Streptococcus thermophilus: A Source of Postbiotics Displaying Anti-Inflammatory Effects in THP 1 Macrophages.

In addition to traditional use in fermented dairy products, S. thermophilus also exhibits anti-inflammatory properties both in live and heat-inactivated form. Recent studies have highlighted that some hydrolysates from surface proteins of S. thermophilus could be responsible partially for overall anti-inflammatory activity of this bacterium. It was hypothesized that anti-inflammatory activity could also be attributed to peptides resulting from the digestion of intracellular proteins of S. thermophilus. Therefore, total intracellular proteins (TIP) from two phenotypically different strains, LMD-9 and CNRZ-21N, were recovered by sonication followed by ammonium sulphate precipitation. The molecular masses of the TIP of both strains were very close to each other as observed by SDS-PAGE. The TIP were fractionated by size exclusion fast protein liquid chromatography to obtain a 3-10 kDa intracellular protein (IP) fraction, which was then hydrolysed with pancreatic enzyme preparation, Corolase PP. The hydrolysed IP fraction from each strain exhibited anti-inflammatory activity by modulating pro-inflammatory mediators, particularly IL-1ß in LPS-stimulated THP-1 macrophages. However, a decrease in IL-8 secretion was only observed with hydrolysed IP fraction from CNRZ-21N, indicating that strain could be an important parameter in obtaining active hydrolysates. Results showed that peptides from the 3-10 kDa IP fraction of S. thermophilus could therefore be considered as postbiotics with potential beneficial effects on human health. Thus, it can be used as a promising bioactive ingredient for the development of functional foods to prevent low-grade inflammation.

Cell Proteins Obtained by Peptic Shaving of Two Phenotypically Different Strains of Streptococcus thermophilus as a Source of Anti-Inflammatory Peptides.

Streptococcus thermophilus, a food grade bacterium, is extensively used in the manufacture of fermented products such as yogurt and cheeses. It has been shown that S. thermophilus strains exhibited varying anti-inflammatory activities in vitro. Our previous study displayed that this activity could be partially due to peptide(s) generated by trypsin hydrolysis of the surface proteins of S. thermophilus LMD-9. Surface protease PrtS could be the source of these peptides during gastrointestinal digestion. Therefore, peptide hydrolysates were obtained by shaving two phenotypically distinct strains of S. thermophilus (LMD-9 PrtS+ and CNRZ-21N PrtS-) with pepsin, a gastric protease, followed or not by trypsinolysis. The peptide hydrolysates of both strains exhibited anti-inflammatory action through the modulation of pro-inflammatory mediators in LPS-stimulated THP-1 macrophages (COX-2, Pro-IL-1ß, IL-1ß, and IL-8) and LPS-stimulated HT-29 cells (IL-8). Therefore, peptides released from either PrtS+ or PrtS- strains in the gastrointestinal tract during digestion of a product containing this bacterium may display anti-inflammatory effects and reduce the risk of inflammation-related chronic diseases.

The Anxiolytic-like Properties of a Tryptic Hydrolysate of Bovine αs1 Casein Containing α-Casozepine Rely on GABAA Receptor Benzodiazepine Binding Sites but Not the Vagus Nerve.

(1) Background: A tryptic hydrolysate of bovine αs1-casein (CH) exerts anxiolytic-like properties in many species, including humans. This is mainly related to the presence of α-casozepine (α-CZP), which yields these properties in rodents. This study evaluates, in a rat model, the roles of the vagus nerve and the benzodiazepine binding site of GABAA receptors in the mode of action of CH. (2) Methods: The conditioned defensive burying test was used to evaluate anxiety. (3) Results: Participation of the vagus nerve in the mode of action of CH was excluded, as the global anxiety score in vagotomised rats was not significantly different from that of non-vagotomised animals. The blocking of the binding sites of benzodiazepines with flumazenil antagonised CH anxiolytic-like properties. (4) Conclusions: The vagus nerve does not play a role in the anxiolytic-like properties of CH. On the other hand, this anxiolytic-like activity relies on the benzodiazepine binding site of the GABAA receptors. This result is consistent with previous in vitro studies and, more specifically with the discovery of α-CZP, the peptide responsible for the anxiolytic-like properties of CH.

In Vitro Anti-Inflammatory Activity of Peptides Obtained by Tryptic Shaving of Surface Proteins of Streptococcus thermophilus LMD-9.

Streptococcus thermophilus, a lactic acid bacterium widely used in the dairy industry, is consumed regularly by a significant proportion of the population. Some strains show in vitro anti-inflammatory activity which is not fully understood. We hypothesized that peptides released from the surface proteins of this bacterium during digestion could be implied in this activity. Consequently, we prepared a peptide hydrolysate by shaving and hydrolysis of surface proteins using trypsin, and the origin of peptides was checked by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Most of the identified peptides originated from bacterial cell surface proteins. The anti-inflammatory activity of peptide hydrolysate was investigated under inflammatory conditions in two cell models. Peptide hydrolysate significantly decreased secretion of pro-inflammatory cytokine IL-8 in lipopolysaccharide (LPS)-stimulated human colon epithelial HT-29 cells. It also reduced the production of pro-inflammatory cytokines IL-8, IL-1ß and the protein expression levels of Pro-IL-1ß and COX-2 in LPS-stimulated THP-1 macrophages. The results showed that peptides released from bacterial surface proteins by a pancreatic protease could therefore participate in an anti-inflammatory activity of S. thermophilus LMD-9 and could prevent low-grade inflammation.

Role of the Sortase A in the Release of Cell-Wall Proteinase PrtS in the Growth Medium of Streptococcus thermophilus 4F44.

Growth of the lactic acid bacterium Streptococcus thermophilus in milk depends on its capacity to hydrolyze proteins of this medium through its surface proteolytic activity. Thus, strains exhibiting the cell envelope proteinase (CEP) PrtS are able to grow in milk at high cellular density. Due to its LPNTG motif, which is possibly the substrate of the sortase A (SrtA), PrtS is anchored to the cell wall in most S. thermophilus strains. Conversely, a soluble extracellular PrtS activity has been reported in the strain 4F44. It corresponds, in fact, to a certain proportion of PrtS that is not anchored to the cell wall but rather is released in the growth medium. The main difference between PrtS of strain 4F44 (PrtS4F44) and other PrtS concerns the absence of a 32-residue imperfect duplication in the prodomain of the CEP, postulated as being required for the maturation and correct subsequent anchoring of PrtS. In fact, both mature (without the prodomain at the N-terminal extremity) and immature (with the prodomain) forms are found in the soluble PrtS4F44 form along with an intact LPNTG at their C-terminal extremity. Investigations we present in this work show that (i) the imperfect duplication is not implied in PrtS maturation; (ii) the maturase PrtM is irrelevant in PrtS maturation which is probably automaturated; and (iii) SrtA allows for the PrtS anchoring in S. thermophilus but the SrtA of strain 4F44 (SrtA4F44) displays an altered activity.

Food protein-derived anxiolytic peptides: their potential role in anxiety management.

About one in three people are affected by anxiety disorders during their lifetime. Anxiety episodes can be brief due to a stressful event, but anxiety disorders can last at least 6 months. A wide variety of therapeutic drugs are available for the treatment of anxiety disorders, but due to the associated side effects of these anxiolytics, it is interesting to find alternatives. Some food protein hydrolysates or active peptide fragments present in such hydrolysates provide a natural and promising mean for preventing certain forms of anxiety. To date, only a small number of hydrolysates or peptides from food proteins with anxiolytic-like activity have been characterized. Most of these hydrolysates or peptides have displayed potent anxiolytic profiles in animal or clinical studies. The results suggest that these molecules may exert their effects at different levels. This paper reviews the data of the structure/activity relationship, physiological effects displayed in in vitro and in vivo assays, bioavailability, and safety profiles of anxiolytic peptides.

Anxiolytic Activity and Brain Modulation Pattern of the α-Casozepine-Derived Pentapeptide YLGYL in Mice.

α-Casozepine (α-CZP) is an anxiolytic-like bioactive decapeptide derived from bovine αs1­casein. The N-terminal peptide YLGYL was previously identified after proteolysis of the original peptide in an in vitro digestion model. Its putative anxiolytic-like properties were evaluated in a Swiss mice model using a light/dark box (LDB) after an intraperitoneal injection (0.5 mg/kg). The effect of YLGYL on c-Fos expression in brain regions linked to anxiety regulation was afterwards evaluated via immunofluorescence and compared to those of α-CZP and diazepam, a reference anxiolytic benzodiazepine. YLGYL elicited some anxiolytic-like properties in the LDB, similar to α­CZP and diazepam. The two peptides displayed some strong differences compared with diazepam in terms of c-Fos expression modulation in the prefontal cortex, the amygdala, the nucleus of the tractus solitarius, the periaqueductal grey, and the raphe magnus nucleus, implying a potentially different mode of action. Additionally, YLGYL modulated c-Fos expression in the amygdala and in one of the raphe nuclei, displaying a somewhat similar pattern of activation as α-­CZP. Nevertheless, some differences were also spotted between the two peptides, making it possible to formulate the hypothesis that these peptides could act differently on anxiety regulation. Taken together, these results showed that YLGYL could contribute to the in vivo overall action of α­CZP.

The X-prolyl dipeptidyl-peptidase PepX of Streptococcus thermophilus initially described as intracellular is also responsible for peptidase extracellular activity.

This study addresses the hypothesis that the extracellular cell-associated X-prolyl dipeptidyl-peptidase activity initially described in Streptococcus thermophilus could be attributable to the intracellular X-prolyl dipeptidyl-peptidase PepX. For this purpose, a PepX-negative mutant of S. thermophilus LMD-9 was constructed by interrupting the pepX gene and named LMD-9-ΔpepX. When cultivated, the S. thermophilus LMD-9 wild type strain grew more rapidly than its ΔpepX mutant counterpart. Thus, the growth rate of the LMD-9-ΔpepX strain was reduced by a factor of 1.5 and 1.6 in milk and LM17 medium (M17 medium supplemented with 2% lactose), respectively. The negative effect of the PepX inactivation on the hydrolysis of ß-casomorphin-7 was also observed. Indeed, when incubated with this peptide, the LMD-9-ΔpepX mutant cells were unable to hydrolyze it, whereas this peptide was completely degraded by the S. thermophilus LMD-9 wild type cells. This hydrolysis was not due to leakage of intracellular PepX, as no peptide hydrolysis was highlighted in cell-free filtrate of wild type strain. Therefore, based on these results, it can be presumed that though lacking an export signal, the intracellular PepX might have accessed the ß-casomorphin-7 externally, perhaps via its galactose-binding domain-like fold, this domain being known to help enzymes bind to several proteins and substrates. Therefore, the identification of novel distinctive features of the proteolytic system of S. thermophilus will further enhance its credibility as a starter in milk fermentation.

New Insights into the Proteolytic System of Streptococcus thermophilus: Use of Isracidin To Characterize Cell-Associated Extracellular Peptidase Activities.

The influence on the hydrolysis of isracidin of cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities in addition to protease PrtS of Streptococcus thermophilus strains was investigated. S. thermophilus LMD-9 (PrtS(+) phenotype) efficiently hydrolyzed the isracidin mainly through the PrtS activity, whereas strain CNRZ1066 (PrtS(-) phenotype) and two mutant strains LMD-9-ΔprtS and LMD-9-ΔprtS-ΔhtrA also displayed substrate hydrolysis, but different from that of the wild type strain LMD-9. Identification by mass spectrometry of breakdown products of isracidin revealed the existence of novel cell-associated extracellular carboxypeptidase and peptidyl dipeptidase activities in all PrtS(-) strains, besides known cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities. Both aminopeptidase and peptidyl dipeptidase activities were not able to cleave the isracidin at peptide bonds with proline residues. No hydrolysis of isracidin was detected in cell free filtrate for all the strains studied, indicating that no cell lysis had occurred. Taken together, these results suggested the presence of cell-associated extracellular peptidase activities in S. thermophilus strains that could be vital for the growth of PrtS(-) strains.

Identification by FT-ICR-MS of Camelus dromedarius α-lactalbumin variants as the result of nonenzymatic deamidation of Asn-16 and Asn-45.

Nonenzymatic deamidation of asparaginyl residues can occur spontaneously under physiological conditions principally when a glycyl residue is at the carboxyl side of Asn and leads to formation of aspartyl and isoaspartyl residues. This modification can change the biological activity of proteins or peptides and trigger an auto-immune response. The α-lactalbumins of members of the Camelidae family are the only of described α-lactalbumins that carry two AsnGly sequences. In the present study, high-resolution mass spectrometry, which enables accurate mass measurement has shown that Asn(16) and Asn(45) underwent a nonenzymatic deamidation, the sequence Asn(45)-Gly(46) being deamidated spontaneously at near-neutral and basic pH and Asn(16)-Gly(17) rather at basic pH. The 16-17 sequence was probably stabilized at near-neutral pH by hydrogen bonds according to the molecular modelisation performed with the camel protein.

Surface plasmon resonance analysis of the binding mechanism of pharmacological and peptidic inhibitors to human somatic angiotensin I-converting enzyme.

Somatic angiotensin I-converting enzyme (ACE) possesses two catalytic domains and plays a major role in the regulation of blood pressure, thus representing a therapeutic target for the treatment of hypertension. We present a comprehensive surface plasmon resonance (SPR) study of the interaction of human somatic ACE with the pharmacological inhibitors captopril and lisinopril, the bradykinin potentiating peptide BPP-11b, and the food peptidic inhibitors from bovine αs2-casein, F(174)ALPQYLK(181) and F(174)ALPQY(179). SPR binding curves recorded with the high potency inhibitors captopril, lisinopril, and BPP-11b were evaluated both by regression analysis and by kinetic distribution analysis. The results indicated that captopril and lisinopril bound ACE with two K(D)'s differing by a factor 10-20 and >30, respectively (lowest K(D) = 0.1-0.3 nM for both inhibitors). This shows, for the first time in a direct binding assay with the two-domain enzyme, the existence of two binding modes of the pharmacological inhibitors, presumably with the two ACE domains. The BPP-11b-ACE binding curves were complex but showed a predominant interaction with K(D) in the nanomolar range. The caseinopeptides, known to inhibit ACE with an IC50 of 4.3 µM, bound to ACE with K(D) = 3-4 µM. Mapping of the F(174)ALPQY(179) binding site on ACE by sequential binding studies using captopril or BPP-11b indicated that it bound to (or near) the two active sites of ACE, in agreement with the stoichiometry of 2 determined from data fitting. Our results provide a detailed characterization of ACE-inhibitor binding modes and validate SPR for predicting the inhibitory potential of new compounds.

Hydrolysis of milk-derived bioactive peptides by cell-associated extracellular peptidases of Streptococcus thermophilus.

The trend to confer new functional properties to fermented dairy products by supplementation with bioactive peptides is growing in order to encounter the challenge of health-promoting foods. But these functional ingredients have not to be hydrolysed by proteases of bacteria used in the manufacture of these products. One of the two yoghurt bacteria, Streptococcus thermophilus, has long been considered as weakly proteolytic since its only cell wall-associated subtilisin-like protease, called PrtS, is not always present. Nevertheless, a recent study pointed out a possible peptidase activity in certain strains. In this present study, the stability of milk-derived bioactive peptides, e.g. the anxiolytic peptide, αs1-CN-(f91-97), in the presence of two different S. thermophilus strains with PrtS+ or PrtS− phenotype was studied. Both strains appeared to be capable of hydrolysing the αs1-CN-(f91-97) and other bioactive peptides by recurrent removal of N-terminal residues. The hydrolysis was neither due to intracellular peptidases nor to HtrA protease. Results obtained showed that the observed activity originates from the presence at the surface of both strains of an extracellular aminopeptidase activity. Moreover, a cell wall-associated X-prolyl dipeptidyl peptidase activity was also highlighted when ß-casomorphin-7 was used as substrate. All of these findings suggest that, in order to use fermented milks as vector of bioactive peptides, the stability of these bioactive peptides in this kind of products implies to carefully characterize the potential action of the surface proteolytic enzymes of S. thermophilus.

Binding of divalent metal ions to 1-25 ß-caseinophosphopeptide: an isothermal titration calorimetry study.

To better understand the mechanism of metal ion transport through the gastrointestinal tract to their absorption sites, isothermal titration calorimetry (ITC) was used to investigate the binding of dicationic metals to ß-CN(1-25)4P, a ß-casein tetraphosphorylated peptide. ITC technology was found suitable for studying weak bonds between metal ions and phosphopeptides and provided a direct means of thermodynamic and stoichiometric characterisation of complex formation. Thus, one mole of ß-CN(1-25)4P binds two moles of Ca(2+), Mg(2+) or Zn(2+) under experimental conditions close to those of the ileum (pH 8, 37°C), with rather low binding affinity constants (K=4900-11,200M(-1)). These low affinities should facilitate the release of metal ions during intestinal absorption. By contrast, Cu(2+) did not bind to ß-CN(1-25)4P at pH 8, despite its reported significant affinity towards ß-casein and the 1-25 peptide at near-neutral pH.

Variability of hydrolysis of ß-, αs1-, and αs2-caseins by 10 strains of Streptococcus thermophilus and resulting bioactive peptides.

Milk proteins contain numerous potential bioactive peptides, which may be released by digestive proteases or by the proteolytic system of lactic acid bacteria during food processing. The capacity of Streptococcus thermophilus to generate peptides, especially bioactive peptides, from bovine caseins was investigated. Strains expressing various levels of the cell envelope proteinase, PrtS, were incubated with α(s1)-, α(s2)-, or ß-casein. Analysis of the supernatants by LC-ESI-MS/MS showed that the ß-casein was preferentially hydrolyzed, followed by α(s2)-casein and then α(s1)-casein. Numbers and types of peptides released were strain-dependent. Hydrolysis appeared to be linked with the accessibility of different casein regions by protease. Analysis of bonds hydrolyzed in the region 1-23 of α(s1)-casein suggests that PrtS is at least in part responsible for the peptide production. Finally, among the generated peptides, 13 peptides from ß-casein, 5 from α(s2)-casein, and 2 from α(s1)-casein have been reported as bioactive, 15 of them being angiotensin-converting enzyme inhibitors.

Transport across Caco-2 cell monolayer and sensitivity to hydrolysis of two anxiolytic peptides from αs1-casein, α-casozepine, and αs1-casein-f91-97: effect of bile salts.

α-Casozepine and f91-97, peptides from α(s1)-casein, display anxiolytic activity in rats and may have to cross the intestinal epithelium to exert this central effect. We evaluated their resistance to hydrolysis by the peptidases of Caco-2 cells and their ability to cross the cell monolayer. To mimic physiological conditions, two preparations of bile salts were used in noncytotoxic concentrations: porcine bile extract and an equimolar mixture of taurocholate, cholate, and deoxycholate. The presence and composition of bile salts appeared to modulate the peptidase activities of the Caco-2 cells involved (i) in the hydrolysis of α-casozepine, leading to much higher formation of fragments f91-99, f91-98, and f91-97, and (ii) in the hydrolysis of f91-97, leading to lower degradation of this peptide. Transport of α-casozepine across Caco-2 monolayer increased significantly, in the presence of bile extract, and of fragment f91-97, in the presence of bile salts.

In vitro digestibility of α-casozepine, a benzodiazepine-like peptide from bovine casein, and biological activity of its main proteolytic fragment.

α-Casozepine is a peptide, corresponding to the sequence 91-100 of the bovine α(s1)-casein, displaying anxiolytic activity in the rat. The α(s1)-casein tryptic hydrolysate containing this peptide decreases stress effects after oral administration in various species including man. Therefore, the stability of this peptide toward gastric and pancreatic proteases has been assessed by using pepsin, chymotrypsin/trypsin, Corolase PP, pepsin followed by chymotrypsin/trypsin or pepsin followed by Corolase PP. α-Casozepine was slowly degraded by chymotrypsin, much more sensitive to pepsin and Corolase PP but not completely destroyed after 4 h kinetics. The bonds in the region 91 to 95 of the α-casozepine were totally resistant to hydrolysis by all studied proteases. Surprisingly, a fragment, corresponding to the sequence 91-97 and found in all the hydrolysis media in significant amount, possessed an anxiolytic activity in three behavioral tests measuring this parameter. This peptide could participate in the in vivo activity of α-casozepine.

Identification of phosphorylation sites of equine beta-casein isoforms.

Equine beta-casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P-7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion-exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro-column. This method turned out to be an efficient tool to separate the CPPs Arg(1)-Lys(34) and Glu(4)-Lys(34) from non-phosphorylated peptides. Purification was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) and each CPP was hydrolyzed by endoproteinase Glu-C. Finally, the digests were analyzed by RP-HPLC/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) and identified by nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) to locate the phosphorylated sites of the beta-casein isoforms 4P-7P with accuracy. Thus, the isoform 4P was found to be phosphorylated on residues Ser(9), Ser(23), Ser(24), and Ser(25). Addition of phosphate groups on Ser(18), Thr(12), and Ser(10) led to the formation of the isoforms 5P-7P, respectively. The results indicated that the in vivo phosphorylation of the equine beta-casein follows a sequential way and is not randomly performed.

The primary structure of a low-Mr multiphosphorylated variant of beta-casein in equine milk.

Highly phosphorylated casein with a low molecular mass was isolated from Haflinger mare's milk by RP-HPLC. It accounts for 4.0% of the casein content. Its mass was determined by LC-ESI-MS before and after treatment by alkaline phosphatase. The molecular mass found for the apo-form (10,591 +/- 2 Da) is in agreement with its primary structure, which was established by ESI-MS/MS from tryptic peptides. It appeared that this short protein (94 amino acid residues) is an internally truncated form of the full-length equine beta-casein (226 residues). This low-Mr variant of equine beta-casein displays a large deletion (residues 50-181), due to a cryptic splice site usage occurring within exon 7 during the course of primary transcripts processing. The phosphorylation pattern of this equine beta-casein variant was investigated by LC-ESI-MS and 2-DE. Seven phosphorylation forms were identified with one to seven phosphate groups with pIs ranging between 4.67 and 4.01. The major isoforms carry five and six phosphate groups.

Determination of the phosphorylation level and deamidation susceptibility of equine beta-casein.

beta-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine beta-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's beta-casein (25 514 +/- 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27-34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the beta-casein isolated from Haflinger mare's milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30. Moreover, the equine beta-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif (135)Asn-Gly(136). Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions.

Angiotensin-I-converting enzyme inhibitory peptides from tryptic hydrolysate of bovine alphaS2-casein.

Angiotensin-I-converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine alpha(S2)-casein (alpha(S2)-CN) was extensively investigated. Forty-three peptide peaks were isolated and tested. Seven casokinins (i.e. CN-derived ACE inhibitory peptides) were identified and their IC50 values were determined. Four peptides exhibited an IC50 value lower than 20 microM. Peptides alpha(S2)-CN (f174-181) and alpha(S2)-CN (f174-179) had IC50 values of 4 microM. Surprisingly, deletion of the C-terminal dipeptide of two of these casokinins did not significantly alter their inhibitory activity.