Nutritional and health benefits of soy proteins

Soy protein is a major component of the diet of food-producing animals and is increasingly important in the human diet. However, soy protein is not an ideal protein because it is deficient in the essential amino acid methionine. Methionine supplementation benefits soy infant formulas, but apparently not food intended for adults with an adequate nitrogen intake. Soy protein content of another essential amino acid, lysine, although higher than that of wheat proteins, is still lower than that of the milk protein casein. Adverse nutritional and other effects following consumption of raw soybean meal have been attributed to the presence of endogenous inhibitors of digestive enzymes and lectins and to poor digestibility. To improve the nutritional quality of soy foods, inhibitors and lectins are generally inactivated by heat treatment or eliminated by fractionation during food processing. Although lectins are heat-labile, the inhibitors are more heat-stable than the lectins. Most commercially heated meals retain up to 20% of the Bowman-Birk (BBI) inhibitor of chymotrypsin and trypsin and the Kunitz inhibitor of trypsin (KTI). To enhance the value of soybeans in human nutrition and health, a better understanding is needed of the factors that impact the nutrition and health-promoting aspects of soy proteins. This paper discusses the composition in relation to properties of soy proteins. Also described are possible beneficial and adverse effects of soy-containing diets. The former include soy-induced lowering of cholesterol, anticarcinogenic effects of BBI, and protective effects against obesity, diabetes, irritants of the digestive tract, bone, and kidney diseases, whereas the latter include poor digestibility and allergy to soy proteins. Approaches to reduce the concentration of soybean inhibitors by rearrangement of protein disulfide bonds, immunoassays of inhibitors in processed soy foods and soybean germplasm, the roles of phytoestrogenic isoflavones and lectins, and research needs in all of these areas are also discussed. This integrated overview of the widely scattered literature emphasizes general concepts based on our own studies as well as recent studies by others. It is intended to stimulate interest in further research to optimize beneficial effects of soy proteins. The payoff will be healthier humans and improved animal feeds.     

Synergistic action of an X-prolyl dipeptidyl aminopeptidase and a non-specific aminopeptidase in protein hydrolysis

Non-specific monoaminopeptidase (AP; E.C. 3.4.11) and X-prolyl dipeptidyl aminopeptidase (X-PDAP; E.C. 3.4.14.5), both from Aspergillus oryzae, demonstrate strong synergism in hydrolyzing proline-containing peptides. Incubation of AP alone with the peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe does not generate free amino acids. However, when AP and X-PDAP are added in combination, complete and immediate hydrolysis of all peptide bonds, other than X-Pro bonds, is observed. In the enzymatic hydrolysis of casein, soy, and gluten, degree of hydrolysis (DH) values of 54, 54, and 47% were achieved, respectively, when subtilisin (E.C. 3.4.21.62) was supplemented with AP. Addition of a third enzyme, X-PDAP, resulted in significantly higher DH values of 69, 72, and 64%, respectively, establishing the utility of this synergism in protein hydrolysis.     

Nutritional responses of rats to diets based on chickpea ( Cicer arietinum L.) seed meal or its protein fractions

The aim of this study was to isolate the protein fractions from chickpea, var. IAC-Marrocos, as well as to evaluate its in vivo nutritional protein quality. Among the proteins, albumins showed better nutritional value in the in vivo assays and amino acid contents, despite their higher trypsin inhibitor contents. Trypsin inhibitors were found to be heat labile in all samples, but the digestibility results for unheated and heated flour and albumins suggest that their contents are not very decisive. The PER values for casein (not supplemented) were very similar to those of heated flour and unheated or heated albumin and total globulins. The albumin and glutelin fractions showed the best results for PDCAAS, however, lower than those of casein. Despite the high digestibility of the globulin the very low essential amino acid content lowered its PDCAAS, and it had the lowest values.     

Controlled Stepwise Reduction of Disulfide Bonds and Heat-Induced Modification of Wheat Dough Proteins

A reducing solution of 2-mercaptoethanol and its oxidized form 2-hydroxyethyl disulfide, whose variable concentrations set variable disulfide reduction potentials, was applied to progressively reduce the disulfide bonds of proteins extracted from doughs made from Meneba and Robin Hood flour. Several dough proteins had disulfide bonds stronger than those of other dough proteins. A SDS-sedimentation method was applied to monitor the baking of dough into bread. Dough proteins susceptible to heat (baking) were studied by SDS-fractionation, extraction with reducing alcoholic solution, SDS-PAGE, and N-terminal protein sequencing. High or low molecular weight glutenins, α, β, and γ-gliadins, α-amylase inhibitor, and α-amylase trypsin inhibitor were identified among the dough proteins modified by heat (as shown by reduced solubility in aqueous-SDS solution). The heat-induced modification of the gliadins and glutenins might contribute to the coagulation of dough proteins, while the heat-induced modification of the amylase or trypsin inhibitors might contribute to the regulation of endogenous or exogenous amylolytic or proteolytic activities in dough or bread.     

Immunochemical and structural analysis of pepsin-digested egg white ovomucoid

Ovomucoid, an egg protein comprising approximately 10% egg white, was digested using the enzyme pepsin, and fragments were isolated by anion-exchange and reverse phase HPLC. Four distinct fragments were identified by analysis with SDS-PAGE, including three large fragments with molecular weights of around 24, 18, and 14 kDa. N- and C-terminal and amino acid sequencing analyses identified the fragments as V134-C186 (domain 3), V21-A133, and A1-A133 (domain 1+2). Further separation and sequencing of the fraction composed of small peptides, to determine their exact makeup and location in the protein, remained to be carried out and identified a peptide G51-Y73. All four fragments showed IgE-binding activity, as measured by ELISA, using human sera from egg-allergic individuals. Little change in the digestibility of ovomucoid by trypsin and chymotrypsin was observed following digestion with pepsin, indicating that pepsin-digested ovomucoid retains its trypsin (protease) inhibitor activities. Reduced carboxymethylated ovomucoid was prepared, and digestion with pepsin produced significantly more peptides than did the digestion of the native ovomucoid, indicating that the disulfide bonds play a significant role in the digestive resistance of ovomucoid. The reduction of ovomucoid enhanced its digestibility and lower allergenicity of the protein.     

beta-lactoglobulin hydrolysis. 1. Peptide composition and functional properties of hydrolysates obtained by the action of plasmin, trypsin, and Staphylococcus aureus V8 protease.

beta-Lactoglobulin (betaLg) was subjected to limited hydrolysis by trypsin, plasmin, and endoproteinase from Staphylococcus aureus V8 (S.aur.V8) to degrees of hydrolysis (DH) of 1, 2, and 4%. The several hydrolysates had different peptide compositions (determined by reversed-phase HPLC and gel-permeation chromatography [GPC]). GPC under nondenaturing, denaturing, and denaturing plus reducing conditions showed that the peptides formed were linked by hydrophobic interactions or by disulfide bonds or were not linked at all. At very low protein concentration, some differences in emulsion-forming properties were observed: only the plasmin hydrolysates could form emulsions with a uniform particle-size distribution. The emulsions formed with S.aur.V8 hydrolysates had poor emulsion-stabilizing properties. Some hydrolysates showed increased foam-forming properties in comparison with the intact protein. All foams formed were stable. Overall, the plasmin hydrolysate (DH4) contained relatively much larger molecules and/or hydrophobic molecules. Many molecules were disulfide-linked peptides. This hydrolysate also had the best functional properties.     

15N‐Labeling of Egg Proteins for Studying Protein Network Formation During Pound Cake Making

Proteins from wheat and egg are important for pound cake texture, but their exact role is insufficiently understood. A clear, analytical distinction between proteins from wheat flour, egg white, or egg yolk has been a main challenge. However, this can be addressed by using egg proteins carrying ¹⁵N. Therefore, egg white and yolk protein were enriched in ¹⁵N by mixing ¹⁵N‐labeled leucine into hen feed. Incorporation of egg and flour proteins in the protein network was monitored based on changes in their extractability during cake making. The relative contribution of different noncovalent and covalent bonds could be determined by using different extraction media. We for the first time distinguished between the contribution of egg white, egg yolk, and wheat protein in network formation during pound cake making. Our results show that during batter mixing hardly any intermolecular disulfide bonds are formed and that baking induces tremendous changes in protein extractability. A protein network based on both disulfide bonds and hydrophobic interactions is formed during baking. This covalent network includes almost all egg white protein and most of the yolk and wheat flour protein. The remaining protein fraction most probably lacks sulfhydryl groups and/or intramolecular disulfide bonds.     

Bowman-Birk and Kunitz Protease Inhibitors among Antinutrients and Bioactives Modified by Germination and Hydrolysis in Brazilian Soybean Cultivar BRS 133

Soybean contains constituents that have antinutritional and bioactive properties. Enzymatic hydrolysis and germination can enhance the biological activity of these compounds in soybean. The objective of this study was to investigate the effect of germination, Alcalase (protease) hydrolysis, and their combination on the concentrations of antinutritional and bioactive compounds in Brazilian soybean cultivar BRS 133. A combination of germination and Alcalase hydrolysis resulted in the degradation of Bowman-Birk inhibitor (BBI), Kunitz trypsin inhibitor (KTI), and lunasin by 96.9, 97.8, and 38.4%. Lectin was not affected by any of the processing treatments when compared to nongerminated and nonhydrolyzed soy protein extract. Total isoflavones (ISF) and total saponins (SAP) increased by 16.2 and 28.7%, respectively, after 18 h of germination, while Alcalase hydrolysis led to the reduction of these compounds. A significant correlation was found between concentrations of BBI and KTI, BBI and lunasin, BBI and ISF, KTI and lunasin, KTI and ISF, KTI and SAP, lunasin and ISF, and ISF and SAP. Germination and Alcalase hydrolysis interacted in reducing BBI, ISF, and SAP. This study presents a process of preparing soy flour ingredients with lower concentrations of antinutritional factors and with biologically active constituents, important for the promotion of health associated with soybean consumption. In conclusion, 18 h of germination and 3 h of Alcalase hydrolysis is recommended for elimination of protease inhibitors, while bioactives are maintained by at least 50% of their original concentrations.     

Comparison of interlaboratory variation in amino acid analysis and rat growth assays for evaluating protein quality

Estimates of inter- and intralaboratory variation of protein efficiency ratio (PER), relative PER (RPER), net protein ratio (NPR), relative NPR (RNPR), and nitrogen utilization (NU) were compared with those of amino acid analysis in the same batches of 7 protein sources (ANRC casein, egg white solids, minced beef, soy assay protein, rapeseed protein concentrate, pea flour, and whole wheat flour). Interlaboratory variation (estimated as between-laboratories coefficients of variation, CV) of NPR and RNPR (up to 6.0%) was lower than that of PER (up to 20.2%) and RPER (up to 18.5%). The interlaboratory determination of NPR and RNPR was also more reproducible than that of most essential amino acids (CV up to 10.0%), especially tryptophan (CV up to 23.7%), cystine (CV up to 17.6%), and methionine (CV up to 16.1%). Intralaboratory variation (estimated as within-laboratories CV) of amino acid analysis (up to 4.7%), however, was comparable to that of protein quality indices in most protein sources (up to 6.0%). The significant (P less than 0.01) positive correlations (r = 0.68-0.74) between amino acid scores and protein quality indices based on rat growth were further improved when amino acid scores were corrected for digestibility of protein (r = 0.73-0.78) or individual amino acids (r = 0.79-0.82).     

不同种类大豆蛋白粉对面包加工特性的影响

为探索大豆蛋白作为营养补充剂在面包中应用时,对面团物理特性和焙烤特性产生的影响,该文考察了不同种类的大豆蛋白制品,包括大豆分离蛋白、灭酶全脂粉、活性全脂粉、活性脱脂粉、灭酶脱脂粉对面团粉质特性、拉伸特性和焙烤特性的影响。结果表明,面粉的吸水率与大豆蛋白粉氮溶解指数显著相关,面团的抗拉阻力受大豆蛋白添加量的影响明显。大豆蛋白粉的加入,对面包比体积产生不利影响,下降趋势与大豆蛋白粉对面团拉伸特性的影响显著相关。大豆蛋白粉有软化面包质地的作用,活性全脂粉表现最为明显。大豆蛋白粉的加入量占面粉质量分数的3%时,对面包口感影响不明显,当加入量超过面粉质量分数的7％时,容易出现发粘和豆腥味等现象。     

Effect of Dehulling and Germination on Physicochemical and Pasting Properties of Black Beans (Phaseolus vulgaris L.)

Dehulled and/or germinated black bean flours were physicochemically characterized, including pasting properties, along with the trypsin inhibitor and antioxidant phenolics. To our best knowledge, this is the first study that, using nonparametric correlations and principal component analysis, identifies the parameters affecting the pasting properties of germinated black bean flour. The carbohydrate loss observed after black bean germination was indirectly correlated with the crude fiber content. Therefore, germination increased the protein and crude fiber contents compared with raw seeds (from 19.1 and 2.4% to 24.0 and 5.1%, respectively). Additionally, the highest protein digestibility was obtained in dehulled germinated black bean flour (78.4%), followed by whole germinated seed flour (74.1%). The dehulling process increased the total starch content 13.5 and 18.8% compared with raw and germinated whole bean flours, respectively. Dehulling decreased both trypsin inhibitor activity and antioxidant phenolics. Germination reduced by twofold the peak and final viscosities of black bean flours. Interestingly, both viscosities were negatively correlated with protein and positively correlated with fat and insoluble dietary fiber. Although resistant starch content was not affected by germination or dehulling, its interactions with fat and insoluble dietary fiber were responsible of the changes observed in pasting properties of germinated black bean flour.     

Effects of grinding and thermal treatments on hydrolysis susceptibility of pea proteins (Pisum sativum L.)

The effects of three particle sizes with two types of grindings and two thermal treatments on pea protein extraction (PE) and susceptibility to in vitro enzymatic hydrolysis (pepsin plus trypsin) were studied. Degrees of hydrolysis (DH) were calculated. Remaining peptides were detected by SDS-PAGE and identified by immunoblotting and MS/MS spectrometry. The increase in particle size decreased PE and DH due to a restricted access of solvents and enzymes to proteins. The thermal treatment induced a decrease in PE but did not modify DH. Heating improved legumin (alphaM) and convicilin pepsin hydrolyses but reduced the pea albumin 2 (PA2) hydrolysis. After pepsin and trypsin hydrolysis, only peptides from vicilin and lectin were identified by LC-MS/MS analyses, whatever the treatment.     

Influence of Cooking Conditions on the Protein Matrix of Sorghum and Maize Endosperm Flours

To understand the influence of the sorghum and maize endosperm protein matrix honeycomb structure on starch hydrolysis in flours, three‐dimensional fluorescence microscopy was applied to floury and vitreous endosperm flours cooked under various conditions. Cooking caused the collapse and matting of the sorghum and maize vitreous endosperm matrices, with the effect being greater in sorghum. The effect of cooking was rather different in the floury endosperm in that the protein matrices expanded and broke up to some extent. These effects were a consequence of expansion of the starch granules through water uptake during gelatinization. Cooking in the presence of 2‐mercaptoethanol caused an expansion of the vitreous endosperm matrix mesh due to breakage of disulfide bonds in the protein matrix. Mercaptoethanol also caused an increase in the proportion of β‐sheet structure relative to α‐helical structure of the endosperm proteins. Increased energy of cooking caused collapse of the sorghum matrix. Disulfide bonding and an increase in β‐sheet structure occurred with cooking, with the increase in disulfide bonding being greatest in sorghum vitreous endosperm. The tendency for the sorghum protein matrix to collapse and mat more with cooking than the maize matrix appears to be due to greater disulfide bonding. This is responsible for the observed low starch digestibility of cooked sorghum flour as a result of the more disulfide‐bonded protein matrix limiting the expansion of the starch granules and hence amylase access.     

大豆蛋白限制性酶解模式与产品胶凝性的相关性

为了改善大豆蛋白的胶凝性,对大豆浓缩蛋白、大豆分离蛋白进行限制性酶解处理,并考察相应产品的蛋白酶酶解模式与胶凝性变化的相关性。该研究以蛋白质的水解度为指标,通过中性蛋白酶、胰蛋白酶的酶解作用,水解大豆浓缩蛋白、大豆分离蛋白至蛋白质水解度(DH)为1%、2%,考察酶性质、蛋白质的DH对产品胶凝性影响,并利用SDS-凝胶电泳进行确认。结果表明:大豆浓缩蛋白经中性蛋白酶、胰蛋白酶的酶解后,产品胶凝性均显著下降;大豆分离蛋白经中性蛋白酶的酶解后,产品胶凝性在DH为1%时增加,但在DH为2%时下降;大豆分离蛋白经胰蛋白酶酶解后,胶凝性显著改善。SDS-凝胶电泳确认,蛋白质的酶水解模式和水解度不同是导致产品胶凝性产生不同变化的原因。     

Microbial Transglutaminase as a Tool to Restore the Functionality of Gluten from Insect‐Damaged Wheat

In some wheat‐growing countries, considerable quantities of commercial wheat are rendered unusable in standard baking because of preharvest damage of the grain by protease‐injecting bugs. In the present study, we studied the ability of transglutaminase (TG) treatment of damaged wheat flour to return the functionality of the gluten network. To confirm the TG cross‐linking, the degree of protein hydrolysis, the amount of free thiol groups, and the electrophoresis properties of glutenin subunits were determined. The effectiveness of the TG treatment on insect‐damaged wheat was analyzed by measuring the dough mixing behavior and the gluten quality. A decrease in the degree of hydrolysis (or free amino groups), a reduction in thiol group concentration, and a decrease of extractable high molecular weight glutenin subunits (HMW‐GS) (measured by high‐performance capillary electrophoresis) confirmed the protein cross‐linking catalyzed by TG, the simultaneous formation of disulfide bonds by the proximity of the cross‐linked polypeptide chains, and the formation of aggregates of high molecular weight. The TG treatment of the damaged wheat flour led to a recovery of the consistograph parameters and gluten index value, and the covalent nature of the bonds ensured the stability of the protein changes.     

In vitro digestion rate and estimated glycemic index of oat flours from typical and high β-glucan oat lines

The in vitro starch digestion rate and estimated glycemic index (GI) of oat flours and oat starches from typical and high β-glucan oat lines were evaluated along with the impact of heating on starch digestion. Flour from oat lines ('Jim', 'Paul', IA95, and N979 containing 4.0, 5.3, 7.4, and 7.7% β-glucan, respectively) was digested by pepsin and porcine pancreatin. To determine the impact of heating on starch digestion, oat slurries were prepared by mixing oat flour and water (1:8 ratio) and heating for 10 min prior to digestion. Viscosity, as measured on a Rapid Visco Analyzer, increased with increases in concentration and molecular weight of β-glucan. The in vitro starch digestion of oat flours and a control, white bread made from wheat flour, increased as the digestion time increased. Starch digestion of oat flour was slower than that of the control (p < 0.05). Heat treatment of oat-flour slurries increased the starch digestion from a range of 31-39% to a range of 52-64% measured after 180 min of in vitro digestion. There were no differences in starch digestibility among oat starches extracted from the different oat lines. The GI, estimated by starch hydrolysis of oat flours, ranged from 61 to 67, which increased to a range of 77-86 after heating. Oat-flour slurries prepared from IA95 and N979 lines with high β-glucan concentrations had lower GI values than did slurries made from Jim and Paul lines. Starch digestion was negatively correlated with β-glucan concentrations in heated oat-flour slurries (R(2) = 0.92). These results illustrate that the oat soluble fiber, β-glucan, slowed the rate of starch digestion. This finding will help to develop new food products with low GI by using oat β-glucan.     

脱酰胺与双酶协同作用提高小麦面筋蛋白酶解效率

为了探讨了不同脱酰胺处理和双酶协同作用方式对小麦面筋蛋白酶解效率及其产物抗氧化活性的影响,该文研究了小麦面筋蛋白在各种预处理方式和酶解条件下的蛋白回收率、水解度、抗氧化性能及肽分子量分布情况。结果显示,单独热处理(90℃,30 min)小麦面筋蛋白对其酶解效率无显著影响,而采用添加0.5 mol/L柠檬酸溶液进行热处理(质量分数为5%,90℃,30 min)可显著(P0.05)提高其蛋白回收率。此外,酶制剂添加顺序及双酶共同水解作用时间对酶解效率均具有较大影响:加入谷氨酰胺酶预先水解对小麦面筋蛋白的深度水解有促进作用;一定时间内的双酶协同作用有利于酶解的进行,但较长时间的双酶作用反而会抑制酶解效率。采用谷氨酰胺酶(质量分数为0.2%)对经柠檬酸加热处理的小麦面筋蛋白作用12 h后再加入胰酶(质量分数为0.6%)共同作用7 h可使蛋白回收率达70.74%,水解度达到9.88%;另外,酶解产物的自由基清除能力ABTS+(2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)+)值与氧化自由基吸收能力(ORAC,oxygen radical absorbance capacity)值分别达到478.95 mmol/g和213.85μmol/g,提示该酶解产物是一种潜在优秀食品抗氧化剂。研究结果可为拓宽小麦面筋蛋白的应用领域,以及高效制备抗氧化活性肽提供方法和理论指导。     

Physicochemical and functional properties of buckwheat protein product

This study was conducted to compare the physicochemical and functional properties of buckwheat protein product (BWP), soy protein isolate (SPI), and casein. BWP was prepared from buckwheat flour by the method including alkaline extraction and isoelectric precipitation. The amino acid composition of BWP was very similar to that of buckwheat flour. The protein solubility (PS) of BWP was much greater than that of SPI at all pH levels (pH 2-10) but lower than that of casein at pH 7-10. The isoelectric point of BWP was around pH 4. The higher aromatic hydrophobicities (ARH) of BWP, SPI, and casein were obtained at lower pH levels (pH 2-3). The emulsifying stability (ES) of BWP was lower than those of SPI and casein at high pH levels (pH 7-10). At all pH levels, BWP formed a thin emulsion. Regression analysis showed that the ARH of BWP was significantly associated with the ES. Although the water holding capacity of BWP was quite lower than that of SPI, its fat absorption capacity was slightly higher than those of SPI and casein. These results indicated that the physicochemical properties of BWP were different from those of SPI or casein. Thus, BWP is a potential source of functional protein for possible food application.     

Hydrophobicity,Solubility, and Emulsifying Properties of Enzyme-Modified Rice Endosperm Protein

Rice endosperm protein was modified to enhance solubility and emulsifying properties by controlled enzymatic hydrolysis. The optimum degree of hydrolysis (DH) was determined for acid, neutral, and alkaline type proteases. Solubility and emulsifying properties of the hydrolysates were compared and correlated with DH and surface hydrophobicity. DH was positively associated with solubility of resulting protein hydrolysate regardless of the hydrolyzing enzyme, but enzyme specificity and DH interactively determined the emulsifying properties of the protein hydrolysate. The optimum DH was 6–10% for good emulsifying properties of rice protein, depending on enzyme specificity. High hydrophobic and sulfhydryl disulfide (SH-SS) interactions contributed to protein insolubility even at high DH. The exposure of buried hydrophobic regions of protein that accompanied high-temperature enzyme inactivation promoted aggregation and cross-linking of partially hydrolyzed proteins, thus decreasing the solubility and emulsifying properties of the resulting hydrolysate. Due to the highly insoluble nature of rice protein, surface hydrophobicity was not a reliable indicator for predicting protein solubility and emulsifying properties. Solubility and molecular flexibility are the essential factors in achieving good emulsifying properties of rice endosperm protein isolates.     

Kinetic study on the changes in the susceptibility of egg white proteins to enzymatic hydrolysis induced by heat and high hydrostatic pressure pretreatment

A kinetic study was conducted on the effect of heat pretreatment in the temperature range of 50-85 degrees C at atmospheric pressure and of high hydrostatic pressure pretreatment (100-700 MPa) at four temperatures (10, 25, 40, and 60 degrees C) on the susceptibility of egg white solutions (10% v/v, pH 7.6) to subsequent enzymatic hydrolysis by a mixture of trypsin and alpha-chymotrypsin at 37 degrees C and pH 8.0. Both heat pretreatment at atmospheric pressure and high-pressure pretreatment resulted in an increase in degree of hydrolysis (DH) after 10 min of enzymatic reaction (DH10) of egg white solutions, as measured using the pH-stat method, which could be described by a fractional conversion model (based on an apparent first-order reaction kinetic model). The temperature dependence of the corresponding rate constants could be described by the Arrhenius equation. At elevated pressure, a negative apparent activation energy was obtained, implying an antagonistic effect of pressure and temperature. The pressure dependence of the rate constants could be described by the Eyring equation, and negative activation volumes were observed, which demonstrates the positive effect of pressure on the susceptibility of egg white solutions to subsequent enzymatic hydrolysis.