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Researchers have devoted a lot of time and efforts to demonstrate the energy storage mechanism and improve the performance of biomass-based porous carbon materials for the past decades. But little attention has been paid to the cleanliness and low energy consumption of the manufacturing process, which is very important for the widely application in the practical production of porous carbon materials. As a fishery industry by-product, squid pen value-added utilizations have attracted attentions in recent years. This study reports an iron doped porous carbon derived from squid pen for supercapacitor application. The fabrication process are designed in a scientific and reasonable pathway, in which the activator—FeCl3 transforms into iron oxide improving the electrochemical performance meanwhile avoiding the repeated washing-centrifugation process of residual Fe. Optimal parameters of the synthesis process are Fe doped dosage (Squid pen: FeCl3 = 10:1, w/w), carbonization temperature 700 °C for 1 h and activation temperature 700 °C for 1 h. The specific capacitance of Fe doped carbonized squid pen powder (CSPP) has increased more than 4 times to 131.7 F/g at a current density of 0.2 A/g comparing to the CSPP. Fe doped CSPP also shows a relatively high yield of 30.6%, which is significantly higher than that of biomass based porous carbon material in previous reports. This high yield and cost-effective method is beneficial for the high value transformation of squid pen as electrode material application. [ABSTRACT FROM AUTHOR]

This study aimed to optimize the medium compositions and cultural conditions for improved chitinase production by a potential strain of Bacillus isolated from the marine environment and determine the antifungal activity of its chitinase against plant pathogenic fungi. Five potential isolates were cultured for chitinase production by submerged fermentation using colloidal chitin in a liquid medium. In this study, chitinase activity was determined by measuring reducing sugars, which were determined by the 3,5-dinitrosalicylic acid (DNS) assay. The most potential isolate, B26, showed similarity to Bacillus paramycoides based on the 16S rRNA gene sequence. The maximum chitinase production was achieved at 6.52±0.02 U/mL after 72 h of incubation in a medium containing 2% squid pen powder, supplemented with 0.5% sodium nitrate and 2% NaCl, with an initial pH of 7. It was observed that the optimization of cultural conditions resulted in 2.83 times higher chitinase production than an unoptimized medium. The antifungal activity of crude chitinase against phytopathogenic fungi was evaluated by a well-diffusion method. The chitinase of B. paramycoides B26 effectively inhibited the growth of Fusarium solani TISTR 3436 (83.4%) and Penicillium chrysogenum TISTR 3554 (80.12%).

This study aimed to optimize the medium compositions and cultural conditions for improved chitinase production by a potential strain of Bacillus isolated from the marine environment and determine the antifungal activity of its chitinase against plant pathogenic fungi. Five potential isolates were cultured for chitinase production by submerged fermentation using colloidal chitin in a liquid medium. in this study, chitinase activity was determined by measuring reducing sugars, which were determined by the 3,5-dinitrosalicylic acid (DNS) assay. the most potential isolate, B26, showed similarity to Bacillus paramycoides based on the 16S rRNA gene sequence. the maximum chitinase production was achieved at 6.52±0.02 U/ml after 72 h of incubation in a medium containing 2% squid pen powder, supplemented with 0.5% sodium nitrate and 2% NaCl, with an initial pH of 7. it was observed that the optimization of cultural conditions resulted in 2.83 times higher chitinase production than an unoptimized medium. the antifungal activity of crude chitinase against phytopathogenic fungi was evaluated by a welldiffusion method. the chitinase of B. paramycoides B26 effectively inhibited the growth of Fusarium solani tiStR 3436 (83.4%) and Penicillium chrysogenum tiStR 3554 (80.12%). [ABSTRACT FROM AUTHOR]

APS is a cheap and eco-friendly oxidant which enables one-step extraction of nanochitin (NCh) from fishery wastes. However, it is challenging to improve the preparation efficiency and NCh quality simultaneously, owing to the uneven or uncontrollable oxidation. Herein, we propose a simple and controllable way to isolate chitin nanofibers (ChNFs) from squid pen by gradient heating activated (GHA)- ammonium persulfate (APS) oxidation. Compared to the isothermal activated (ITA)-APS oxidation, our strategy reduced the mass ratio of squid pen to APS from 1:45 to 1:6 and reaction time from 15 h to 8 h. Meanwhile, the as-prepared ChNFs exhibited high yield (91.5 %), light transmittance (98 % at 500 nm), crystallinity index (96.9 %), and carboxyl content (1.53 mmol/g). GHA-APS oxidation involved multiple continuous heating and isothermal stages. The former stimulates a moderate activation of APS and enhances the oxidation rate, while the latter provides a duration for surface chemistry. This non-isothermal heating facilitates the continuous decomposition of APS at a relatively high and consistent rate, thereby enhances its oxidation efficiency. Furthermore, green assessments indicate this method is simple, time-saving, eco-friendly and cost-effective. Overall, this work introduces a novel perspective for the industrial extraction of high-efficiency and high-quality nanomaterials. [Display omitted] [ABSTRACT FROM AUTHOR]

We report on the extraction of β-chitin from pens (or Gladius) of Uroteuthis edulis , a squid species prevalent in the Pacific coastal regions of East Asia. In particular, we employ cryogenic mechanical grinding (or cryomilling) as a pre-treatment process for the raw squid pens. We show that the cryomilling step enables an effective pulverization of the raw materials, which facilitates the removal of protein residues allowing the extraction of high-purity β-chitin with a high acetylation degree (∼97 %) and crystallinity (∼82 %). We also demonstrate that the Uroteuthis edulis extract β-chitin affords a free-standing film with excellent optical transmittance and mechanical properties. [ABSTRACT FROM AUTHOR]

Squid pen (SP) is a valuable source of protein and β-chitin. However, current research has primarily focused on extracting β-chitin from SP. This study innovatively extracted both SP protein hydrolysates (SPPHs) and SP β-chitin (SPC) simultaneously using protease hydrolysis. The effects of different proteases on their structural characteristics and bioactivity were evaluated. The results showed that SP alcalase β-chitin (SPAC) had the highest degree of deproteinization (DP, 98.19 %) and SP alcalase hydrolysates (SPAH) had a degree of hydrolysis (DH) of 24.47 %. The analysis of amino acid composition suggested that aromatic amino acids accounted for 17.44 % in SPAH. Structural characterization revealed that SP flavourzyme hydrolysates (SPFH) had the sparsest structure. SPC exhibited an excellent crystallinity index (CI, over 60 %) and degree of acetylation (DA, over 70 %). During simulated gastrointestinal digestion (SGD), the hydroxyl radical scavenging activity, ABTS radical scavenging activity, Fe2+ chelating activity, and reducing power of the SPPHs remained stable or increased significantly. Additionally, SPFC exhibited substantial inhibitory effects on Staphylococcus aureus and Escherichia coli (S. aureus and E. coli), with inhibition circle diameters measuring 2.4 cm and 2.1 cm. These findings supported the potential use of SPPHs as natural antioxidant alternatives and suggested that SPC could serve as a potential antibacterial supplement. [ABSTRACT FROM AUTHOR]

Abstract Chitooligosaccharides (COS) from squid pen produced using amylase, lipase and pepsin were characterized. COS produced by 8% (w/w) lipase (COS-L) showed the maximum FRAP and ABTS radical scavenging activity than those prepared using other two enzymes. COS-L had the average molecular weight (MW) of 79 kDa, intrinsic viscosity of 0.41 dL/g and water solubility of 49%. DPPH, ABTS radical scavenging activities, FRAP and ORAC of COS-L were 5.68, 322.68, 5.66 and 42.20 μmol TE/g sample, respectively. Metal chelating activity was 2.58 μmol EE/g sample. For antibacterial activity, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of COS-L against the targeted bacteria were in the range of 0.31–4.91 mg/mL and 0.62–4.91 mg/mL, respectively. Sardine surimi gel added with 1% (w/w) COS-L showed the lower PV, TBARS and microbial growth during 10 days of storage at 4 °C. COS-L from squid pen could inhibit lipid oxidation and extend the shelf-life of refrigerated sardine surimi gel. Graphical abstract

Background: Tooth loss is a dental and oral health problem that often arises in the community because it often interferes with the function of mastication, speech, aesthetics, and even social relations. Purpose: This study aims to examine the content of chitosan in squid pen as an antibacterial against Streptococcus mutans. Methods: This study is an experimental laboratory with a Post Test Only Control Group Design research design. The sample was the Streptococcus Mutans bacteria obtained from the Research Center of the Faculty of Dental Medicine, Universitas Airlangga, Surabaya. The results were analyzed using the Oneway ANOVA and Post hoc Tukey test. Result: The results showed that at concentrations of 3.13% with the mean value of 12.57 bacterial colonies and at a concentration of 1.56% with the mean value of 30.85 bacterial colonies. Conclusion: that the chitosan extract from squid pen has an antibacterial power against Streptococcus mutans at a concentration of 6.25%. [ABSTRACT FROM AUTHOR]

Chitosan is a natural material that can be obtained from fishery waste such as crustacean shells and squid pens. Squid pen is a waste produced from the processing of squid with transparent structures with feather-like shapes attached to the dorsal inside a squid mantle. Chitosan from squid pen has been developed in the medical field because it has biocompatible properties, biodegradable, non- toxic, and also has antifungal activity. Being able to eliminate the Candida albicans fungus which is a predisposing factor for the occurrence of denture stomatitis due to the use of long-term dentures. This study aims to determine the antifungal activity of chitosan extracted from squid pen against Candida albicans. Candida albicans was planted in Saboraud broth liquid media tubes and given Chitosan suspension 1.5% (w / v) with each sample group consisting of 50%, 25%, 12.5%, 6.25%, 3.125%, 1.56%, 0.78% and there are two control groups namely positive and negative controls. All samples were incubated for 24 hours at 37°C Then a visual observation of turbidity on liquid Saboraud broth media was then carried out by planting culture on SDA media with 7 replications, incubated for 2x24 hours at 37 ° Gmd counted the number of living colonies. The results were analyzed using Kruskal-Wallis and Post hoc tukey test. At the concentration of 25% there was no fungal growth in saboraud dextrose agar media. Chitosan extracted from squid pens has antifungal activity against Candida albicans fungi with a minimum concentration of 25%. [ABSTRACT FROM AUTHOR]

Background and Objectives: Chitosan, a polysaccharide derived from squid pens – the squid waste, is gaining considerable interests in biomedical engineering due to the biodegradability, biocompatibility, nontoxicity, and antibacterial activity. It is necessary to eradicate the bacteria from root canal in endodontic treatment, including Porphyromonas gingivalis. P. gingivalis is one of the most prevalently found bacteria in root canals and its presence can cause endodontic treatment failure. This study was conducted to find the antibacterial effect of chitosan from squid pen against P. gingivalis at a certain concentration. Materials and Methods: Chitosan 1.5% (w/v) was diluted in several tubes. The lowest concentration with no bacterial growth was considered to have antibacterial activity against P. gingivalis. Results: There was no bacterial growth in nutrient agar media at the concentration of 10.75%. Conclusion: Chitosan that was made from squid pens has antibacterial activity against P. gingivalis.

Summary: Effect of chitooligosaccharide from squid pen prepared using lipase (COS‐L) at various concentrations (0–30 g kg−1) on gel properties of sardine surimi gel was investigated. Breaking force (BF) and deformation (DF) of gel were increased, when COS‐L level was increased up to 10 g kg−1 (P < 0.05). Water holding capacity and whiteness of gel were improved with the addition of COS‐L than those of control. Gel added with 10 g kg−1COS‐L had denser network with higher likeness score for all sensory attributes, compared to control. When gel incorporated with 10 g kg−1COS‐L was stored at 4 °C, BF, DF and whiteness were maintained during 10 days of storage. Textural properties of surimi gel added with COS‐L were higher than those of control throughout storage. Thus, incorporation of 10 g kg−1COS‐L could improve gel properties of sardine surimi gel and retarded the deterioration of gel properties during refrigerated storage. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Carboxylated chitin nanofibers were fabricated directly from the squid pens. • Ammonium persulfate was applied to oxidize chitin. • Thus-prepared nanofibers were 2–4 nm in width and a few microns in length. • The ultrafine nanofibers can be transformed to transparent and stiff hydrogels. Abstract TEMPO-mediated oxidation has been successfully used to prepare carboxylated chitin nanofibers (ChNFs) with purified chitins originating from the outer shells of crab and shrimp (α-form) or tubeworm (β-form). However, the method for obtaining carboxylated ChNFs with squid pen chitin (hydrated β-form) has not been developed yet. It might be due to the existence of the small amount of partial deacetylation (DD ≈ 9%) in the squid pen β-chitins. Herein, ultrafine (2–4 nm in width and several micrometers in length) and carboxylated β-ChNFs were fabricated directly from the squid pen with simultaneously removing of protein, CaCO3 and other non-chitin components by one-step oxidation procedure using ammonium persulfate (APS) and followed ultrasonic disintegration under acid conditions. When 45 wt% APS was used to react with squid pen, the carboxylate content of ChNFs reaches 0.802 mmol/g. Therefore, the β-ChNFs with anionically charged groups (COO−) can be dispersed stably in aqueous solution under basic conditions. Meanwhile, thus-obtained β-ChNFs aqueous solution even with very low concentration (0.8%) can be transformed to transparent, robust and moldable hydrogels by gas coagulation of acetic acid. [ABSTRACT FROM AUTHOR]

Background and Objectives: Chitosan, a polysaccharide derived from squid pens – the squid waste, is gaining considerable interests in biomedical engineering due to the biodegradability, biocompatibility, nontoxicity, and antibacterial activity. It is necessary to eradicate the bacteria from root canal in endodontic treatment, including Porphyromonas gingivalis. P. gingivalis is one of the most prevalently found bacteria in root canals and its presence can cause endodontic treatment failure. This study was conducted to find the antibacterial effect of chitosan from squid pen against P. gingivalis at a certain concentration. Materials and Methods: Chitosan 1.5% (w/v) was diluted in several tubes. The lowest concentration with no bacterial growth was considered to have antibacterial activity against P. gingivalis. Results: There was no bacterial growth in nutrient agar media at the concentration of 10.75%. Conclusion: Chitosan that was made from squid pens has antibacterial activity against P. gingivalis. [ABSTRACT FROM AUTHOR]

Chitosan from squid (Loligo formosana) pens were prepared and characterized. First, ultrasonication condition was optimized for deproteinization of squid pens using central composite design (CCD) of response surface methodology (RSM). Squid pens were ultrasonicated at amplitude 69% for 41.46 min at the solid/solvent ratio of 1:18 yielded 34% (w/w) chitin with the lowest remaining protein. Therefore, ultrasonication effectively reduced the extraction time for chitin production from squid pens as compared to traditional method (5 hr). When the resultant chitin was subjected to deacetylation at different temperatures and times, yield and degree of deacetylation (DDA) of chitosan were in the range of 50% to 65% (w/w) and 78% to 90%. Intrinsic viscosity and molecular weight (MW) of chitosan were in the range of 3.2 to 6.52 dL/g and 1.2 × 105 to 3.2 × 105 Da, respectively. All the chitosans with different DDA were able to bind oil droplets under the mimicked pH condition of gastrointestinal tract. Chitosan produced by deacetylation at 130 °C for 2 hr (CH130‐2) showed the optimum yield (54%) and had medium MW (1.5 × 105 Da). DDA of CH130‐2 determined using 1H‐NMR was 89%, which was similar to that (87%) obtained from FTIR. XRD results showed destruction of chitin structure and decreased crystallinity index from 55% to 27% after deacetylation. CH130‐2 stabilized the emulsion under the simulated gastrointestinal conditions. Therefore, it could be used as dietary fiber to control the adsorption of fat/oil in the human digestive tract. Practical Application: Chitin and chitosan are marketable products manufactured from crustacean shells. However, extraction of chitin is time consuming. Ultrasonication has been used for extraction of various biomolecules from different sources. It effectively lowers the processing time and enhances extraction yield. Therefore, application of ultrasonication with optimized condition using RSM could reduce extraction time and enhance yield of chitin from squid pen. Chitin from squid pen could be further converted to chitosan with high DDA. Chitosan was able to act as a dietary fiber and reduce fat absorption in gastrointestinal tract. Thus, this information is of benefit for squid processing industry to exploit squid pen, a processing byproduct. [ABSTRACT FROM AUTHOR]

The aim of this work was to produce chitosanase by fermenting from squid pen, and recover the fermented squid pen for dye removal by adsorption. One chitosanase induced from squid pen powder (SPP)-containing medium by Bacillus cereus TKU034 was purified in high purification fold (441) and high yield of activity recovery (51%) by ammonium sulfate precipitation and combined column chromatography. The SDS-PAGE results showed its molecular mass to be around 43 kDa. The TKU034 chitosanase used for the chitooligomers preparation was studied. The enzyme products revealed that the chitosanase could degrade chitosan with various degrees of polymerization, ranging from 3 to 9, as well as the chitosanase in an endolytic manner. Besides, the fermented SPP was recovered and displayed a better adsorption rate (up to 99.5%) for the disperse dyes (red, yellow, blue, and black) than the water-soluble food colorants, Allura Red AC (R40) and Tartrazine (Y4). The adsorbed R40 on the unfermented SPP and the fermented SPP was eluted by distilled water and 1 M NaOH to confirm the dye adsorption mechanism. The fermented SPP had a slightly higher adsorption capacity than the unfermented, and elution of the dye from the fermented SPP was easier than from the unfermented. The main dye adsorption mechanism of fermented SPP was physical adsorption, while the adsorption mechanism of unfermented SPP was chemical adsorption.

This review provides a comprehensive summary of the most recent developments of various aspects (i.e., production, purification, structure, and bioactivity) of the exopolysaccharides (EPSs) from Paenibacillus spp. For the production, in particular, squid pen waste was first utilized successfully to produce a high yield of inexpensive EPSs from Paenibacillus sp. TKU023 and P. macerans TKU029. In addition, this technology for EPS production is prevailing because it is more environmentally friendly. The Paenibacillus spp. EPSs reported from various references constitute a structurally diverse class of biological macromolecules with different applications in the broad fields of pharmacy, cosmetics and bioremediation. The EPS produced by P. macerans TKU029 can increase in vivo skin hydration and may be a new source of natural moisturizers with potential value in cosmetics. However, the relationships between the structures and activities of these EPSs in many studies are not well established. The contents and data in this review will serve as useful references for further investigation, production, structure and application of Paenibacillus spp. EPSs in various fields.

One of the most promising applications of chitosanase is the conversion of chitinous biowaste into bioactive chitooligomers (COS). TKU033 chitosanase was induced from squid pen powder (SPP)-containing Bacillus cereus TKU033 medium and purified by ammonium sulfate precipitation and column chromatography. The enzyme was relatively more thermostable in the presence of the substrate and had an activity of 93% at 50 °C in a pH 5 buffer solution for 60 min. Furthermore, the enzyme used for the COS preparation was also studied. The enzyme products revealed various mixtures of COS that with different degrees of polymerization (DP), ranging from three to nine. In the culture medium, the fermented SPP was recovered, and it displayed a better adsorption rate (up to 96%) for the disperse dyes than the water-soluble food colorants, Allura Red AC (R40) and Tartrazne (Y4). Fourier transform-infrared spectroscopic (FT-IR) analysis proved that the adsorption of the dyes onto fermented SPP was a physical adsorption. Results also showed that fermented SPP was a favorable adsorber and could be employed as low-cost alternative for dye removal in wastewater treatment.

The squid pen (gladius) from the Loligo vulgaris was used for preparation of β-chitin materials characterized by different chemical, micro- and nano-structural properties that preserved, almost completely the macrostructural and the mechanical ones. The β-chitin materials obtained by alkaline treatment showed porosity, wettability and swelling that are a function of the duration of the treatment. Microscopic, spectroscopic and synchrotron X-ray diffraction techniques showed that the chemical environment of the N-acetyl groups of the β-chitin chains changes after the thermal alkaline treatment. As a consequence, the crystalline packing of the β-chitin is modified, due to the intercalation of water molecules between β-chitin sheets. Potential applications of these β-chitin materials range from the nanotechnology to the regenerative medicine. The use of gladii, which are waste products of the fishing industry, has also important environmental implications.

Chitosan is produced by thermochemical alkaline deacetylation of chitin, but the process is usually environmentally problematic. In the present study, Illex argentinus squid pen chitin, after de-proteinization and demineralization, was pretreated with a compressional-puffing (CP) process under various puffing pressures. The CP process facilitated the increase of the crystalline index and degree of deacetylation of chitins. The CP-treated chitins were subjected to further extraction of chitosan, and four chitosan isolates (CI1–CI4) were obtained. The CP process was found to have beneficial effects in terms of increased extraction yield and increased antibacterial activity of the extracted chitosans. Moreover, the antibacterial property of the extracted chitosans seemed to be negatively related to their molecular weight (MW). Our findings showed that CI4 exhibited the highest extraction yield and the greatest antibacterial activity, and thus we recommend it as a safe and potent antibacterial agent for food, biomedicine, and other industrial usages. [ABSTRACT FROM AUTHOR]

Squid pens were subjected to alkali hydrolysis to extract chitin and chitosan. Proteins present in the alkaline extraction wastewater were recovered at pH 3, 4, 5 and 6, and were subjected to hydrolysis by trypsin, pepsin and a bacterial protease called HT for 1, 2, 4 and 24 h. Hydrolysis of the extracted proteins with either trypsin or HT generated more antioxidant activity than hydrolysis with pepsin. Higher ACE-inhibitory activity was generated in the trypsin and pepsin hydrolysates than in the HT hydrolysate. Squid pen protein recovered from chitosan processing waste alkaline solution can be a potential source of bioactive peptides for addition to foods. The antioxidant and ACE-inhibitory activities of the extracted proteins were initially low and increased upon incubation with the proteases. Pepsin generated significantly lower (P < 0.05) antioxidant activities compared to trypsin and HT, while trypsin and pepsin hydrolysates exhibited higher ACE-inhibitory activity than HT (P < 0.05). [ABSTRACT FROM AUTHOR]

Byproducts generated in high levels by marine processes have been recognized for their value as recyclable or reclaimable waste. Among marine byproducts, shrimp shells, crab shells, and squid pens have the highest chitin content. The chemical treatments of these chitin-containing byproducts for preparing chitin and chitosan create waste disposal problems because neutralization and detoxification of the discharged wastewater are necessary. Therefore, the cost of chitin and chitosan preparations was far higher than those of their raw materials, marine chitin-containing byproducts. Chitin and chitosan have been widely used as the major carbon source of bacteria for producing chitinolytic enzymes. In 1997, the bifunctional chitinase/lysozymes from Pseudomonas aeruginosa K-187 using shrimp and crab shells as the sole carbon/nitrogen (C/N) source was first reported. Thereafter, the use of squid pens as the only C/N source for producing enzymes and bioactive materials had also been studied. The use of shellfish chitin waste as the sole C/N source not only solves environmental problems, it decreases the production costs for microbial conversion. This review summarizes our recent research of microbial reclamation of these marine byproducts for producing enzymes and bioactive materials; the characterization and applications of these products were also studied. [ABSTRACT FROM AUTHOR]

The antibacterial activity of squid pen chitosans (deacetylation degree (DD) of 91%) with Mw varying from 93-1740 kDa was tested on Erwinia carotovora that causes soft rot disease in tomatoes. Specifically the inhibition level of chitosan to bacterial colonies development (in vitro) and disease lesion on tomato fruits (in vivo) was evaluated. Sigma's chitosan (Mw of 50-90 kDa with DD of 80%) was used as positive control. In vitro results showed that the antibacterial action of chitosan depended greatly on the Mw of chitosan. The chitosan with a low Mw exhibited a higher inhibition activity on Erwinia carotovora than higher molecular weight commercial chitosan. The minimum inhibitory concentration of the chitosan with a Mw of 93 kDa was 1.25% (w/v). High Mw chitosan (Mw > 1000 kDa) did not show any inhibition activity in vitro. [ABSTRACT FROM AUTHOR]

This study intends to investigate the potentials of chitosan- g-PMMA [poly(methyl methacrylate)] graft copolymers in corneal tissue engineering. The polymerization reaction between chitosan and methyl methacrylate (MMA) was standardized by varying temperature, pH, initiator and monomer concentrations. 113 % grafting yield was obtained at 60 °C, pH 6, 2.1 × 10 mol/L of sodium metabisulfite and 1.48 × 10 mol/L of potassium persulphate and 1.99 × 10 mol/L of monomer concentration. Fourier transform infrared spectroscopy confirmed the grafting of PMMA onto chitosan. The grafts possessed better thermal stability compared to chitosan films. The rough-surfaced (as evidenced by scanning electron microscopy) grafts were able to degrade under in vitro conditions in presence of lysozyme. The grafts exhibited 88 % optical clarity and also supported the proliferation of human corneal epithelial cell line. The above results indicate the potentials of chitosan- g-PMMA and its possible use in corneal tissue engineering applications. [ABSTRACT FROM AUTHOR]

The culture supernatant of Paenibacillus sp. TKU036, a bacterium isolated from Taiwanese soils, showed high antioxidant activity (85%) when cultured in a squid pen powder (SPP)-containing medium at 37° C for three days. Homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) was isolated and found to be the major antioxidant in the culture supernatant of the SPP-containing medium fermented by Paenibacillus sp. TKU036. Tryptophan was also present in the culture supernatant. The results of high-performance liquid chromatography (HPLC) fingerprinting showed that HGA and tryptophan were produced via fermentation but did not pre-exist in the unfermented SPP-containing medium. Neither HGA nor tryptophan was found in the culture supernatants obtained from the fermentation of nutrient broth or other chitinous material, i.e., medium containing shrimp head powder, by Paenibacillus sp. TKU036. The production of HGA via microorganisms has rarely been reported. In this study, we found that squid pen was a potential carbon and nitrogen source for Paenibacillus sp. Tryptophan (105 mg/L) and HGA (60 mg/L) were recovered from the culture supernatant. The isolated HGA was found to have higher antioxidant activity (IC50 = 6.9 µg/mL) than α-tocopherol (IC50 = 17.6 µg/mL). The anti-inflammatory activity of the isolated HGA (IC50 = 10.14 µg/mL) was lower than that of quercetin (IC50 = 1.14 µg/mL). As a result, squid pen, a fishery processing byproduct, is a valuable material for the production of tryptophan and the antioxidant and anti-inflammatory HGA via microbial conversion. [ABSTRACT FROM AUTHOR]

The objectives of this investigation were to produce a novel chitosanase for application in industries and waste treatment. The transformation of chitinous biowaste into valuable bioactive chitooligomers (COS) is one of the most exciting applications of chitosanase. An amphiprotic novel chitosanase from Bacillus mycoides TKU038 using squid pen powder (SPP)-containing medium was retrieved from a Taiwan soil sample, which was purified by column chromatography, and characterized by biochemical protocol. Extracellular chitosanase (CS038) was purified to 130-fold with a 35% yield, and its molecular mass was roughly 48 kDa. CS038 was stable over a wide range of pH values (4-10) at 50°C and exhibited an optimal temperature of 50°C. Interestingly, the optimum pH values were estimated as 6 and 10, whereas CS038 exhibited chitosan-degrading activity (100% and 94%, respectively). CS038 had Km and Vmax values of 0.098 mg/mL and 1.336 U/min, separately, using different concentrations of water-soluble chitosan. A combination of the high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer data revealed that the chitosan oligosaccharides obtained from the hydrolysis of chitosan by CS038 comprise oligomers with multiple degrees of polymerization (DP), varying from 3-9, as well as CS038 in an endolytic fashion. The TKU038 culture supernatant and COS mixture exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities. The COS activities were dose dependent and correlated to their DP. The COS with high DP exhibited enhanced DPPH radical scavenging capability compared with COS with low DP. Furthermore, the COS exhibited inhibitory behavior on nitric oxide (NO) production in murine RAW 264.7 macrophage cells, which was induced by Escherichia coli O111 lipopolysaccharide (LPS). The COS with low DP possesses a more potent anti-inflammatory capability to decrease NO production (IC50, 76.27 ± 1.49 µg/mL) than that of COS with high DP (IC50, 82.65 ± 1.18 µg/mL). Given its effectiveness in production and purification, acidophilic and alkalophilic properties, stability over ranges of pH values, ability to generate COS, antioxidant activity, and anti-inflammatory, CS038 has potential applications in SPP waste treatment and industries for COS production as a medical prebiotic. [ABSTRACT FROM AUTHOR]

The management of fishery residues and plastics is considered to be a vital strategy for conserving resources and maintaining the quality of the environment. Poly(L-lactic acid) (PLA) is a commercially promising, renewable, and biodegradable plastic. In this study, a PLA depolymerase was produced in a squid pen powder (SPP) and recycled plastic waste (PLA powder)-containing medium by Pseudomonas tamsuii TKU015, a bacterial strain isolated from Taiwanese soil. This PLA depolymerase had a molecular weight of 58 kDa and was purified to homogeneity from the supernatant of a TKU015 culture. The optimum pH of TKU015 PLA depolymerase is 10, and the optimal temperature of the enzyme is 60 °C. In addition to PLA, TKU015 PLA depolymerase degraded fibrinogen and tributyrin, but did not hydrolyze casein, triolein, and poly(β-hydroxybutyrate). Taken together, these data demonstrate that P. tamsuii TKU015 produces a PLA depolymerase to utilize SPP and polylactide as carbon/nitrogen sources. [ABSTRACT FROM AUTHOR]

Natural polysaccharides have received much attention due to their wide range of applications. Although most microbial exopolysaccharides (EPSs) use sugars as the major carbon source, such as glucose or sucrose, in this study, EPSs were induced from a squid pen powder (SPP)-containing medium by Paenibacillus mucilaginosus TKU032, a bacterial strain isolated from Taiwanese soil. Under the optimal culture conditions, the maximum EPS yield (14.8 g/L) was obtained. MALDI-TOF MS analysis of an EPS fraction purified by gel filtration revealed two mass peaks with molecular weights of ~1.05 x 104 and ~1.35 x 104 Da, respectively. The analysis of the hydrolysates of TKU032 EPS with cellulase, pectinase or amylase indicated that the glycosidic bond of TKU032 EPS is most likely an α-1,4 glycosidic bond and the hydrolysates are similar to those of starch. In addition, the purified EPS demonstrated strong antioxidant abilities. [ABSTRACT FROM AUTHOR]

Various chitin substrates were used to investigate the properties of enzymes produced from the chitinase-producing bacterium Paenibacillus chitinolyticus MP-306 against phytopathogens. The MP-306 bacterium was incubated in nine culture media [crab shell powder chitin (CRS), chitin-protein complex powder (CPC), carboxymethyl-chitin powder (CMC), yeast extract only (YE), LB (Trypton, NaCl, and yeast extract), GT (Trypton, NaCl, and glucose), crab shell colloidal chitin (CSC), squid pen powder chitin (SPC), and cicada slough powder chitin (CSP)] at 30 °C for 3 days. Chitinase isozymes in CPC medium were expressed strongly as CN1, CN2, CN3, CN4, CN5, and CN6 bands on native-PAGE gels. Chitinase isozymes in CPC and CMC medium were expressed as 13 bands (CS1–CS13) on SDS-PAGE gels. Chitinase isozymes were expressed strongly on SDS-PAGE gels as two bands (CS6 and CS8) on YE and LB medium and 13 bands (CS1–CS13) on SPC medium. In crude enzyme, chitinase isozymes at pH 7 and pH 9 in chitin media appeared strongly on SDS-PAGE gels. Partial purified enzyme indicated high stability of enzyme activity at various temperatures and pHs in chitin medium, while these enzymes indicated low activity staining of enzyme on electrophoresis gels at various temperatures and pHs condition of chitin medium. [ABSTRACT FROM AUTHOR]

Pseudomonas tamsuii TKU015 produced insecticidal materials using squid pen as the sole carbon and nitrogen source. The insecticidal activity of the produced metabolites was evaluated against Drosophila melanogaster larvae. The growth of P. tamsuii TKU015 was accompanied by increased mortality. SDS-PAGE showed the molecular weight of the insecticidal toxin of P. tamsuii TKU015 to be 94.7 kDa, corresponding to the TccC insecticidal protein from P. taiwanensis TKU015. These results showed that P. tamsuii TKU015 could be applicable in controlling Drosophila for developing novel biopesticides in organic agriculture. [ABSTRACT FROM AUTHOR]

Reports of tyrosinase inhibitors from microorganisms are rare. The tyrosinase inhibitory activity was found from the culture supernatant of Burkholderia cepacia TKU025 with 1 % squid pen as the sole carbon/nitrogen source. The fourth-day supernatant showed the strongest tyrosinase inhibitory activity (500 U/ml) and the highest total phenolic content (789 μg/ml GAE). The supernatant was further purified by liquid-liquid partition, and it was found that the methanol extract exhibited the lowest IC value of 2 μg/ml in the tyrosinase inhibitory activity assay and was compared with the positive control kojic acid and vitamin C. Significant associations between the tyrosinase inhibitory potency and the total phenolic content were found for the supernatant, extract, and fractions. The tyrosinase inhibitory activity persisted even when it was treated with acidic or alkaline conditions of pH 2 or 12. The tyrosinase inhibitory activity remained at 100 %, even after treatment at 100 °C for 60 min. The tyrosinase inhibitor also showed antimicrobial activity against Escherichia coli, Bacillus cereus, Aspergillus fumigatus, and Fusarium oxysporum. [ABSTRACT FROM AUTHOR]

In the present study, chitosan/hydroxyapatite (HA)/β-tircalcium phosphate (β-TCP) composites were produced using squid pen derived chitosan (CHS) and commercial crab derived chitosan (CHC). CHS was prepared from squid pens by alkaline N-deacetylation. HA and β-TCP were extracted from mussel shells using a microwave irradiation method. Two different composites were prepared by incorporating 50% (w/w) HA/(β-TCP) in CHS or CHC followed by lyophilization and cross-linking of composites by tripolyphosphate (TPP). The effect of different freezing temperatures of −20, −80 and −196 °C on the physicochemical characteristics of composites was investigated. A simulated body fluid (SBF) solution was used for preliminary in vitro study for 1, 7, 14 and 28 days and the composites were characterized by XRD, FTIR, TGA, SEM, μ-CT and ICP-MS. Porosity, pore size, water uptake; water retention abilities and in vitro degradations of the prepared composites were evaluated. The CHS composites were found to have higher porosity (62%) compared to the CHC composites (porosity 42%) and better mechanical properties. The results of this study indicated that composites produced at −20 °C had higher mechanical properties and lower degradation rate compared with −80 °C. Chitosan from the squid pen is an excellent biomaterial candidate for bone tissue engineering applications. [ABSTRACT FROM AUTHOR]

The aim of this work was to produce chitosanase by fermenting from squid pen, and recover the fermented squid pen for dye removal by adsorption. One chitosanase induced from squid pen powder (SPP)-containing medium by Bacillus cereus TKU034 was purified in high purification fold (441) and high yield of activity recovery (51%) by ammonium sulfate precipitation and combined column chromatography. The SDS-PAGE results showed its molecular mass to be around 43 kDa. The TKU034 chitosanase used for the chitooligomers preparation was studied. The enzyme products revealed that the chitosanase could degrade chitosan with various degrees of polymerization, ranging from 3 to 9, as well as the chitosanase in an endolytic manner. Besides, the fermented SPP was recovered and displayed a better adsorption rate (up to 99.5%) for the disperse dyes (red, yellow, blue, and black) than the water-soluble food colorants, Allura Red AC (R40) and Tartrazine (Y4). The adsorbed R40 on the unfermented SPP and the fermented SPP was eluted by distilled water and 1 M NaOH to confirm the dye adsorption mechanism. The fermented SPP had a slightly higher adsorption capacity than the unfermented, and elution of the dye from the fermented SPP was easier than from the unfermented. The main dye adsorption mechanism of fermented SPP was physical adsorption, while the adsorption mechanism of unfermented SPP was chemical adsorption. [ABSTRACT FROM AUTHOR]

The squid pen (gladius) from the Loligo vulgaris was used for preparation of ß-chitin materials characterized by different chemical, micro- and nano-structural properties that preserved, almost completely the macrostructural and the mechanical ones. The ß-chitin materials obtained by alkaline treatment showed porosity, wettability and swelling that are a function of the duration of the treatment. Microscopic, spectroscopic and synchrotron X-ray diffraction techniques showed that the chemical environment of the N-acetyl groups of the ß-chitin chains changes after the thermal alkaline treatment. As a consequence, the crystalline packing of the ß-chitin is modified, due to the intercalation of water molecules between ß-chitin sheets. Potential applications of these ß-chitin materials range from the nanotechnology to the regenerative medicine. The use of gladii, which are waste products of the fishing industry, has also important environmental implications. [ABSTRACT FROM AUTHOR]

Highlights: [•] This research aimed for the microbial reclamation of squid pen wastes. [•] Purified and characterised a chitosanase, induced by SPP from Penicillium janthinellum D4. [•] Discovered some synthetic small boron-containing molecules as chitosanase activators. [•] The chitosan was depolymerized by D4 chitosanase. [Copyright &y& Elsevier]

In the present work, a chitosanase was induced from a squid pen powder-containing Bacillus cereus TKU031 medium, and the addition of 0.05 % (w/v) boric acid or sodium tetraborate resulted in 195 and 177 % enhancement, respectively, in TKU031 chitosanase production. The purified TKU031 chitosanase exhibited optimum activity at pH 5 and 50 °C and was stable at pH 5-9 and <50 °C. The TKU031 chitosanase that was used for chitooligomers preparation was studied. The enzyme products revealed various chitooligomers with different degrees of polymerisation from 3 to 8, as determined by a MALDI-TOF-mass spectrometer, confirming the endo-type nature of the TKU031 chitosanase. [ABSTRACT FROM AUTHOR]

Reports of tyrosinase inhibitors from microorganisms are rare. A tyrosinase inhibitor- and insecticidal materials-producing bacterium, strain TKU026, was isolated from Taiwanese soil and identified as Burkholderia cepacia. Among the tested chitin-containing materials, squid pen best enhanced the production of tyrosinase inhibitors and insecticidal materials. The tyrosinase inhibitory activity (5,000 U/mL) and insecticidal activity (81 %) against Drosophila larvae was maximised after cultivation on 1 % squid pen-containing medium for 3 days. The tyrosinase inhibitory activity persisted even when the culture was treated with acidic or alkaline conditions of pH 3 or 11. The activities of both tyrosinase inhibitors and insecticide remained at 100 %, even after treatment at 100 °C for 30 min. The culture supernatant after 3 days of cultivation also showed antifungal activity against Aspergillus fumigatus and Fusarium oxysporum with maximal activities of 100 and 80 %, respectively, but no antibacterial activity against Escherichia coli was observed. The tyrosinase inhibitors were assumed to be polyphenolic compounds according to the results of chromatography. [ABSTRACT FROM AUTHOR]

A chitinase and a chitosanase were induced from a squid pen powder (SPP)-containing medium of Bacillus cereus TKU030 and purified by precipitation with ammonium sulphate and combined column chromatography. The purified chitinase and chitosanase exhibited optimum activity at 60°C, pH 5–6 and 40°C, pH 4, respectively. The chitinase and chitosanase were stable at 25–60°C, pH 4–7 and 25–50°C, pH 3–7, respectively. The chitinase and chitosanase showed the highest activity toward β-chitin and 60% DD chitosan, respectively. The chitinase was significantly inhibited by Mn2+ and EDTA but activated by Cu2+, Fe2+ and Ca2+. The chitosanase was significantly inhibited by Cu2+, Fe2+, Zn2+, Mn2+ and EDTA. The chitinase showed high stability in the presence of various surfactants, such as SDS, Tween 20, Tween 40 and Triton X-100. In contrast, these surfactants were inhibitors of the chitosanase. The chitinase and chitosanase were also inhibited by TKUPSP017, a small synthetic boron-containing molecule with a BF3K side-chain. However, TKUPSP017 enhanced the growth of B. cereus TKU030 in SPP-containing medium. [ABSTRACT FROM AUTHOR]

Paenibacillus macerans TKU029 can produce exopolysaccharides (EPSs; 3.46 g/L) and a biosurfactant (1.78 g/L) in a medium with 2 % ( w/ v) squid pen powder as the sole carbon/nitrogen source. The biosurfactant can reduce the surface tension of water from 72.30 to 35.34 mN/m at a concentration of 2.76 g/L and reach an emulsification index of 56 % after a 24-h reaction with machine oil. This biosurfactant is stable at 121 °C for 20 min, over a pH range from 3 to 11, and in <5 % salt solutions. It also shows significant antimicrobial activity, which remains active after treatment at 121 °C and at pH values from 4 to 10, against Escherichia coli BCRC13086, Staphylococcus aureus BCRC10780, Fusarium oxysporum BCRC32121 and Aspergillus fumigatus BCRC30099. Furthermore, human skin shows from 37.3 to 44.3 % hydration after being treated with TKU029 EPSs for 180 min. These results imply that EPSs and the biosurfactant from this strain have potential in cosmetics, for removal of oil contamination, and as antimicrobial agents. [ABSTRACT FROM AUTHOR]

Prodigiosin (PG) has been reported to have various biological activities. With the aim of increasing Serratia marcescens TKU011 PG production on squid pen powder (SPP)-containing medium, the effects of phosphate and ferrous ion supplementation, autoclave treatment, and aeration were studied. Autoclave treatment showed positive results for PG productivity (2.48 mg/mL), which increased 2.5-fold when the organism was incubated in 50 mL of 40-min autoclaved medium in a baffle-based flask (250 mL) containing 1.5% SPP at 30 °C for 1 day and then at 25 °C for 2 additional days. Furthermore, the use of pigments including PG and the food colorants Allura Red AC (R40) and Tartrazine (Y4) as insecticides was also investigated. The lethal concentrations causing 50% Drosophila larval mortality (LC50) of PG, Y4, and R40 using a 5-d exposure period were 230, 449, and 30000 ppm, respectively. The results indicated that the biopigment PG and the food colorant Y4 were potentially toxic to Drosophila larvae. [ABSTRACT FROM AUTHOR]

Squid pen β-chitosans prepared under various deacetylation conditions (30%, 35%, 40% and/or 45% Na OH for 15, 30 and/or 60 min) were characterised. β- Chitosans (deacetylated with 35-45% Na OH for 15-60 min) had 87.1-96.2% degree of deacetylation ( DD), 93.5-96.7% solubility and 120.5-654.9 mPa s viscosity. Treatment with 30% Na OH for 15-60 min yielded inadequately deacetylated β-chitosans ( DD = 51.9-80.2%). Two chitosans prepared under 35% Na OH for 15 min and 45% Na OH for 30 min (designated as 35%-15 and 45%-30, respectively) were further compared. Drying (sun-drying vs. oven-drying) methods did not affect DD. 35%-15 chitosan exhibited lower nitrogen, DD and bulk density, but higher viscosity compared with 45%-30 chitosan. Higher water- and fat-binding capacity but lower DPPH radical scavenging activity were observed for 35%-15 chitosan compared with 45%-30 chitosan. Compared with 45%-30 chitosan, 35%-15 chitosan exhibited higher antibacterial activity against Salmonella Enteritidis and Listeria monocytogenes, but lower antibacterial activity against Escherichia coli. [ABSTRACT FROM AUTHOR]

Squid ( Todarodes pacifica) pen was an excellent source of β-chitin with 25.5% yield. The optimal condition to prepare squid pen β-chitin was established: deproteinisation with 3% NaOH for 30 min at 15 psi/121 °C and a solid/solvent ratio of 1:10 (w/v) and a subsequent demineralisation with 1 N HCl for 30 min at room temperature and a solid/solvent ratio of 1:10 (w/v). Squid pen β-chitin contained 6.29% nitrogen, 0.25% ash, and negligible fat with degree of acetylation of 94.02%, residual amino acid of 0.499 g/100 g and bulk density of 0.28 g mL−1. Depending on its particle size, squid pen β-chitin visually looked white ( L* = 82.82, a* = −0.67, b* = 6.31; particle size of 0.15-0.18 mm) or light grey ( L* = 62.88, a* = 0.33, b* = 10.66; particle size of 0.425-0.841 mm). Water, fat and dye-binding capacity of squid pen β-chitin was 694.67%, 194.03% and 79.81%, respectively. [ABSTRACT FROM AUTHOR]

Abstract: A transparent nanofibril suspension could be readily obtained by treating purified squid pen powder in water with ultrasonic irradiation. The obtained suspension is consisted of β-chitin nanofibrils (CNF) with 3–10nm in width and several micrometers in length. The degree of acetylation (DA) of CNF was found to be 84% which is about 10% lower than that of untreated sample. The CNF suspension could be transformed into a durable 3-D hydrogels (CH) by simply heating to 180°C for 1–4h in an autoclave. Hydrophobic interaction between CNF was believed to play the major role for CNF self-assembling into hydrogels, since the as-prepared chitin hydrogels readily dissolved in a typical chaotropic solution (8M urea) under room temperature. The hydrothermal duration and CNF concentration (0.3–2% (w/v)) strongly affected the physical properties of CH. The suspension of 1% (w/v) CNF treated with 4h, 180°C hydrothermal heating generated a CH with 99.3% water content, CNF with 87% crystallinity and an mechanical strength of 0.7N breaking force. [Copyright &y& Elsevier]

Abstract: Byproducts generated in great amounts by marine processes have been recognized for their value as recyclable or reclaimable waste. Squid have been investigated to extract β-chitin from squid pen (SP), produce peptides with angiotensin-I-converting enzyme (ACE)-inhibitory, antihypertensive, and antibacterial activities from squid skin, and isolate polysaccharides with antitumor and antibacterial activities from squid ink. To further meliorate the utilization of SP, we have recently explored the bioconversion of SP for the production of bioactive materials. Rather than endeavoring to review the literatures, this paper recapitulated our recently published efforts in the region of microbial reclamation of SP. [Copyright &y& Elsevier]

Abstract: Bacillus licheniformis TKU004 produced biosurfactants in a medium with 1% squid pen powder as the sole carbon/nitrogen source at 37°C for 1 day by using a baffled base flask, showing a growth-associated production. The biosurfactant was extracted from the culture supernatant by acid precipitation, extraction of ethyl acetate and methanol with the yield of 0.55g/L. The biosurfactant could reduce surface tension of water from 72.30 to 25.42mN/m at a concentration of 350mg/L and the emulsification index of machine oil reached 68% after 24h. The biosurfactant showed stability including thermal (121°C, 20min) and pH (7–11) stability regarding the surface tension reduction and the emulsification capacity of machine oil, and tolerance under salt concentrations (<2%). The biosurfactant also showed antimicrobial activity significantly against cell growth of E. coli BCRC13086 and S. aureus BCRC10780. The results obtained in this work are noteworthy for possible biosurfactant production from SPP with potential of application in removal of oil and in the future to gasoline-contaminated ocean shores for ecological protection purposes. Besides, it is also possible to be applied as a biocontrol agent. [Copyright &y& Elsevier]

Abstract: The exploitation of chitinous materials seems to be an infinite treasure. The oligosaccharides of chitin/chitosan, prepared by hydrolyzing chitin/chitosan with chitinase/chitosanase, have various potential applications in the fields of food, agricultural, and pharmaceutical industries. Almost all of the chitinase/chitosanases-producing strains use chitin/chitosan (or colloidal chitin/chitosan) as a major carbon source. However, the preparation of chitin involves demineralization and deproteinization of shellfish waste with the use of strong acids or bases. The utilization of shellfish waste as the sole carbon/nitrogen source not only solves environmental problems but also decreases the production costs of microbial chitinases. To further enhance the utilization of chitin-containing seafood processing wastes, we have recently investigated the bioconversion of shellfish chitin wastes (shrimp shell, crab shell, and squid pen) for the production of enzymes and bioactive materials. Rather than attempting to review the literature, this paper summarized our recently published works in the field of microbial reclamation of these unutilized marine bioresources. [Copyright &y& Elsevier]

Chitin from squid pen (Loligo sp.) and kiddi shrimp shell (Parapenaeopsis stylifera) were treated at room temperature (30 ± 2°C) with four different concentrations of sodium hydroxide: 20, 30, 40, and 50% w/w. With 50% sodium hydroxide solution, within 108 h, the chitin from squid pen was deacetylated to give chitosan. But it required 126 h at 40% and 144 h at 30% concentration of sodium hydroxide. In the case of chitin from Parapenaeopsis stylifera, complete deacetylation took place after 120 h and 168 h at 50 and 40% concentrations of sodium hydroxide, respectively. But shrimp shell on treatment with 20 and 30% sodium hydroxide solutions and squid pen kept at 20% sodium hydroxide were not sufficiently deacetylated even after 480 h. Properties like degree of deacetylation, viscosity and molecular weight of the prepared chitosan samples were studied. Minimum alkali concentration required for the formation of chitosan at room temperature was found to be 30% for squid chitin and 40% for shrimp chitin. With the increase in the time of deacetylation, decreases in molecular weight and viscosity were observed in chitosan from both sources. Maximum viscosity was recorded by chitosan prepared from squid pen using 30% sodium hydroxide solution at room temperature. [ABSTRACT FROM AUTHOR]

A protease-producing bacterium, strain TKU010, was isolated from infant vomited milk and identified as Lactobacillus paracasei subsp. paracasei. A surfactant-stable protease, purified 64-fold from the third day culture supernatant to homogeneity in an overall yield of 11%, has a molecular weight of about 49,000. The enzyme degraded casein and gelatin, but did not degrade albumin, fibrin, and elastin. The enzyme activity was increased about 1.5-fold by the addition of 5mM Ba2+. However, Fe2+ and Cu2+ ions strongly inhibited the enzyme. The enzyme was maximally active at pH 10 and 60°C and retained 94% and 71% activity in the presence of Tween 20 (2% w/v) and SDS (2mM), respectively. The result of identification of TKU010 protease showed that nine tryptic peptides were identical to Serratia protease (serralysin) (GenBank accession number gi999638) with 35% sequence coverage. In comparison with the tryptic peptides of L. paracasei subsp. paracasei TKU012 protease, TKU010 protease possessed two additional peptides with sequences of AATTGYDAVDDLLHYHER and QTFTHEIGHALGLSHPGDYNAGEGNPTYR. The fourth day culture supernatant of TKU010 showed maximal activity of about 5-fold growth enhancing effect on lettuce weight, which was not shown with L. paracasei subsp paracasei TKU012. [Copyright &y& Elsevier]

Abstract: The main purpose of this research was to investigate the antitumor and antimicrobial activities of the chitooligosaccharides containing hydrolyzates obtained from the hydrolysis of chitinous materials (such as chitin, chitosan, and squid pen) by bromelain. The optimum preparations were gained in the hydrolysates of squid pen powder hydrolyzed at pH 5, 37°C for 2 days by 0.1% bromelain. The hydrolysates had an 80% inhibitory activity on phyto-pathogenic mold Fusarium oxysporum. Chitooligosaccharides were recovered from the hydrolysates and were used for tumor cell surviving test. Surviving rate of the human leukemic U937 cells was reduced to 69% by the chitooligosaccharides. The solution of 0.1% of water-soluble chitosan was also hydrolyzed for 1 day at pH 5, 37°C by bromelain. The resultant hydrolysates contained the highest chitooligosaccharides, which had inhibitory effect on Bacillus subtilis and also had 40% inhibitory activity on human pathogenic mold Aspergillus fumigatus. Surviving rate of mouse CT26 colorectal adenocarcinoma cells was reduced to 57% by the chitooligosaccharides. This is the first publication of enzymatic reclamation of squid pen (fishery processing waste) for the preparation of antitumor and antimicrobial materials. [Copyright &y& Elsevier]