-fibres, which were derived from rice straw, were aminated via a consecutive reaction with epichlorohydrin and ammonium hydroxide. The aminated fibres were then activated via 0.5% GN at pH 7.4. The procured GN activated cellulose nano-fibres (GN-CF) were then exploited to immobilize acetylcholinesterase (AChE). The immobilized AChE presented outstanding operational stability as it demonstrated 92% during its 20th cycle. Moreover, the immobilized AChE demonstrated 82% activity on its 60th storage day whereas its free analogue lost its entire activity [29].

GN cross-linked enzymes aggregates (GN-CLEA)

Fabrication of CLEA is a carrier-free immobilization protocol which comprises an initial enzyme precipitation step. This precipitation step is followed by a cross-linking step in which the precipitated enzyme is cross-linked into insoluble CLEA via altered chemical cross-linkers, such as GA and GN. Eliminating the need for immobilization carriers would lower the cost of the CLEA. Furthermore, their large-scale fabrication is simple [92, 93]. Thus, CLEA fabrication would be encouraged. Nonetheless, the minute sized pores of the CLEA would lower the diffusion rates within theses entities [92]. Such lowered diffusion rates would negatively influence the transport of the substrates and products, and consequently, would reduce the enzyme activity. This hurdle could be overcome by lowering the size of the CLEA. If the size of the CLEA was lowered, the surface area/volume ratio would be incremented. Hence, the cross-linked enzymes would be more accessible for the substrates [92]. The GN-CLEA size was formerly reported to be minute. For instance, the GN-CLEA of urease presented a size < 10μm [93]. On another occasion, the sizes of the GN-CLEA and the GA-CLEA, which were fabricated during laccase immobilization, were compared. The GN-CLEA size was only 0.5μm whereas the size of the GA-CLEA was 60 μm. The smaller size of the GN-CLEA promoted its substrate interactions and elevated its substrate affinity. Thus, the GN-CLEA presented a reduced Km of 1.72 mM whereas a 6.96 mM Km was presented by GA-CLEA [92].

Further inspecting the GA-CLEA and the GN-CLEA, which immobilized laccase, revealed that the GA-CLEA provided loftier laccase activity recovery. Nonetheless, the GN-CLEA presented more incremented thermal stability as compared to the GA-CLEA. The laccase 50°C half life was incremented from 40 to 360 min following its cross-linking as GN-CLEA whereas only a slight increment was recorded in case of the GA-CLEA. The GN introduced intra and inter-molecular covalent cross-links rigidified laccase construction and averted its conformational alterations. As regards to the operational stability, the GN-CLEA presented lower operational stability than the GA-CLEA. The GN-CLEA smaller size caused their retrieving process to be more challenging. Thus, lower amounts of GN-CLEA were retrieved following each re-usability cycle and this lowered the observed activity. Nonetheless, when the size of the GN-CLEA was incremented from 0.5 μM to 0.7 μM, after incorporating bovine serum albumin (BSA) as an amino-acid feeder, the operational stability was meliorated and became comparable to that achieved by the BSA-GA-CLEA where 86% and 88% activities, respectively, were evident during the 7th cycles [92]. Thus, GN could be regarded as auspicious for the fabrication of CLEA. Noteworthy, amino-acid feeders, such as BSA are frequently incorporated during the CLEA fabrication. These feeders serve to avert the incidence of excessive cross-linking within the enzyme construction as such excessive cross-linking might negatively influence the enzyme activity [92, 93]. On the other hand, these feeders could provide the lysine entities, required for sufficient cross-linking, if the enzyme surface was deficient in them, and this would also escalate the recovered enzyme activity [92].

Kahoush et al. [16] postulated to combine both GN-CLEA fabrication and carrier based immobilization. In order to do so, the glucose oxidase (GOX) powder was put into an aqueous GN solution (30 or 60 mM). After stirring for 30 min at ambient temperature, a 1cm2 carbon felt was soaked into the GN-GOX mixture for 24 h at 4°C. This protocol was assumed to allow the fabrication of GN-CLEA of GOX. These GN-CLEA would then bind to either the native carbon felt or the plasma processed carbon felt. The binding of the GN-CLEA to the native carbon felt would involve physical interactions, such as hydrogen bonding. Additional bonds might also be involved in case of the plasma processed carbon felt as the GN in the GN-CLEA might bond with the processed felt secondary amino entities. At 30 mM GN, the plasma processed carbon felt presented somewhat loftier initial GOX activity and finer operational stability than did the native carbon felt. Nonetheless, the reusability of the native carbon felt physically bound GN-CLEA of GOX was superior to that of the GOX, which was physically bound to native carbon felt in absence of GN, as the former was successfully re-used for 6 cycles whereas the latter was reused for 4 cycles only [16]. Such superior performance could be regarded to GN and its fabricated CLEA as both GOX specimens were physically immobilized. Noteworthy, inspecting the influence of GN concentration on the performance of the plasma processed carbon felt bound GN-CLEA revealed that incrementing GN concentration from 30 to 60 mM slightly lowered the initial GOX observed activity but meliorated the operational stability. Incrementing GN would induce more cross-linkings, and this would diminish the GOX flexibility and lower its activity [16]. Nonetheless, such lowered flexibility indicated the incremented rigidity of the enzyme which would boost its stability [54].

Conclusions and future perspective

GN was proven to be efficient in constructing covalent immobilizers and also in meliorating the physical and chemical aspects of the biopolymers intended for the biomedical and packaging applications. Nonetheless, GN concentration should be carefully selected in order to acquire the required traits and also to ensure the bio-comptability of the procured biomaterials. Despite the quantity of research performed on GN, more research is still needed. For instance, GN possible interactions with other entities other than the primary amine entities should be further inspected as it was already shown that interactions occurred amidst GN and secondary and tertiary amines. Moreover, research reports should consider the GN mediated physical interactions as they were only briefly mentioned. It could also be seen that most of the GN research involved the three biopolymers; chitosan, gelatin, and collagen. Other biopolymers, such as soy protein isolate, pea protein, and keratin were only involved in limited studies despite their promising traits and their availability. More research should be performed on the GN cross-linking of these biopolymers in order to inspect their potential in altered applications. Furthermore, the colorimetric and fluorescent aspects of the GN blue complexes also need to be further exploited in analysis.