Effect of isoelectric solubilization/precipitation and titanium dioxide on whitening and texture of proteins recovered from dark chicken-meat processing by-products

Processing of chicken generates by-products containing muscle proteins attached to bones and skin that, if recovered, could be a functional ingredient in restructured food products. However, color of restructured products made of proteins recovered from chicken processing by-products is poor. The by-products contain bones, skin, fat, etc. that affect color of restructured products. Therefore, color properties need to be improved. The objectives of this study were to determine effects of isoelectric solubilization/precipitation (ISP) and TiO₂ on instrumental color and texture properties of heat-set gels made of proteins recovered from dark chicken-meat processing by-products as compared to gels made of chicken breast meat. Skin-on bone-in chicken drumsticks were used as a model dark chicken-meat processing by-products. TiO₂ at 0-1 g/100 g and canola oil at 10 g/100 g were added to the ISP-recovered proteins followed by cooking. Due to higher (P < 0.05) yellowness (b∗) and lower (P < 0.05) lightness (L∗), the whiteness of drumstick gels without TiO₂ was lower (P < 0.05) than breast gels. TiO₂ at 1 g/100 g with canola oil resulted in slightly better (P < 0.05) whiteness of drumstick gels than breast gels. TiO₂ did not deteriorate gel texture, which was generally comparable to breast gels. This research indicates that ISP allows recovery of proteins from skin-on bone-in dark chicken-meat processing by-products without removal of bones, skin, and fat prior to processing. Addition of TiO₂ to proteins recovered from these by-products allows development of heat-set gels with color and texture comparable to chicken breast gels. Although this study shows the potential for a novel, marketable food product, sensory tests are recommended.     

Color improvement by titanium dioxide and its effect on gelation and texture of proteins recovered from whole fish using isoelectric solubilization/precipitation

Whiteness is a critical attribute for restructured fish products such as surimi seafood. However, the whiteness of gels made from proteins recovered from fish processing by-products or whole fish using isoelectric solubilization/precipitation is poor. The by-products and whole fish contain bones, scales, skin, etc. that affect gel color. Therefore, whiteness needs to be improved if marketable products are to be developed from recovered proteins. The objectives of this study were to determine effects of titanium dioxide (TiO₂) on: (1) color; (2) texture; and (3) viscoelasticity (G′) of gels made from isolated carp proteins and Alaska pollock surimi. Carp proteins were recovered with isoelectric solubilization/precipitation. TiO₂ was added to carp proteins at 0-0.5 g/100 g. TiO₂ was not added to surimi. Due to much higher (P < 0.05) yellowness (b^∗) and lower (P < 0.05) lightness (L^∗), the whiteness of carp gels without TiO₂ was lower (P < 0.05) than surimi gels. TiO₂ at ≥ 0.2 g/100 g resulted in better (P < 0.05) whiteness of carp gels than surimi gels without chalky and artificially white appearance. TiO₂ did not affect texture or viscoelasticity. This research demonstrates that whiteness of restructured fish products based on proteins recovered from whole fish via isoelectric solubilization/precipitation can be similar to the whiteness of surimi seafood.     

Functional and nutritional characteristics of proteins and lipids recovered by isoelectric processing of fish by-products and low-value fish: A review

Several fisheries are over-exploited and may collapse; yet the amounts of fish processing by-products containing muscle proteins and ω-3-rich oil are staggering. The by-products are land-filled, ground and discarded or otherwise diverted from human consumption. Due to the lack of technology to recover proteins and lipids from by-products or low-value species, this tremendous resource is unavailable for human consumption. Isoelectric solubilisation/precipitation (ISP) allows efficient recovery of fish proteins and oil which retain functionality and nutritional value of food products. Isoelectric point (pI) is a pH where protein maintains zero electrostatic charge. At pI, protein–protein hydrophobic attraction overcomes protein–water electrostatic attraction resulting in isoelectric precipitation. Conversely, isoelectric solubilisation occurs at a pH different from pI, whereby protein–water attraction and protein–protein electrostatic repulsion are favoured. Therefore, protein solubility/insolubility is induced by ISP, respectively. Consequently, ISP allows selective protein recovery. Lipids are also recovered during ISP processing. This article reviews recent ISP developments to recover proteins and lipids from by-products and low-value marine species.     

Amino acid, fatty acid, and mineral profiles of materials recovered from rainbow trout (Oncorhynchus mykiss) processing by-products using isoelectric solubilization/precipitation

ABSTRACT:  Protein, lipid, and insolubles (bones, skin, scales, fins, insoluble protein, and more) were recovered from rainbow trout processing by-products by means of isoelectric solubilization/precipitation at basic pH and acidic pH. Isoelectric solubilization/precipitation of the trout processing by-products resulted in the recovery of protein that was higher ( P < 0.05) in essential amino acids (EAAs), non-EAAs, and total EAA/total AA ratio when compared to the processing by-products. Basic pH treatments yielded a higher ( P < 0.05) content of EAAs than the acidic pH treatments. Nutritional quality of the recovered protein was high based on EAAs meeting the FAO/WHO/UNU recommendations for adults. The presence of omega-3 and omega-6 fatty acids (ω-3, ω-6 FAs) and the ω-3/ω-6 ratio in the recovered lipids were similar to the trout processing by-products, indicating that the pH treatments had no effect on these FAs. Ca and P contents of the processing by-products exceeded the recommended dietary allowances (RDA), but Fe and Mg did not. Basic pH treatments yielded protein with the lowest ( P < 0.05) amount of minerals and the highest ( P < 0.05) amount of Ca, P, and Mg in the insolubles when compared to acidic pH. The isoelectric solubilization/precipitation of the processing by-products effectively removed minerals from the recovered protein without removal of the bones, skin, scales, fins, and so on, prior to processing. The results indicated that isoelectric solubilization/precipitation, particularly at basic pH, permitted recovery of high-quality protein and lipids from fish processing by-products for human food uses; also, the recovered insolubles may be used in animal feeds as a source of minerals.     

A three-prong strategy to develop functional food using protein isolates recovered from chicken processing by-products with isoelectric solubilization/precipitation

BACKGROUND: Skin‐on bone‐in chicken drumsticks were processed with isoelectric solubilization/precipitation to recover muscle proteins. The drumsticks were used as a model for dark chicken meat processing by‐products. The main objective of this study was conversion of dark chicken meat processing by‐products to restructured functional food product. An attempt was made to develop functional food product that would resemble respective product made from boneless skinless chicken breast meat. A three‐prong strategy to address diet‐driven cardiovascular disease (CVD)with a functional food was used in this study. The strategy included addition of three ingredients with well‐documented cardiovascular benefits: (i) ω‐3 polyunsaturated fatty acid‐rich oil (flaxseed‐algae, 9:1); (ii) soluble fiber; and (iii) salt substitute. Titanium dioxide, potato starch, polyphosphate, and transglutaminase were also added. The batters were formulated and cooked resulting in heat‐set gels. RESULTS: Color (L*a*b*), texture (torsion test, Kramer shear test, and texture profile analysis), thermal denaturation (differential scanning calorimetry), and gelation (dynamic rheology) of chicken drumstick gels and chicken breast gels were determined and compared. Chicken drumstick gels generally had comparable color and texture properties to the gels made from chicken breast meat. The endothermic transition (thermal denaturation) of myosin was more pronounced and gelation properties were better for the drumstick gels. CONCLUSION: This study demonstrated a feasibility to develop functional food made of muscle proteins recovered with isoelectric solubilization/precipitation from low‐value dark chicken meat processing by‐products. The functional food developed in this study was enriched with CVD‐beneficial nutrients and had comparable instrumental quality attributes to respective products made of chicken breast meat. Although the results of this study point towards the potential for a novel, marketable functional food product, sensory tests and storage stability study are recommended. Copyright © 2012 Society of Chemical Industry     

Determination of the nutritional value,protein quality and safety of krill protein concentrate isolated using an isoelectric solubilization/precipitation technique

Despite its abundance, krill is not utilized for human consumption due to the lack of proper technology for protein recovery. The objectives of this study were to isolate krill protein concentrate (KPC) and to determine the nutritional value, health benefits, and safety of KPC for human consumption. Proximate analysis of protein recovered by isoelectric solubilzation/precipitation indicated that KPC was composed of approximately 78% protein and 8% fat (dry weight). In vivo analysis of protein quality indicated that protein digestibility corrected for amino acid score and protein efficiency ratio was equal to casein. KPC safety was indicated by the absence of differences in clinical measures of kidney function compared to casein. Fatty acid analysis of KPC showed that approximately 27% were omega-3 polyunsaturated fatty acids (ω-3 PUFAs). Based on our study, KPC appears to be a promising protein source for human consumption with the advantage of being a rich source of ω-3 PUFAs.     

Functional properties of proteins recovered from silver carp (Hypophthalmichthys molitrix) by isoelectric solubilization/precipitation

Isoelectric solubilization/precipitation at acidic and basic pH ranges was applied to whole gutted silver carp (Hypophthalmichthys molitrix) in order to recover muscle proteins. Thermal denaturation (T_onset, T_max, and ΔH), viscoelasticity (G′), and texture properties (shear stress) of proteins recovered from carp as affected by functional additives (beef plasma protein, potato starch, exogenous transglutaminase, polyphosphate, and titanium dioxide) were determined and compared to Alaska pollock surimi. Proteins recovered from carp showed typical endothermic transitions only when functional additives were used. Similar to endothermic transitions, viscoelasticity in carp proteins increased only when the additives were used. Typical endothermic peaks and viscoelasticity increase were recorded for Alaska pollock surimi. Carp protein-based gels with functional additives had lower (P < 0.05) shear stress than their surimi counterparts, but greater (P < 0.05) or similar (P > 0.05) when compared to surimi gels without functional additives. In addition, generally higher shear stress was measured for carp protein-based gels developed from basic pH treatments than the acidic counterparts. The present study indicates that proteins can be recovered from whole gutted carp using isoelectric solubilization/precipitation. However, if the recovered proteins are used for subsequent development of restructured food products, functional additives should be used.     

酸碱处理技术在肌肉蛋白质分离加工中的应用

酸碱处理技术作为一种蛋白分离、加工技术在肉类加工领域得到了广泛的研究和应用。本文介绍了酸碱处理技术的基本原理及对蛋白质结构和加工特性的影响,其次综述了该技术在鱼类去腥、改善禽类产品凝胶品质以及制备功能食品中的应用及相关研究进展,同时对该技术存在的问题及发展趋势进行探讨,以期对该技术在肌肉蛋白质加工中的应用提供参考。      

鱼糜废水中蛋白质等电沉淀的规律研究

为寻找鱼糜废水中鱼浆蛋白的优化回收方法,实现鱼糜废水净化及蛋白回收再利用的双重目的,对鱼糜废水中蛋白质的等电沉淀规律进行了研究。在初步了解鱼糜废水中蛋白成分性质的前提下,考察了不同pH、酸种类、离子强度下的蛋白质沉淀规律,并尝试采用分级等电沉淀方法提高蛋白总回收率。结果表明,鱼浆蛋白成分的等电点主要密集分布在4.3和5.9两个位置;多元酸的沉淀效果较优,离子强度提高也有促进蛋白沉淀作用;分级等电沉淀可将蛋白总回收率从传统方法60%左右高至74.36%。本研究所做的基础研究及提出的分级等电沉淀技术思路,将为蛋白沉淀回收技术基础理论的丰富和思路的开拓提供有益补充。      

Pilot plant scale production of protein from catfish treated by acid solubilization/isoelectric precipitation

ABSTRACT:  Catfish fillets were minced and acid (pH 3.0) solubilized in dilute conditions (1:9). Insolubles were removed by centrifugal force (3000 × g ) and the soluble protein was recovered by isoelectric precipitation (pI approximately 5.4). Moisture and NaCl of the batters were equilibrated to 78% and 2%, respectively. Samples were stuffed into casings, cooked, and chilled. Proximate composition, color, cook yield, water holding ability (WHA), and texture profile analysis (TPA) were determined. Treatment of catfish protein by the solubilization process removed 74% of the fat. In addition, gels from treated protein were less red (−1.61 compared with −1.06) and yellow (6.58 compared with 7.09). There was no difference in whiteness (72.0 compared with 71.8) of gels or in percent cook yield (94.6% compared with 95.0%) between treated protein and the control, respectively. The WHA of gels made from control protein was 0.73 g/g protein higher than treated. The texture analysis indicated that treated proteins had significantly higher hardness (1193 g compared with 1046 g ), springiness (0.919 compared with 0.897), chewiness (0.701 compared with 0.584), and cohesiveness (0.637 mJ compared with 0.619 mJ). The resilience was equivalent for both treated and control gels. The results indicated the protein solubilization process produced a low fat protein product with good gel strength properties.     

Comparison between isoelectric precipitation and ultrafiltration processes to obtain Amaranth mantegazzianus protein concentrates at pilot plant scale

The aim of this study was to compare protein yield, protein concentration and physicochemical characteristics of Amaranth mantegazzianus protein concentrates (APC) obtained at pilot-scale by a conventional process (CP) (alkaline extraction and isoelectric precipitation) and two alternative processes (AP): (1) acid pre-treatment process combined with isoelectric precipitation and (2) acid pre-treatment process combined with ultrafiltration. Although AP resulted in higher protein concentration, protein yield was lower than in CP. SDS–PAGE and size-exclusion chromatography showed high molecular weight fractions only for isoelectric precipitation concentrates (obtained by CP and AP). The amino acids concentration, especially phenylalanine, isoleucine and methionine, increased in all protein concentrates respect to the amaranth flour. Particularly, the product obtained by ultrafiltration was rich in phenylalanine and lysine, and presented no limiting amino acid with respect to the recommendation of the Food and Agriculture Organization of the United Nations (FAO).     

鮰鱼下脚料蛋白质的回收及其凝胶特性研究

通过回收并制备鮰鱼下脚料的蛋白质凝胶,考察溶解pH、离心力、离子强度对鱼下脚料中蛋白质的溶解性及凝胶理化性质的影响。结果表明,溶解pH、离心力、离子强度对鱼下脚料中的蛋白质的溶解性能均有显著影响(P<0.05)。pH=2.5或pH=11.5,离心力为5 500r/min,离子强度为0.6M时,蛋白质溶解性能较好,pH=5.5时蛋白质溶解性最差。溶解pH=11.5时,制备的鱼肉凝胶蛋白质、灰分含量较高,脂类含量较低,并且凝胶的破断强度值、凹陷度以及凝胶强度值较高,但凝胶色泽较差。     

Gelation properties of goose liver protein recovered by isoelectric solubilisation/precipitation process

Isoelectric solubilisation/precipitation (ISP) process was applied to goose liver (GL) for protein extraction. The gelation properties of proteins extracted by acid processes (ACP, pH 2.0, 2.5 and 3.0) and alkaline processes (ALP, pH 11.0, 11.5 and 12.0) were estimated, where the unextracted ground GL was set as the control. Nearly 58.39~79.00% of GL proteins were recovered by ISP treatments. High molecular weight (100~250 kDa) proteins were found to be partially hydrolysed by ACP, while few changes in proteins occurred during ALP. As evidenced by rheological and textural measurements, ALP proteins formed gels with high elasticity and superior texture, whereas ACP proteins had inferior gelation properties. Moreover, ALP proteins were able to form a highly interconnected and homogeneous three‐dimensional microstructure. Predominantly, gels produced by 11.0 had optimal texture and the lowest cooking loss (P < 0.05). These results suggested that the ISP process (ALP) is a potential method to improve the economic value of GL.     

The effect of using citric or acetic acid on survival of Listeria monocytogenes during fish protein recovery by isoelectric solubilization and precipitation process

Isoelectric solubilization and precipitation (ISP) is a protein recovery process effective at reducing Listeria innocua, a nonpathogenic bacterium typically used as a surrogate for L. monocytogenes in recovered trout protein. The response of L. monocytogenes to ISP processing was determined and compared to the response of L. innocua. Headed and gutted rainbow trout were inoculated with L. monocytogenes (10.16 log CFU/g), homogenized, and pH-adjusted with granular citric acid (pH 2.0 and 2.5) or glacial acetic acid (pH 3.0 and 3.5). Proteins were solubilized and centrifugation was used to remove insoluble components (skin, insoluble protein, so on). The supernatant was returned to the protein isoelectric point (pH 5.5) with NaOH and centrifuged to remove precipitated protein. Microbial load was enumerated on both growth and selective media; recovery was not significantly different (P > 0.05). Surviving cells from each component (protein, insoluble, and water) were compared to initial inoculum numbers. Significant reductions were detected at all pH (P < 0.05). The greatest reductions were at pH 3.0 with acetic acid, with a mean log reduction of 3.03 in the combined components, and a 3.53 log reduction in the protein portion. Data were compared to results from a previous study using L. innocua. Significant differences (P < 0.05) in recovery were found between the 2 species at pH 2.0 and 3.0 with greater recovery of L. monocytogenes, regardless of processing pH or acid type. These results demonstrate the variability in resistance between species and indicate that L. innocua is not an appropriate surrogate for L. monocytogenes for ISP processing with organic acids.     

A review: Protein isolates recovered by isoelectric solubilization/precipitation processing from muscle food by-products as a component of nutraceutical foods

It is possible to recover muscle protein isolates from food processing by-products and under-utilized or difficult to process sources that otherwise would be discarded or diverted from direct human consumption by using isoelectric solubilization/precipitation (ISP). ISP selectively induces water solubility of muscle proteins by changing pH. When muscle proteins are dissolved, they are separated from lipids and other insoluble fractions such as skin, bones, scales, etc. Following separation, the dissolved proteins are subjected to subsequent pH change that causes protein precipitation and yields protein isolate. ISP processing efficiently recovers protein isolates of high quality from both nutritional and technological stand point. However, attempts at commercializing food products developed from the ISP-recovered protein isolates have been very limited. Results from laboratory-scale product development research demonstrate the potential for the use of ISP-recovered protein isolates as a base and functional ingredient for prototypes of nutraceutical foods with specific health benefits. This article reviews ISP as an innovative means to recover functional protein isolates from low-value sources. It also covers recent attempts to develop prototypes of nutraceutical food products using the ISP-recovered protein isolates targeting diet-driven cardiovascular disease.     

Texture and colour properties of proteins recovered from whole gutted silver carp (Hypophthalmichthys molitrix) using isoelectric solubilisation/precipitation

BACKGROUND: According to an FAO report, carp are the cheapest and by far the most commonly consumed fish in the world. Carp have minimal growth requirements, yet rapid growth rates. Although carp are generally considered unsuitable for human consumption in the USA, they have rapidly started populating major bodies of fresh water in the USA to the extent that commercial processing becomes of interest. However, typical mechanical means of meat recovery from carp are impractical owing to the bony nature of the carp carcass. Therefore the aim of the present study was to devise processing strategies to recover fish meat from carp that could be used in the development of human food products. RESULTS: Isoelectric solubilisation/precipitation at acidic and basic pH values was applied to whole gutted silver carp. Depending on the solubilisation pH, protein and fat recovery yields were approximately 420–660 and 800–950 g kg^?1 respectively. The process effectively removed impurities such as bones, scales, skin, fins, etc. from whole gutted carp. The proteins were concentrated to approximately 900 g kg^?1, while the fat was reduced by 970–990 g kg^?1. Functional additives (potato starch, beef plasma protein, transglutaminase and polyphosphate) improved (P < 0.05) the texture of carp protein‐based gels such that it was generally comparable to the texture of Alaska pollock surimi gels. Although titanium dioxide improved (P < 0.05) the whiteness of carp gels, it was lower (P < 0.05) than the whiteness of Alaska pollock surimi gels. CONCLUSION: Isoelectric solublisation/precipitation allows protein and lipid recovery from whole gutted carp. However, if the proteins are used as a gelling ingredient in fish food products, functional additives are recommended. Copyright © 2008 Society of Chemical Industry     

Dynamic rheology and endothermic transitions of proteins recovered from chicken-meat processing by-products using isoelectric solubilization/precipitation and addition of TiO2

Chicken-meat processing generates large quantities of by-products (backs, necks, etc.). Dark chicken-meat processing by-products present the lowest value and greatest challenge. Therefore, recovery of functional proteins from this source for inclusion in food products resembling those from light chicken-meat presents the greatest value addition and opportunity. Novel isoelectric solubilization/precipitation (ISP) was applied to model, dark chicken-meat processing by-products (skin-on bone-in chicken drumsticks) to recover muscle proteins. Thermal denaturation (endothermic transitions), gelation (elasticity, G′), and fundamental texture properties (shear stress and strain at mechanical fracture) of the ISP-recovered proteins were determined with differential scanning calorimetry (DSC), dynamic rheometer, and torsion test, respectively; and compared to boneless skinless chicken breast. Endothermic transition of myosin was not detected only when TiO₂ was not added, while the ISP-recovered proteins with TiO₂ showed small myosin peak and large actin peak. However, the level of TiO₂ addition did not affect thermal transition/denaturation of the ISP-recovered proteins. The ISP-recovered proteins had a greater transition for actin compared to chicken breast, suggesting that ISP predisposes this protein to thermal denaturation. Similar to endothermic transitions, elasticity (G′) generally increased when TiO₂ was added to the ISP-recovered proteins. Gels made of chicken breast had the highest (P < 0.05) shear stress (i.e., gel strength), but gels made of the ISP-recovered chicken proteins had greater (P < 0.05) shear strain (i.e., gel cohesiveness). Addition of TiO₂ to the ISP-recovered proteins resulted in increased (P < 0.05) gel strength. Based on the present study, addition of TiO₂ is suggested for the development of restructured food products based on proteins recovered from dark chicken-meat processing by-products using ISP. Although the results of this study point towards a novel food product, further studies are recommended.     

Effect of microbial transglutaminase and setting condition on gel properties of blend fish protein isolate recovered by alkaline solubilisation/isoelectric precipitation

The effect of microbial transglutaminase (M-TGase) (0–0.6 units g⁻¹ sample) and setting condition (25 °C/180 min, 30 °C/120 min, 35 °C/60 min and 40 °C/30 min) on gel properties of blend protein isolate of gutted kilka and silver carp was studied. The protein isolate provided a good substrate for M-TGase activity so that a low amount of M-TGase (0.2 unit g⁻¹ sample) substantially improved textural properties and water holding capacity (WHC) of the gels. Breaking force of the gels was positively affected by M-TGase up to 0.6 unit g⁻¹ sample, but it negatively affected their WHC. Prior setting at 25–35 °C increased the breaking force of proteins compared to directly heated gel, resulting in maximum breaking force at 35 °C/60 min. However, the setting at 40 °C/30 min caused proteolysis, which was reflected in higher amounts of TCA-soluble peptides and gel weakening. Denser microstructure and higher myosin heavy chain polymerisation observed in the gels which experienced the setting was well correlated with improvement in textural properties.     

谷氨酰胺转氨酶对碱溶酸沉法提取南极磷虾(Euphausia superba)蛋白质得率的影响

通过碱溶酸沉法提取了南极磷虾蛋白质,研究了谷氨酰胺转氨酶的应用对蛋白质得率的影响。结果表明:碱溶酸沉法能够较好地回收南极磷虾蛋白质;在酸沉过程中,合理添加谷氨酰胺转氨酶能够提高蛋白质得率约5%。     

Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from heads and viscera of sardinelle (Sardinella aurita) by treatment with various proteases were investigated. Protein hydrolysates were obtained by treatment with Alcalase^®, chymotrypsin, crude enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina pilchardus) viscera. All hydrolysates exhibited inhibitory activity towards ACE. The alkaline protease extract from the viscera of sardine produced hydrolysate with the highest ACE inhibitory activity (63.2 ± 1.5% at 2 mg/ml). Further, the degrees of hydrolysis and the inhibitory activities of ACE increased with increasing proteolysis time. The protein hydrolysate generated with alkaline proteases from the viscera of sardine was then fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Biological functions of all fractions were assayed, and P₄ was found to display a high ACE inhibitory activity. The IC₅₀ values for ACE inhibitory activities of sardinelle by-products protein hydrolysates and fraction P₄ were 1.2 ± 0.09 and 0.81 ± 0.013 mg/ml, respectively. Further, P₄ showed resistance to in vitro digestion by gastrointestinal proteases. The amino acid analysis by GC/MS showed that P₄ was rich in phenylalanine, arginine, glycine, leucine, methionine, histidine and tyrosine. The added-value of sardinelle by-products may be improved by enzymatic treatment with visceral serine proteases from sardine.