In an ageing society, polypharmacy has become a major public health and economic issue. Overuse of medications, especially in patients with chronic diseases, carries major health risks. One common consequence of polypharmacy is the increased emergence of adverse drug events, mainly from drug–drug interactions. The majority of currently available drugs are metabolized by CYP450 enzymes. Interactions due to shared CYP450-mediated metabolic pathways for two or more drugs are frequent, especially through reversible or irreversible CYP450 inhibition. The magnitude of these interactions depends on several factors, including varying affinity and concentration of substrates, time delay between the administration of the drugs, and mechanisms of CYP450 inhibition. Various types of CYP450 inhibition  have been observed clinically, and interactions of these types require a distinct clinical management strategy. This review focuses on mechanism-based inhibition, which occurs when a substrate forms a reactive intermediate, creating a stable enzyme–intermediate complex that irreversibly reduces enzyme activity. This type of inhibition can cause interactions with drugs such as omeprazole, paroxetine, macrolide antibiotics, or mirabegron. A good understanding of mechanism-based inhibition and proper clinical management is needed by clinicians when such drugs are prescribed. It is important to recognize mechanism-based inhibition since it cannot be prevented by separating the time of administration of the interacting drugs. Here, we provide a comprehensive overview of the different types of mechanism-based inhibition, along with illustrative examples of how mechanism-based inhibition might affect prescribing and clinical behaviors. In clinical practice, the need for the use of multiple drugs is common, as patients often present with numerous chronic diseases. To improve commodity and drug adherence, several medications are often administered concomitantly. Although this may represent a preferred clinical strategy, the administration of two or more drugs at overlapping times increases the likelihood of drug–drug interactions ,2. As thInhibition of cytochrome P450 (CYP450) enzymes is the most common mechanism leading to drug–drug interactions  is the substrate concentration. In most clinical situations, liver enzymes are rarely saturated so that, generally, the substrate concentration is much smaller than the mK and the equation can be simplified to:Binding affinity to an enzyme is measured by the mK is increased for the victim drug such that its intCL is decreased).The binding affinity of a substrate can be modified by the presence of other molecules  CYP450-mediated metabolism is the major route of elimination for a large number of drugs, and (2) multiple drugs can compete for the same CYP450 active site. Mechanisms of CYP450 inhibition can be categorized as reversible (including competitive or non-competitive) or irreversible/quasi-irreversible (mechanism-based inhibition).Reversible inhibition is a result of rapid association and dissociation between the substrate drugs and the enzyme and can be categorized as competitive or non-competitive. A third category, uncompetitive inhibitor, also considered as a reversible inhibition type, is a very rare phenomenon and will not be considered in this current review; this type of inhibitor binds only the enzyme–substrate complex, leading to a dead-end complex.The ability of a single CYP450 isoform to metabolize multiple substrates is responsible for several drug interactions associated with reversible competitive inhibition. Competitive inhibition occurs when two substrates compete for the same active site—such as the prosthetic heme iron or substrate-binding region—of CYP450s. The competition is a function of the respective affinities of the two substrates for the binding site and their concentrations in the proximity of the enzyme. First, the most clinically relevant situation will be discussed.mK of the victim drug (decreased affinity) and reducing the extent of its breakdown (decrease in its intCL) over a period of time.This situation is often encountered in clinical practice. Under a competitive inhibition condition, a substrate with strong affinity (acting as a perpetrator) can displace a weaker substrate (behaving as a victim) from the active site , thus inintCL of one of its metabolic pathways can lead to a decrease in the total clearance of the drug (capacity for eliminating the drug) and can result in increased plasma concentrations, potentially precipitating adverse effects. However, for prodrugs, this interaction can instead result in a decrease in the formation of the active metabolite, reducing drug efficacy. The magnitude of changes observed in the overall disposition of the victim drug (increase in its plasma levels) will be a function of the relative contribution of the inhibited metabolic pathway to the clearance of this drug. For example, if 15% of a drug is excreted unchanged in urine—35% by enzyme 1 and 50% by enzyme 2—a 50% decrease in the total CL of the victim drug is expected if enzyme 2 is inhibited:For an active drug, the decrease in the Since,AUC) measured over a dosing interval (τ) is equal to AUC0–∞. Since the average concentration over a dosing interval (avC) at steady state can be estimated by AUCτ0-/τ, the equation could be rearranged in a simpler manner to yield:Under steady-state conditions, the area under the drug concentration curve  towards another substrate metabolized by the same enzyme. Competitive inhibition is almost immediate and the degree of inhibition of the enzyme does not change with time if the concentration of the two substrates remains the same.intCL), leading to increased plasma levels of theophylline and possibly side effects . To minimize competitive inhibition, two competing substrates should be administered with as much time apart as possible.In an example illustrating this scenario, theophylline (weak CYP1A2 substrate) is largely metabolized by CYP1A2 by binding to its active site.  If that As mentioned previously, the competitive inhibition process is sensitive to substrate concentrations. If concentrations of the weaker affinity substrate are much higher than concentrations of the stronger affinity substrate, the weaker affinity substrate can displace the stronger affinity substrate and overcome the enzyme inhibition, which is why this type of inhibition is deemed reversible.  The greaintCL of the substrate due to a decrease in its maxV is observed. Similar to competitive inhibitors, non-competitive inhibitors also have an almost immediate effect. As long as the concentration of the inhibitor is not changed, the amount of inhibition will not increase over time. This type of inhibition does not require the involvement of NADPH as a cofactor, i.e., the inhibitor is not metabolized by the enzyme, but merely sits in an allosteric site. Other non-competitive inhibition conditions may involve CYPb5 and/or CYP450 oxidoreductase as these factors have been shown to modulate CYP450 activities, at least in in vitro systems [The non-competitive inhibitor does not generally have any structural resemblance to the substrate as it binds to an allosteric site. The non-competitive inhibitor will cause a conformational change in the structure of the active site such that the active site loses its affinity for the substrate.  Thus, th systems ,14. Sepa systems ,16,17.In the case of mixed inhibition, both competitive and non-competitive inhibition occur. Mixed inhibitors can simultaneously bind to both the heme iron atom (at the active site) and lipophilic regions of the protein . Mixed inhibitors are usually more potent inhibitors than competitive or non-competitive inhibitors. Ketoconazole and fluconazole, both imidazole antifungals, exhibit potent mixed reversible inhibition of CYP3As. However, fluconazole is a weaker mixed reversible inhibitor compared to ketoconazole, mainly due to its lower lipophilicity .For a CYP3A substrate like midazolam, concomitant use of non-competitive or mixed CYP3A inhibitors will reduce its transformation to α-hydroxy midazolam, increasing midazolam plasma levels and augmenting the risk of adverse drug events .Several clinically important pharmacokinetic drug interactions result from a decrease in the metabolic clearance of a substrate due to CYP450 irreversible inhibition. Mechanism-based inhibition is a condition often encountered with irreversible CYP450 inhibitors.max)V. , resulting in irreversible long-lasting loss of enzyme activity (decrease in Vmax).  Several In the case of quasi-irreversible inhibition, the metabolites form very stable complexes with the heme prosthetic site (metabolite–intermediate complex), so that the enzyme is sequestered in a functionally inactive state. This phenomenon is called quasi-irreversible since, in theory, this complex can be disrupted. In the case of irreversible inhibition, the metabolites covalently bind to the heme prosthetic site or the protein part of the CYP450, leading to irreversible inactivation ,20.Hence, mechanism-based inhibition is active site mediated, and the allosteric site is not involved. In contrast to reversible inhibition mechanisms, mechanism-based inhibition is time dependent and NADPH dependent. This means that the enzyme has to start breaking down the substrate in order for inhibition to proceed. As more drug molecules are metabolized, more complexes are stably formed in the active sites, increasing inhibition over time before it reaches a plateau. Mechanism-based inhibition is therefore also saturable. New enzyme formation is necessary to restore activity: the relationship between the amount of intermediate complex formed and the speed of new enzyme synthesis dictate the equilibrium and extent of enzyme inhibition.Mechanism-based inhibitors can be classified into two categories: metabolic–intermediate complex formation inhibitors and protein and/or heme alkylation inhibitors.Such a condition occurs when a stable intermediate metabolite formed during the normal metabolic cycle forms covalent bonds at the active site. This stable intermediate–enzyme complex is not easily broken by increasing substrate concentration. Since the enzyme structure remains otherwise unchanged, theoretically this reaction is reversible with time. However, in in vivo conditions, with this metabolic intermediate complex being excessively stable, the metabolic intermediate cannot be displaced and the enzyme remains inaccessible for metabolism so the reaction seems irreversible.An example of alternate substrate inhibition is observed with paroxetine as its methoxy diene carbon moiety was found to be responsible for the formation of covalent bonds at the active site of CYP2D6 ,22. AnotThis situation takes place when a latent highly reactive  intermediate is formed in the catalysis process. The reactive intermediate forms covalent bonds (strong irreversible bonds) with the enzyme in a step that is not part of the normal metabolic pathway. This process can change the conformational structure of the enzyme significantly—it can even destroy the enzyme in some cases—making it functionally unviable. For example, inhibition of CYP2C19 by esomeprazole was found to be mediated by crosslinking the heme and apoprotein moieties in the enzyme, changing its conformational structure .It is important to note that since mechanism-based inhibitors are substrates of the enzyme, they can also cause acute competitive inhibition when co-administered with other sensitive substrates. The difference between competitive inhibition and mechanism-based inhibition is that as the time period of exposure to mechanism-based inhibitors increases, the degree of inhibition also increases. Proton pump inhibitors (PPIs) are commonly prescribed along with antiplatelet drugs like clopidogrel to reduce the incidence of gastric bleeding during treatment with antiplatelet therapy ,26. OmepSince omeprazole is a strong affinity substrate for CYP2C19, an “immediate” competitive inhibition is expected between these two drugs. Since competitive inhibition was expected, separating time of administration was considered a logical mitigation strategy to avoid or alleviate the extent of the drug interaction . Others Multiple studies have been conducted to determine the clinical impact of the potential reduced antiplatelet efficacy resulting from this interaction. In two retrospective studies looking at interactions between clopidogrel and PPIs and the effects on clinical outcomes, it was reported either that PPIs were associated with increased cardiac adverse events in acute coronary syndrome patients, or that cardiac adverse events were less common in PPI non-users ,36. ShorSeveral studies also report no change in the frequency of cardiovascular adverse events with omeprazole administration during clopidogrel treatment ,40,41,42maxC and AUC of paroxetine were increased 5.2- and 7-fold after 2 weeks of paroxetine administration, respectively [The antidepressant paroxetine is a known substrate of CYP2D6, but also a potent mechanism-based inhibitor of this enzyme. As such, paroxetine is expected to inhibit its own metabolism over time. This has been illustrated in two clinical studies, where the ectively ,47. In tectively .As paroxetine is a strong CYP2D6 affinity substrate, it can exhibit acute competitive inhibition when co-administered with sensitive substrates like nortriptyline.  Though sAnother example that illustrates the concept of saturability in mechanism-based inhibition is observed with the commonly used macrolide antibiotics erythromycin  or clariAUC of midazolam increased 2.3-fold after 2 days of erythromycin pretreatment, compared to control. Following 4 days of pretreatment, midazolam’s AUC increase was 3.38-fold. After a 7-day pretreatment, a similar increase (3.38-fold) was observed, indicating an increase in inhibition with repeated administration of erythromycin and that a plateau effect had been reached after 4 days of exposure [In another study with 12 healthy volunteers, the effects of erythromycin on midazolam metabolism were studied . It was exposure .AUC and 0.6-fold increase in maxC of desipramine was observed [AUC was observed with mirabegron pretreatment compared to control [AUC of desipramine was much larger than the increase observed with duloxetine, a “purely” potential competitive inhibitor. It is important to note that in the short term, mechanism-based inhibitors can act as competitive inhibitors if the other drug has lower affinity for the metabolizing enzyme. Duloxetine and mirabegron are both substrates of CYP2D6 with moderate affinity, exhibiting potential competitive inhibition over desipramine; therefore, following a single dose of each drug, a similar level of CYP2D6 inhibition is expected towards the CYP2D6 victim drug. . This variability on CYP450 expression/activities translates into intersubject variability in drug disposition and drug response. Often, the impact of genetic polymorphisms and drug–drug interactions on CYP450s have been studied separately. However, an interaction exists between these factors. Genetic polymorphisms could also contribute to variability observed in the magnitude of drug–drug interactions observed between two drugs. So, genetic polymorphisms in drug-metabolizing enzymes can affect the occurrence of phenoconversion induced by drug inhibitors. As reported by Storelli et al., differences in CYP2D6 inhibition observed in vitro with paroxetine (mechanism-based inhibitor) or duloxetine (competitive inhibitor) across CYP2D6 genotypes were not related to their inhibition parameters but likely due to a differential level of functional enzymes as a function of the CYP2D6 genotype [In addition to drug–drug interactions, high variability in terms of CYP450 expression and/or activities can be explained by genetic polymorphisms in genes encoding specific isoforms (such as genotype ,58.Polypharmacy in many cases is deemed to be required and elderly patients are particularly prone to this phenomenon. Aging is associated with the presence of multiple independent chronic diseases and is almost always accompanied by multiple drug regimens. Polypharmacy has been associated with many adverse clinical outcomes, such as drug–drug interactions, leading to adverse drug events. Among these, mechanism-based inhibitor–victim drug combinations like clopidogrel and omeprazole are commonly prescribed. Drugs for overactive bladder like mirabegron and tricyclic antidepressants like paroxetine are also commonly prescribed in elderly populations. Polypharmacy is not necessarily synonymous with inappropriate treatment, but in several situations, it can lead to significant drug–drug interactions especially in the presence of mechanism-based inhibitor drugs, as described in the current review. In these cases, polypharmacy might cause problems like blunted efficacy of clopidogrel due to the co-administration of omeprazole, or increased toxicity of other drugs co-administered with paroxetine or mirabegron. Clinicians must be able to recognize and intervene appropriately based on the mechanism of these interactions.As highlighted in this current review, mechanism-based inhibition can cause severe clinical interactions. The magnitude of these interactions and their impacts depend on the duration of the mechanism-based inhibitor exposure and the exact time-point when the victim drug is introduced into the drug regimen. Without a comprehensive understanding of this mechanism, healthcare professionals might find it difficult to diagnose and mitigate these interactions. Separating the time of administration of interacting drugs cannot mitigate an interaction caused by mechanism-based inhibition. A careful titration of dose, monitoring of clinical effects, or switching to an alternate drug with a different metabolic pathway may become necessary to avoid such interactions. Advanced clinical decision support systems that consider and distinguish competitive versus mechanism-based inhibition drug–drug interactions would help identify potential interactions mediated by enzyme inhibition. Using such tools can help pharmacists quickly identify and mitigate drug interactions, thus helping reduce preventable drug interactions.


Torpedo californica, as well as Lymnaeastagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing α7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca2+ rise, but inhibited the acetylcholine-evoked Ca2+ rise with IC50 9 to 12 μM. In the A549 lung cancer cells, where α7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with iK= 16.7 ± 1.5 μM and iαK= 51.4 ± 4.1 μM for AChE and iK= 70.5 ± 6.3 μM and iαK= 214 ± 17 μM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both α7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from  A system for the synthesis, reception, transport, and hydrolysis of acetylcholine (ACh) is present in most types of human cells . In addiDue to a low production rate and a lower affinity of these atypical esters for the ACh receptors, they are considered to be modulators of cholinergic signaling due to the desensitization of the receptors against the dominant signal molecule, acetylcholine .Recently, novel ACh analogs with the chain length of 18 to 22 carbon atoms and various degree of unsaturation of fatty acid residue were discovered. Normal human plasma and urine contain choline derivatives with arachidonic, docosahexaenoic, oleic, linoleic, linolenic, and palmitic fatty acid residues . More imStrongylocentrotus droebachiensis and S. purpuratus embryo development model. Unfertilized sea urchin eggs express ACh receptors resembling the neuronal nicotinic ACh receptors (nAChR)  and the values of inhibition constants Tested AA-CHOL solution was injected into the triceps muscles of the mouse forelimbs at a volume of 0.5 mL/kg per each limb or subcutaneously. For all animals, their forelimb grip strength was recorded before the substance administration with a 1027 grip strength meter . Their grip strength was measured 15, 30, 60,120, and 240 min after the AA-CHOL or saline administration.6 evaluations, 27 × 104 generations, and a population size of 3000.Geometry of AA-CHOL was quantum-mechanically optimized with Gamess-US  softwarewww.pymol.org).Figures were prepared with PyMol p}, where IC50 is the concentration at which 50% of the binding sites are inhibited, and p is the Hill coefficient. Data in the radioligand assay are presented as mean with 95% confidence interval (CI).The radioligand assay results were analyzed with ORIGIN 8.0  fitting to a one-site dose-response curve by the equation: % response  =  100/{1  +  , oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) fatty acids  were syn125I-αBgt binding to the orthosteric sites of the nAChRs. At first, we tested the binding of acylcholines with the L. stagnalis AChBPs, which is the model for the ligand-binding domains of the whole family of Cys-loop receptors [50 2.3 µM) (50~13 to 20 μM). Using a radioligand assay, we studied acylcholines for their ability to compete with eceptors ,25,26. L 2.3 µM) , whereas125I-αBgt binding. In the presence of 40 μM SDS, the binding of 125I-αBgt to Torpedo membranes or GH4C1 cells did not differ from the control values (data not shown). After that, we studied the ability of acylcholines to compete with 125I-αBgt for binding to the full-size receptors, muscle-type nAChR from T. californica electric ray (50 18.7 µM). At the same time, α7 nAChR was most efficiently inhibited by Ol-CHOL (IC50 14.2 µM).Since acylcholines could be expected to exhibit detergent properties to the nAChRs in the cell membranes, we tested the effect of the widely used detergent sodium dodecyl sulfate (SDS) on the tric ray B or humatric ray . The Ln-vice versa, inhibition of the activation due to competition with endogenous agonists acetylcholine and choline (in the case of α7 nAChR). To find out whether the acylcholines act as agonists or antagonists, we performed fluorimetric measurements of cytoplasmic calcium evoked by activation of nAChR. The ability of the compounds to induce calcium mobilization was studied in the SH-SY5Y cells endogenously expressing α7 nAChR. Neither of the three tested compounds  induced a rise in the intracellular Ca2+, and thus showed no agonistic properties towards this receptor. To test their antagonistic activity against α7 nAChR, they were applied with ACh in the presence of PNU120596, a positive allosteric modulator of this homooligomeric nAChR subtype. All tested compounds at micromolar concentrations inhibited the acetylcholine-evoked Ca2+ rise in a concentration-dependent manner  that α-The observation of muscle nAChR inhibition by AA-CHOL with micromolar affinity led us to the hypothesis that acylcholines acts as muscle relaxants at high doses. A possible muscle relaxant effect of the AA-CHOL was studied by measuring the mouse front legs strength at test points of 15 min, 30 min, 1 h, 2 h, and 4 h, according to the protocol we previously used for azemiopsin, a peptide inhibitor of the muscle nAChR . AA-CHOLLung cancer cells endogenously express α7 nAChR, and their activation on these cells leads to stimulation of proliferation . BecauseOn the one hand, the specific blocker of nAChR methyllycaconitine did not affect A549 proliferation up to the concentration of 10 μM. Nicotine, on the other hand, slightly decreased A549 proliferation at the concentration 15 μM but did not affect the cytotoxicity of 90 or 100 μM of Ol-CHOL D.Because long-chain acylcholines are structurally similar to the acetylcholine, we hypothesized that the enzymes which metabolize the latter could also degrade the long-chain acylcholines. We tested the ability of purified AChE and BChE to hydrolyze AA-CHOL. ATCh, as ACh analogue for spectrophotometric Ellman assay was used as a control for the enzyme activity, and incubations with the heat-inactivated enzyme were used for non-enzymatic degradation controls. Under experimental conditions, ATCh was fully hydrolyzed by both enzymes. AA-CHOL was not hydrolyzed by AChE, but with BChE the hydrolysis of arachidonoylcholine accounted for up to 30% during 40 min. With the heat-inactivated enzymes, no hydrolysis of either ATCh or AA-CHOL was detected. 50 values were quite high.To further check if acylcholines possess the ability to interact with these enzymes, we tested their inhibition of AChE and BChE catalyzed ATCh hydrolysis . Only AAVmax and Km values. Such alterations are consistent with mixed-type inhibition  are known to act as nAChR-like agonists in physiological tests [To study the functional response to the acylcholines, we measured their ability to induce Car nAChRs . The effal tests . The binal tests . The speal tests . Fatty aA somewhat contradictory activity has been earlier described for AA-CHOL and DHA-CHOL on the unfertilized sea urchin eggs, which expressed receptors somewhat similar to the nAChRs . In thisDistinct nAChR subtypes are involved in the stimulation of cancer cell proliferation . BecauseWith regards to the in vitro tests, the acylcholines behaved as nAChR blockers, and we supposed that they could also act as neuromuscular blocking myorelaxants in vivo. It is noteworthy that such activity was recently demonstrated for azemiopsin, a linear peptide from the snake venom inhibiting the muscle nAChR at micromolar concentrations ,44. HoweIn addition to the action on the nAChR, we tested the acylcholines activity on another part of the acetylcholine signaling system, namely on the acetylcholine hydrolyzing enzymes. AChE did not hydrolyze the tested acylcholines, but AA-CHOL inhibited its activity as a mixed-type inhibitor with quite high inhibition constants, which agrees with molecular docking results. Neither Ln-CHOL nor DHA-CHOL had noticeable inhibitory activity. BChE was also rather weakly inhibited by AA-CHOL. Molecular docking confirmed the possibility of BChE catalyzed hydrolysis of AA-CHOL and mixed-type mechanism of its inhibition of ATCh hydrolysis. No data on the inhibition of the AChE and BChE by acylcholines could be found in the literature. The existence of other enzymes for degradation of acylcholines seems possible, but testing this hypothesis requires further studies.Therefore, long-chain fatty acid derivatives of choline could be both inhibitors (via the receptor blocking) and weak potentiators (via the inhibition of acetylcholine hydrolysis) of the acetylcholine signaling. However, receptor inhibition appeared at concentrations in the range 10–50 μM, while enzyme inhibition required 100 to 300 μM of the substances. Thus, receptor blocking and acetylcholine signaling inhibition appear as a dominating activity of acylcholines. At present, there are no data on the concentrations and biosynthesis of these compounds, making the correct estimation of their biological role very difficult. The discovered cytotoxicity of Ol-CHOL against the lung cancer cell line A549 points to the existence of additional receptor targets for this substance, and clarification of this issue could be of interest.For the first time, we have demonstrated that arachidonoylcholine and its unsaturated fatty acid analogs with the chain length of 18 and 22 carbon atoms are inhibitors of the neuronal and muscle-type nicotinic acetylcholine receptors and modest inhibitors of AChE and BChE, thus, playing a possible role as endogenous modulators of the acetylcholine signaling system.


Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1. Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47phox, and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.The human cardiovascular system has adapted to function optimally in Earth's 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of  Spaceflight or microgravity conditions are associated with myocardial atrophy, which contributes to the functional depression of the heart , 2, 3. DProtein degradation systems play important roles in muscle atrophy . There aCapn1 and Capn2, respectively, and a common small 28-kDa regulatory subunit encoded by Capns1. The regulatory subunit is indispensable for calpain-1 and calpain-2 activities, and thus, deficiency of Capns1 disrupts calpain-1 and calpain-2  and a cytosolic complex . Activation of NADPH oxidase initiates multiple steps, mainly including phosphorylation of cytosolic subunits (e.g. p47phox), their translocation to the membranes, and the assembly of both cytosolic and membrane-bound complexes  consensus sequence that is phosphorylated by p38 and ERK1/2 of the MAPK family . Tail suspension for 14 days resulted in higher levels of calpain activities in WT mouse heart tissues; the levels of calpain activities were even higher in mouse hearts after 28 days of tail suspension (B); however, calpain activities were not increased in Capns1-KO mouse hearts after 14 and 28 days of tail suspension (B). These results indicated calpain activation in tail-suspended mouse hearts. This time-dependent activation of calpain negatively correlated with heart weight  in tail-suspended mice. Tail suspension for 28 days reduced the mitochondrial DNA copies of NADH dehydrogenase subunit 1 (Mtnd1) but not ATP production in WT mouse hearts , bigger cardiomyocyte size (B), and better myocardial function in Capns1 mice compared with their WT littermates . Notably, deficiency of Capns1 reduced the protein levels of Rac1, p47phox, and p67phox in the cell membranes of tail-suspended mouse hearts, indicating that disruption of calpain-1 and calpain-2 prevents NADPH oxidase activation in tail-suspended mouse hearts (B–E).Our recent study reported that simulated microgravity induced NADPH oxidase activation in tail-suspended mouse hearts . As showFig. 3embranes , and theembranes B. NotablA), as was greater protein carbonyl content and malondialdehyde (MDA) production, indicators of oxidized protein and lipid peroxidation, respectively (B and C). However, ROS production was much less in tail-suspended Capns1-KO mice compared with their WT littermates (A). Similarly, deficiency of Capns1 also resulted in less oxidative damage in hearts of tail-suspended mice as determined by less protein carbonyl and MDA contents (B and C).In line with activation of NADPH oxidase, more ROS production was observed in heart tissues of tail-suspended mice compared with sham animals A, as wasectively , B and Ctermates A. Similacontents , B and CCapns1 did not change the higher protein levels of NOX4 due to tail suspension . Interestingly, in tail-suspended mice, deletion of Capns1 resulted in lower levels of phosphorylation of Ser-345 on p47phox (B), suggesting that calpain promotes phosphorylation of Ser-345 on p47phox in mediating NADPH oxidase activation in tail-suspended mouse hearts.To investigate the potential mechanisms by which calpain promoted NADPH oxidase activation, we focused on the phosphorylation state of p47embranes . Similarspension A. Intere p47phox B, suggesphox phosphorylation, we simulated microgravity in cultured cardiomyocytes using the rotary cell culture system. Simulated microgravity increased calpain activities (C), and this was correlated with a reduction of total protein/DNA ratio (D) and an increase in phosphorylation of Ser-345 on p47phox (E). Incubation with calpain inhibitor-III prevented calpain activation, increased total protein/DNA ratio, and attenuated microgravity-induced phosphorylation of Ser-345 on p47phox in cardiomyocytes (C–E). These results recapitulated the inhibitory effect of calpain disruption on p47phox phosphorylation in tail-suspended mouse hearts.To provide further evidence supporting the role of calpain in promoting p47tivities C, and thNA ratio D and an  p47phox E. Incubamyocytes , C–E. Thphox is mediated by ERK1/2 and p38 , the levels of phosphorylated ERK1/2 and p38 were much lower in Capns1-KO mice compared with their WT littermates 14 days after tail suspension. These results suggest that calpain may promote phosphorylation of ERK1/2 and p38 primarily in the first 2 weeks in tail-suspended mouse hearts.Because phosphorylation of Ser-345 on p47 and p38 , we analD). Incubation with calpain inhibitor-III prevented ERK1/2 phosphorylation in cardiomyocytes 6 h after simulated microgravity (E).To further address this in cultured cardiomyocytes, we showed that the protein levels of phosphorylated ERK1/2 were higher 6 h but not 24 h after simulated microgravity D. Incubaogravity E.D). We then chose to determine the effect of calpain inhibition on p38 phosphorylation 24 h after simulated microgravity. Similarly, incubation with calpain inhibitor-III prevented p38 phosphorylation (F).The protein levels of phosphorylated p38 were higher 6 h and even higher 24 h after simulated microgravity D. We therylation F.phox in cultured cardiomyocytes after 24 h of simulated microgravity (A and B). Because the time-course analysis for ERK1/2 and p38 phosphorylation showed that ERK1/2 phosphorylation reached the highest levels at 6 h, whereas p38 phosphorylation was at the highest level in cardiomyocytes at 24 h after simulated microgravity (B), we therefore determined whether ERK1/2 mediated p38 phosphorylation induced by microgravity. However, inhibition of ERK1/2 with PD98059 did not affect the phosphorylated levels of p38 in cardiomyocytes following simulated microgravity (C), suggesting that ERK1/2 and p38 independently facilitate p47phox phosphorylation in microgravity, whereas ERK1/2 and p38 exert their roles at early and relatively late phases of microgravity, respectively. In support of this conclusion, co-incubation of PD98059 and SB20358 did not further inhibit p47phox phosphorylation in microgravity compared with PD98059 or SB203580 alone  as a smaller cardiomyocyte may have a lesser number of mitochondria, whereas ATP production remains the same per unit of cell volume. Thus, a reduction of mitochondrial DNA copies is not a potential mechanism for myocardial dysfunction induced by microgravity. Future studies are needed to determine the detailed mechanisms by which microgravity induces myocardial dysfunction.Although myocardial atrophy weakens myocardial contractility, the underlying mechanisms remain to be determined. Our data show that tail suspension for 14 and 28 days reduced mitochondrial In summary, we have demonstrated an important role of calpain in promoting myocardial abnormalities in tail-suspended mice. Thus, targeting calpain may be a useful strategy to protect the heart under conditions of microgravity. Given that pharmacological inhibitors of calpain are under clinical trials, our findings provide important preclinical evidence to support future translational studies.Capns1 (Capns1-KO) were generated by breeding mice bearing the targeted PZCapns1 allele containing loxP sites flanking essential coding exons and mice with cardiomyocyte-specific expression of Cre recombinase under the control of α-myosin heavy chain as we described recently . Breeding pairs of C57BL/6 mice were purchased from the Jackson Laboratory. Mice with cardiomyocyte-specific disruption of recently . Deletioeviously   to simulate microgravity using the methods described previously  and imaged using a 40-MHz linear array transducer attached to a preclinical ultrasound system  with nominal in-plane spatial resolution of 40 μm  × 80 μm . M-mode and two-dimensional parasternal short-axis scans (133 frames/s) at the level of the papillary muscles were used to assess changes in left ventricular (LV) end-systolic inner diameter, LV end-diastolic inner diameter, LV posterior wall thickness in end-diastole and end-systole, fractional shortening (%), and ejection fraction.Mice were euthanized by cervical dislocation under anesthesia using a mixture of ketamine (100 mg/kg)/xylazine . Heart tissues were collected, fixed, processed, and sectioned. For cardiomyocyte size measurement, several cross-sections of the whole heart (5 μm thick) were prepared and stained for membranes and nuclei with FITC-conjugated wheat germ agglutinin (Thermo Fisher Scientific) and Hoechst 33342 (Thermo Fisher Scientific), respectively. Single cardiomyocytes were measured using an image quantitative digital analysis system (NIH Image version 1.6) as described previously . The outTM Protein Assay (Bio-Rad). Genomic DNA was extracted from cultured cardiomyocytes using a Hiyield Genomic DNA Isolation Kit (cultured cells)  and measured by a Nanodrop spectrophotometer (A260).The protein levels were determined by DCThe formation of ROS in heart tissue lysates was measured using the Amplex® Red hydrogen peroxide/peroxidase assay kit (Thermo Fisher Scientific), according to the manufacturer's instructions. Briefly, frozen heart tissues were homogenized in an assay buffer. The homogenates (50 μg of protein) were incubated with a fluorescent probe Amplex® Red and hydrogen peroxide/peroxidase at 37 °C. The fluorescent product formed was quantified using a spectrofluorometer measured at 485/525 nm. Changes in fluorescence were expressed as arbitrary units.Protein carbonyl content was determined using a protein carbonyl colorimetric assay kit  following the manufacturer's instructions. A total of 700 μg of protein was used for each sample.Lipid peroxidation in heart tissue lysates was assessed by measuring MDA production using a TBARS assay kit  following the manufacturer's instructions. A total of 500 μg of protein was used for each sample.Neonatal mice (born within 24 h) were euthanized by decapitation. Neonatal cardiomyocytes were prepared and cultured in Dulbecco's modified Eagle's medium with 10% newborn calf serum and penicillin-streptomycin according to methods described previously .6) were cultured with Cytodex 3 microcarrier beads  for 24 h. To simulate microgravity, we dispersed cardiomyocytes on Cytodex 3 microcarrier beads in 5 ml of Dulbecco's modified Eagle's medium with 10% newborn calf serum and penicillin-streptomycin and then inoculated them through the syringe port inside the 5-ml high-aspect ratio vessels of a rotary cell culture system . After being assembled, they were placed on their rotary base and maintained in a 37 °C incubator with a 5% CO2/air mixture and saturating humidity. Vessel rotation was set at 18 rpm according to our recent report (m), SB23580 (10 μm), and calpain inhibitor-III (10 μm)), the inhibitors or vehicle were added to culture media at the time of inoculation into the rotary device. The cells were incubated under simulated microgravity for 24 h.Right after isolation, cardiomyocytes , p67phox , phosphorylation of Ser-345 and Ser-370 on p47phox and total p47phox , phosphorylated p38 and total p38 , phosphorylated ERK1/2 and total ERK1/2 , and GAPDH , respectively, followed by secondary relevant antibodies conjugated with horseradish peroxidase. The signals were then developed using an enhanced version of the chemiluminescence reaction.40 μg of protein from heart tissue or cell lysates or 10 μg of protein from isolated membrane lysates were loaded onto SDS-polyacrylamide gels. After electrophoresis, the separate proteins were transferred onto Bio-Rad polyvinylidene difluoride membranes. After blocking in 5% nonfat milk for 1 h, the membranes were incubated with antibodies against NOX4 , Rac1 , MuRF1 and atrogin-1 , NaThe protein ladders were purchased from FroggaBio Inc.  for cultured cardiomyocytes  and Thermo Fisher Scientific China Co. Ltd. for heart tissue samples .Mtnd1 region of the mitochondrial genome and β2-microglobulin (B2M) as a nuclear gene reference. The sequences of primers are as follows: Mtnd1, 5′-GAGGGAACCAAACTGAACGC-3′ and 5′-TGGATCCGTTCGTAGTTGGAG-3′; B2M, 5′-CAGACTCTGCGATGTTTCCA-3′ and 5′-GCCTGAGCACTTCCAGAAAC-3′. The mitochondrial DNA copies were expressed as the ratio of Mtnd1 to B2M.Total DNA was extracted using a DNA extraction kit (Qiagen), following the manufacturer's instructions. Real-time PCR was conducted using primers specific to the mitochondrial N-succinyl-LLVY-7-amido-4-methylcoumarin (Cedarlane Laboratories) as described previously  using a fluorescence substrate, eviously .phox, and p67phox to cell membranes. Briefly, cell membranes were isolated from heart tissues using a commercial kit  according to the manufacturer's instructions. The protein levels of Rac1, p47phox, and p67phox in cell membranes and whole-heart lysates were analyzed using Western blotting. Na+/K+-ATPase and GAPDH were used as loading controls for the membranes and whole lysates, respectively.NADPH oxidase activation was determined by measuring the translocation of Rac1, p47Xanthine oxidase activity was determined in heart tissue lysates using a commercial assay kit (Beyotime Biotechnology), following the manufacturer's instructions. A total of 100 μg of protein was used for each sample in the assay.These activities were measured in heart tissue lysates using commercial assay kits (Beyotime Biotechnology), following the manufacturer's instructions. A total of 100 μg of protein was used for each sample in the assay.t test. For multigroup comparisons, ANOVA followed by Newman–Keuls test was performed. A value of p < 0.05 was considered statistically significant.All data are expressed as means ± S.D. Differences between two groups were compared by unpaired Student's tpeng2@uwo.ca or tqpeng@suda.edu.cn).All data presented are available upon request from Tianqing Peng (


This study deals with the mathematical modeling of crystallization kinetics occurring during batch production of the ice cream. The temperature decrease was recorded in-situ through a computerized wireless system. A robust pattern-recognition algorithm of the experimental cooling curves was developed to determine the initial freezing point. The theoretical freezing point was used to calibrate the whole time-temperature profile. Finally, a modified Gompertz’s function was used to describe the main steps of crystallization kinetics. Derivative analysis of the Gompertz’s function allowed to determine the time-temperature physical markers of dynamic nucleation, ice crystal growth and air whipping. Composition and freezing properties were used as input variables in multivariate analysis to classification purposes of the ice cream mixtures as a function of their ability to produce high-quality ice cream. The numerical analysis of the whole cooling curve was used to build predictive models of the ice cream quality indices.  Ice cream is the most widely consumed frozen dairy dessert, with a global market value of $68,072 million in 2016 .From a manufacturing point of view, ice cream can be categorized as industrial and artisanal ice cream as a function of the process technologies and production scale. The artisanal ice cream production involves the following operations: preparation of a liquid mixture generally containing ingredients such as milk fat, milk-solids-not-fat , sugars (or sweeteners), hydrocolloids (stabilizers and emulsifiers); aging of this mixture; simultaneous whipping and freezing under high-shear dynamic conditions; incorporation of flavoring ingredients to this partially frozen mixture; tempering, shaping and serving of the final product. In the case of the industrial production, another freezing step under static conditions is required after shaping. The first freezing step is carried out using scraped-surface freezers, to decrease the mixture temperature below its freezing point  to obtain a soft, semi-frozen texture. In the artisanal process, scraped-surface freezers are batch plants able to incorporate air under atmospheric conditions (overrun below 40%). Instead, in the industrial process, scraped-surface freezers are continuous plants able to incorporate air under the pressure to reach an overrun of 100%. The second freezing step required in the industrial production is carried out using forced-air convection freezers to reduce the internal temperature to −25/−30 °C (below its glass transition) thus resulting in a harder texture and a longer shelf-life. From the qualitative point of view, industrial and artisanal ice creams mainly differ for air content and texture properties . The liqUnder a microstructural point of view, both industrial and artisanal ice creams can be considered three-phase colloidal systems consisting of ice crystals; unfrozen serum phase, that in turn contains clusters of partially crystalline and partially coalesced fat globules, all interacting with hydrocolloids to create a three-dimensional viscoelastic network; air cells, entrapped into the serum phase ,7,8,9,10Sensory quality and melting resistance are two key quality aspects of the ice cream and they are strictly dependent on what takes place in the barrel of the scraped-surface heat exchanger (SSHE) used to produce the ice cream, which is the place in which ice nucleation starts. Many tiny ice crystal nuclei are formed, scraped and melt into the bulk of the ice cream mixture where the larger-size crystals grown. Once the product leaves the SSHE, no further nucleation takes place but the existing crystals grow with time and such phenomenon is accelerated under temperature variation. Ice crystals larger than 40–50 μm result in coarse or grainy texture if present in sufficient number. Both the rate and extent of water crystallization affect the amount and the size distribution of the ice crystals in the ice cream. Water crystallization that takes place under dynamic conditions inside the SSHE is the main cause of the formation of a viscoelastic network inside the unfrozen phase, which in turn limits the ice crystal growth and the stable incorporation of air in the final product. Water crystallization causes the increase of both solute concentration and viscosity of the unfrozen phase remaining at the end of the cooling process. Instead, mechanical shearing causes structure transition from slurry ice cream mixture to soft-solid ice cream by promoting shear-induced destabilization of the milk fat globules and the stable incorporation of finely dispersed air cells (overrun). The increasing of the overall stiffness of the ice cream mixture during freezing is due to the intermolecular interactions occurring under high-shear rate among fats, proteins and hydrocolloids from colloidal to the macroscopic scale.Thus, the quantitative description of the water crystallization kinetics is of fundamental importance to understand and manage the ice cream production but it is difficult to describe by mechanistic models. Currently, there are several gaps in both scientific and technical knowledges on the topic. From the scientific point of view, there is a gap between the theoretical knowledge based on the thermodynamics of water crystallization  and the knowledge about this phase transition within heterogeneous and high-concentrated liquid systems, such as the ice cream formulations. The partial freezing of ice cream mixture occurring under dynamic conditions involves complex thermodynamic and kinetic mechanisms such as heat and mass transfer, supercooling, heterogeneous nucleation, as well as changes in thermo physical properties, surface curvature effects and so forth. Furthermore, multiple re-crystallization/melting and air incorporation must be considered. The equilibrium temperature at which the water crystallization takes place in the ice cream mixtures strictly depends on the ability of colligative solutes to depress the freezing point. Initial freezing point and freezable water fraction are the two parameters required to determine the temperature dependence of the frozen water fraction. The importance of freezing point depression in the ice cream production has induced researchers to develop models based on mix composition. As recently reviewed , some emThe technological aim of freezing in the SSHE is to obtain the rapid formation of small ice crystals and to develop the desired overrun. The drawing temperature of ice cream −5 to −7 °C) from the batch freezer is slightly higher than that used for continuous freezers and residence times in the freezer barrel are longer for batch processing (5–6 min). This results in larger ice crystals. Furthermore, the overrun control in the batch freezer is difficult because the scraping blades continue to operate during unloading of the freezer and the air is present at atmospheric pressure. It is useful point out that subjective evaluation of the time to shut off the refrigeration system as determined by the operator may produce high variability both in the size of the ice crystals and in the product yield (overrun). By prolonging the freezing time beyond the set point of desired stiffness, the amount of the incorporated air reaches a maximum and then decreases as a balance between the rheological, interfacial properties and mechanical stress established inside the freezing mixture ,10,17,18 to −7 °CThe above considerations highlight the need to identify, from one hand, the optimal technological properties of the starting ice cream mixture and from another hand, an objective strategy to manage the freezing/whipping operations in batch ice cream production. Hard work is required to realize simultaneously the two key technological properties, that is, the ability to entrap the finely dispersed air cells (referred as to ”whippability”) and the ability to support fast crystallization of the water molecules aiming to obtain finely dispersed ice crystals. Both these properties depend on the composition of the starting formulations, characteristics of the freezer and desired quality of the ice cream. To optimize the air incorporation and prevent the excessive growth of the ice crystals, an optimal composition of the starting liquid mixture must be considered a technological pre-requisite for ice cream production. The present work provides a new strategy to evaluate in-situ the optimal technological properties under batch freezing conditions. In particular, this paper suggests a suitable setup for in-bulk temperature measurement inside the barrel of the SSHE for batch production of ice cream and describes a numerical approach to analyze the time-temperature history  experienced by the ice cream mixture, aiming to characterize the kinetics of water crystallization and air incorporation. The most significant kinetic properties were correlated to the overrun, melting and sensory properties providing predictive statistic models.-preparation of mixtures characterized by different total solid contents and different level of complexity ;-partial freezing of the starting mixtures inside a batch SSHE;-phenomenological analysis of the behavior of model systems and ice cream mixtures in order to explore the effects on the solute-solvent interactions on the main freezing events registered along the experimental cooling curve ;-numerical analysis of the cooling curves  and a polyol (sorbitol) of interest as sweeteners in the ice cream production were selected for their different colligative properties to modulate both the equilibrium temperature and the crystallization kinetic. The individual and synergistic colligative effects of such sugars were firstly evaluated in the water- and milk-based solutions. Then, the information acquired with these trials were used to clarify the dynamic behavior of the investigated ice cream mixtures. Three water-based model systems, namely “WSUCR,” “WDEXT”; and “WSORB” were produced by adding 300 g of sucrose or dextrose or sorbitol to 1000 g of distilled water. Similarly, three milk-based model solutions were prepared by replacing water with whole milk. In the following they are referred as to “MSUCR,” “MDEXT,” “MSORB,” respectively.Another more complex water-based model system, namely “WSUCR + WSORB, was produced by adding 150 g of sucrose and 150 g of sorbitol to 1000 g of distilled. Similarly, a more complex milk-based model system, namely “MSUCR + MDEXTR,” was produced by adding 150 g of sucrose and 150 g of dextrose to 1000 g of whole milk. All ingredients were purchased from REIRE s.r.l. . About 10 Kg of each of the 5 solutions were prepared according to the following procedure. The ingredients were blended under mechanical agitation carried out with a turbomixer (Robot Coupe-94305 Vincennes Cedex-France) for 1 min, then the solution was heated up to 88 °C (the temperature was reached in about 5 min) and finally cooled under 8 °C (the temperature was reached in about 1.5 min).In order to exactly quantify the initial water content and the colligative and not-colligative compounds derives from the ingredients used, the mass balances of all the model systems were calculated and reported in . As can be inferred from these data, the water concentrations were in the ranges 77.03–78.31 and 69.53–70.81 g/100 g for water-based and milk-based model systems, respectively while sugars were in the range 21.69–22.97 and 25.35–26.62 g/100 g for water-based and milk-based model systems, respectively. In the milk-based systems, the concentrations of fats, milk solids non-fat  and NaCl were 1.19, 2.56 and 0.10 (g/100 g), respectively. All the ice cream mixtures were prepared with variable amounts of sucrose, dextrose, sorbitol but with the same amount of whole milk and of a multicomponent basis. The composition of the multicomponent basis was the following: skimmed-milk powder (30%), dextrose (30%), maltodextrin (18.36%), inulin (10%), milk proteins (5%), sucrose esters (4.35%), carrageenan (0.29%), Tara-seed flour (1.5%) and carboxymethylcellulose (0.5%). All ingredients were purchased from REIRE s.r.l. . First of all, the following 3 ice cream mixtures were prepared with the increasing concentrations of the total sugar content (expressed as g/100 g of water): 16.6% ; 23.1% , whose composition corresponds to that of the traditional recipe for the Italian-style ice cream; and 28.6% . Further 4 MSC ice cream mixtures, namely MSC1, MSC,” MSC3 and MSC4, were prepared by varying the relative amounts of each sugar without changing the total sugar content . The mass balances of all the ice cream mixtures were reported in . The concentrations of water were 76.01 and 65.42 (g/100 g) for LSC and HSC, respectively and ranged from 70.20 to 70.41 within all the MSC mixtures. Sugar concentrations were 18.34 and 29.74 (g/100 g) for LSC and HSC, respectively and were within 24.37–24.58 (g/100 g) range for all MSCs. The concentrations of fats were 1.32 and 1.13 (g/100 g) for LSC and HSC, respectively and 1.22 (g/100 g) for all MSCs. MSNF were 4.20, 3.60 and 3.88 (g/100 g) for LSC, HSC and MSCs, respectively. Finally, NaCl contents were 0.14 and 0.12 and 0.13 (g/100 g) for LSC, HSC all MSCs, respectively.For each of the ice cream mixtures denominated LSC, MSC and HSC, an amount of about 60 Kg was prepared. Instead, for each of the ice cream mixtures denominated MSC1, MSC2, MSC3 and MSC4, an amount of about 30 Kg was prepared. The ingredients were blended under mechanical agitation carried out with a turbomixer  for 1 min, then the solution was heated up to 88 °C (the temperature was reached in about 5 min) and finally cooled under 8 °C (the temperature was reached in about 1.5 min). Model systems and ice cream mixtures were held at 4.0 ± 0.2 °C for 24 h under static conditions in a cabinet . Such operation allowed the partial crystallization of the milk fat globules and the protein hydration. Both physical phenomena concur to the formation of a viscoelastic network inside the unfrozen phase during freezing. The aged products were then homogenized with a turbomixer  for 1 min before freezing.The freezing operation was carried out on batches of about 3 Kg of aged model systems or ice cream mixtures in a SSHE  having the following technical characteristics: capacity 7.5 Kg; max power 7.8 kW; 3 blades rotating at 150 rpm; and 1.1 Kg of R404A as coolant. The temperatures were recorded by using a PT100 probe  with a DIN sensor certified according to the international standard IEC751 for its insensibility to the electrical interferences, response time below 3.5 s , platinum resistance of 100 Ω 0 °C and accuracy of ±0.03 °C at 0 °C. The probe worked at an operative frequency of 1 Hz. The probe was located inside the SSHE at an intermediate radial distance between the axial shaft and surface wall, within the gap between the scraping blades and freezer exit according to the scheme reported in . The probe location was chosen in order to obtain comprehensive information about the ice crystallization kinetics, taking into account that the ice nucleation rate is higher on the surface of the SSHE (which is the colder zone) and that the ice growth and air whipping rates is higher inside the SSHE bulk (which is the warmer zone) .The temperature probe was equipped with a steel-sealing standard clamp (G1/2 units), a fixing element qualified for process connection. It was fixed on the Plexiglas exit window of the SSHE through a horizontal installation . The posIn order to minimize the variability of the freezing experiments, after homogenization, the aged liquid products were immediately transferred to the SSHE dasher, whose temperature was previously regulated at 12 °C.During freezing the mixture mechanical stiffness increased due to temperature lowering, ice crystallization and unfrozen phase concentration. The final product was extracted from the SSHE after it reached a mechanical stiffness corresponding to the 65% of the maximum motor electric power.Tempering operation was necessary to stabilize the ice cream texture before melting and sensory analysis. To this aim, immediately after extraction from the SSHE, ice creams were held at −13 ± 0.2 °C for 24 h in a ventilated ice-cream cabinet .The theoretical freezing point is usually defined as the sub-zero temperature at which the water begins to crystallize in a solution of colligative solutes. Freezing point depression of both model systems and ice cream mixtures was estimated considering their contents in colligative solutes, according to the well-established sucrose equivalent method . In ordeThen, the contribution of sugars to the freezing point depression was calculated using the polynomial equation with intercept through zero derived from the regression model where g sucrose/100 g water is graphed against freezing point depression . The contributions of MSNF and NaCl to the freezing point depression were calculated according to Goff and Hartel  and PottFats, proteins, large molecular weight carbohydrates , stabilizers and emulsifiers do not contribute to freezing point depression since fats are immiscible with the aqueous phase and proteins and polysaccharides are very large molecules  with a nIn order to evaluate the air volume incorporated within the final product, the following formula was applied:A calibrated metal cup of 235 mL ± 0.1 mL was used for the measurements.Melting tests were carried out in a conditioned chamber at 20 ± 0.1 °C using a known volume of ice cream contained in a calibrated cup. The ice cream sample was transferred on a 2 mm mesh sieve under which there was an empty cup, placed on a precision balance , to collect and weigh the ice cream that melts and drops under gravity. The weight of the melted ice cream was registered at the first drop and after 15, 30 and 45 min. A trained panel of 12 judges was selected to perform a rating analysis of the ice cream based on their perception of differences among samples and rating for intensity of the sensory attributes according to attribute-specific hedonic scales as described in :A pre-screening questionnaire was administered for recruitment purposes to 35 undergraduates of the Department of Life Sciences, University of Modena and Reggio Emilia  who had followed a 10 h course on ice cream sensory evaluation. Interest and availability, lactose-tolerance, natural dentition, normal health conditions, abstinence from smoking, alcohol and chewing betel and no dietary restriction on calorie intake were included in the pre-screening questionnaire as major criteria for recruitment. Thirty people compliant with the pre-screening criteria were then recruited as aspiring panelists and attended a one-year training program to improve their capability to identify and rank the intensity of sensory attributes of the ice cream in training tests sessions of 5 days every month. A set of ice cream references were prepared with four levels intensity for each sensory attribute of interest and used for training purposes. Such reference samples were prepared using the same ingredients and freezing conditions used to produce the MSC ice cream if not otherwise specified. However, the total amount of the added sucrose or emulsifiers and stabilizers present in the multicomponent basis was modulated according to a linear increasing function. Concerning sandiness attribute, the reference samples were produced with the same MSC composition but tempering operation was performed under fluctuating temperatures conditions around the tempering target (−13 °C) by shutting off the power supply intermittently for increasing time periods on 24 h. For cool sensation, reference samples were produced with the same MSC composition but were extracted from the SSHE at four drawing temperatures. To evaluate both flavor persistency and intensity as well as off-flavor, reference samples were produced with the same amount of total added sugars of MSC ice cream but with different ratio between whole milk and multicomponent basis.The specifications used to produce the reference ice creams and their intensity levels used for the rating tests are listed in . During training sessions, the reference ice creams were presented in a random order and the judges were asked to pick the samples with different intensity and rank them according to the perceived intensity using the attribute-specific hedonic scale. To compensate for the known variations throughout training test sessions a qualifying test was performed, those who make an acceptable (75 percent correct) were chosen, otherwise the training session was replicated. A series of 12 judgments were obtained from each prospective panelist. Twelve judges were selected as panel components based on their abilities to match at least 5 out 8 of sensory attributes and at least 3 out 4 of rating tests.During both the training and rating tests sessions, the ice creams were removed at −13 ± 0.2 °C from the ventilated cabinet used for tempering and anonym glasses of about 100 mL were prepared containing the same amount of product. No more five ice cream samples were analyzed for each test session. Panelists were asked to firstly evaluate the appearance of the ice cream surface, noting if the ice creams have retained its form and shape. Next, they assessed the in-mouth properties. Immediately after placing a portion of ice cream into the mouth, they roll it between the incisors and bring them together very gently, noting the eventual presence of ice crystals and whether grittiness is apparent between the teeth. A small portion between the incisors revealed the presence of minute traces of a gritty or sandy texture. Meltdown intensity was assessed by evaluating the time required for the product to soften and melt in the mouth when continuously pressed by the tongue against the palate. When the sample was completely liquefied and warmed to body temperature, the detection of the sweetness as well as of flavor intensity or persistency was done. It was accomplished by placing a small teaspoon of the ice cream directly into the mouth, quickly manipulating the sample between the teeth and palate and noting the taste and odor sensations as well as any releasing of after taste. Since the evaluations of each attribute were carried out on evaluation scale specific to that attribute, the scores assigned by the panelists were re-parameterized on a common scale by performing standardization of the original scores to show the overall ice-cream profile in a single figure for comparative purposes. To eliminate both the units and scaling effects each score was divided by the standard deviation, obtaining a variance of 1 but different means and ranges. To evaluate the preference among the investigated ice creams, both the highest scores and optimum score (where defined) were fixed as the highest level of liking degree. The final judgment was calculated by summing all desired sensory attributes  and subtracting all undesired ones . As a consequence, an overall liking level was calculated: Model systems were produced in triplicate. Concerning the ice cream mixtures, 18 replicates were produced for the samples denominated LSC, MSC and HSC and 9 replicates were produced for MSC1, MSC2, MSC3 and MSC4 samples. To reduce the experimental noise, time-temperature data were filtered by using the 3-point Savitzky-Golay numerical method .The reproducibility of the time-temperature data was evaluated by overlapping the cooling profile of the independent replicates. More than 1200 values (400 sampling times for each temperature profile) were used for the descriptive statistical analysis of the differences measured among three replicates. As calculated by doubling the experimental standard error, the uncertainty of temperature measurement always ranged within the +/−0.2 °C.2 with a statistical significance of p < 0.01.Principal component analysis (PCA) was performed to classify the ice cream samples according to composition and freezing properties. Multiple regression analysis (MRA) was carried out to develop predictive models for the following ice cream quality properties: melting rate, overrun, appearance, cool sensation, smoothness, sweetness, meltdown, flavor intensity and flavor persistence. The predictive accuracy of the MRA models was evaluated by performing the cross-validation on an independent dataset. The original dataset was split in two subsets namely “calibration dataset”  from which the MRA models were derived and “validation dataset”  against which the MAR models were validated. The goodness of fit for MRA models and for the linear regression between predicted vs. observed data was evaluated calculating the multiple RAll statistical analyses were carried out using Statistica 10.0 .In this section, the cooling curves registered during the freezing of the model solutions and of the ice cream mixtures were separately analyzed and discussed.rate) of −17.69 ± 0.52 °C/min. The lowest temperature in the cooling curve represents the supercooling (in the figure it was referred to as “S”): it was reached by all the investigated water-based model solutions on average after about 2.4–2.7 min. As can be inferred from figure, a similar trend can be recognized among the model systems with a significant difference. Temperature decreased under 0 °C with a chilling rate  was determined as the slope of the linear regression analysis of the cooling curve in the time domain ranging between TP1 and S. The formation of unstable clusters of ice nuclei started at about 0.8 min and NUR was on average −0.49 °C/min.Starting from supercooling, the formation of new metastable nuclei occurred with phase transition with the release of the latent heat that determines the asymptotical increase of solution temperature up to the equilibrium level, representing the freezing point  and thermodynamic (FP) parameters were sugar-specific and followed the order WSUCR, WSUCR + WSORB, WDEXTR and WSORB. This behavior could be attributed to the different ability of disaccharides, monosaccharides and sugar alcohols to form hydrogen bonds with water molecules (colligative behavior), which is in turn related to the number of hydroxylic groups and the sugar steric hindrance. These results are in agreement with the findings of previous research. The nucleation rate in dextrose solutions shows a low-order dependence upon supercooling, for supercooling in the range −0.25 °C and −1.00 °C but it increases markedly with increasing sugar concentration . SucroseSugars were the only colligative solutes determining the depression of the freezing point in the water-based model systems. The theoretical freezing point FPt for the water-based model systems was calculated according to the sucrose equivalent method  using thAs can be inferred from the data reported in , FPt valAs can be inferred from data, alternate exothermic and endothermic events characterized the cooling process suggesting that more than one apparent nucleation steps occurred with an equal number of supercooling events. Dynamic and synergistic interactions occurring at a molecular scale between water and sugar molecules and at a colloidal scale between ice crystals and between sugars and milk constituents were hypothesized as the main causes of the observed temperature profiles and they could be due to the formation of hydrogen bonds. The formation of large agglomerates of ice crystals was observed in freezing experiments concerning sugar solutions . rate of −16.00 ± 0.52 °C/min and was significantly lower than that observed in water-based model systems. The presence of milk fat and proteins is responsible for a relatively higher liquid viscosity and a lower heat transfer coefficient of the milk-based model systems. Due to the occurrence of several apparent nucleation events, the temperature corresponding to the first event was determined as initial supercooling, IS. Starting from the initial supercooling, the formation of new metastable nuclei occurred during phase transition with the release of latent heat that determines an increase of solution temperature up to the first equilibrium level, that is, the experimental initial freezing point, IFP. The theoretical freezing point, IFPt, for the milk-based model systems were significantly lower than the experimental ones  was determined as the slope of the linear regression analysis of the cooling curve in the time domain ranging between TP1 and IS; while the “Heterogeneous Nucleation time, HNT” was calculated as the time length between TP1 and IFP. NUR was on average −3.25 °C/min and the HNT values were in the range 0.28 ± 0.07–0.39 ± 0.10 °C/min indicating that the IFP point was reached faster than in water-based models. Both the kinetic (IS) and thermodynamic (IFP) parameters highlighted sugar-specific effects on the water crystallization kinetics and were in the following decreasing order MSUCR, MSUCR + DEXTR, MDEXTR and MSORB. As for the milk-based systems, more than one apparent nucleation steps occurred with an equal number of supercooling events but they occurred in a defined time and with lower temperature fluctuations due to the presence of emulsifiers, which promoted the destabilization of the milk fat globules and stabilizers, responsible for the higher initial viscosity. After heterogeneous nucleation, that is, an unstable crystallization period during which temperature fluctuation may be registered due to more than ones supercooling and nucleation, the ice nuclei reach a minimum stable size, enough to growth at a macroscopic scale size without changes in temperature. Such equilibrium temperature was kept for very little time, because the significant subtraction of water as ice from the liquid mixture caused the concentration of the unfrozen phase that becomes slurry. Water crystallization required lower temperatures to progress when the unfrozen phase concentrates significantly.In order to simplify the quantitative analysis of the water crystallization kinetics, the following main steps of the cooling curve were individuated:Chilling, corresponding to the linear part of the cooling curve above 0 °C.Initial Freezing Point (IFP) corresponding to the highest temperature  reached after the first supercooling (kinetic event)Steady-State, part of the cooling curve where the equilibrium temperature remains constant for a little time since the ice crystallization occurs without significant increase of the concentration of the unfrozen phase of the ice cream mixture.eff) corresponding to the equilibrium temperature, at which the water crystallization kinetics is at its acceleration point and the ice starts to accumulate significantly and extensively in the ice-cream mixtures. Effective Freezing Point (IFPeff corresponding to the multiple heterogeneous nucleation (supercooling) events.Dynamic Nucleation, part of the cooling curve below 0 °C and IFPeff and temperature at which the ice cream exits (Texit) from the SSHE where the ice crystals growth is limited by the length of the freezing time as a balance between the mechanical shearing and technological properties of the ice cream mixture Dynamic Ice Crystallization, part of the cooling curve between IFPAs an example,  reports rate (−15.41 ± 1.60 °C/min) was not significantly different from those referred to the milk-based model systems, while NUR (−7.80 ± 1.42 °C/min) and HNT (0.79 ± 0.16 min) were significantly higher. Regarding IS (−2.34 ± 0.18 °C) and IFP (−2.23 ± 0.35 °C), no differences were highlighted between HSC and MSUCR, while the values found for the ice cream were significantly lower than those observed for MSUCR + MDEXTR, MDEXTR and MSORB. The temperature gradient (0.70 ± 0.17 °C), expressed in terms of the ∆IFP parameter was not significantly different to that (0.65 ± 0.27 °C) measured on in milk-based model system. However, HSC samples showed the highest average value (1.39 ± 0.17 °C) of ∆IFP: it was not significantly different from that (1.29 ± 0.37 °C) measured in the water-based model systems. The behavior observed for the HSC samples was attributed to the highest concentration of the hydrocolloids of the multicomponent basis used in their formulations. The stable incorporation of finely disperse air cells surrounded by milk proteins and partially-destabilized fat globules concurred to the heterogeneous nucleation process, resulting in a decreasing of the supercooling required, so that water crystallization occurred at higher equilibrium temperatures than in the static freezing conditions considered to calculate the theoretical freezing point.Since more than one nucleation events were observed in the ice cream mixtures during cooling, both the NUR and HNT were calculated as for the milk-based model systems. The kinetic and thermodynamic parameters of the HSC ice cream mixtures were compared to those of the milk-based model systems since they were prepared with the same amounts of added sugars. CLThe theoretical values of IFPt for the ice cream mixtures were estimated considering the presence of sugars, MSNF and NaCl, whose individual contributions are listed in .x-axis accounts for the inverse absolute values of the experimental freezing point ). The lower values of the x-axis correspond to the higher freezing point depression. The y-axis accounts for the ratio Mw/Ms  and the concentration of sucrose equivalents (expressed as g/100 g of water) corresponding to the colligative solutes sugars, MSNF and NaCl. The strength of colligative solute-solvent interaction is represented by the intercept of the linear regression plot while the degree of dependence between the freezing point and Mw/Ms is represented by the slope. As can be inferred from the figure, the linear Weast’s equation is satisfactorily applied to all the investigated mixtures: the inverse value of the freezing point increased linearly with Mw/Ms (with R2 > 0.876 at least). The absolute values of both the slope and intercept from ILPs were 2.8419 and 0.0159, 3.4962 and 0.0147, 4.5621 and 0.1805 for water-based model systems, ice cream mixtures and milk-based model systems, respectively. Intercept data suggested that the greatest contribution to the overall strength of the interaction between the colligative solutes and the free water molecules was observed in milk-based model systems, more likely caused by the milk proteins and partially destabilized fat globules at a colloidal scale. From one hand, both milk proteins and partially destabilized fat globules behave as hydrophilic colloids: they can subtract free water molecules, resulting in an increasing of the concentration of sucrose equivalents in the unfrozen serum phase and therefore in a decreasing of the initial freezing point. Furthermore, an additive sugar-specific colligative effect arising from lactose, MSNF and milk salts concurs to the degree of dependence of Mw/Ms on freezing point depression. On the other hand, the colloids can also promote the heterogeneous nucleation step of the water crystallization kinetics that takes place inside the SSHE, resulting in a decreasing of supercooling and therefore in an increasing in the initial freezing point. As suggested by the experimental cooling curves, both the NUR and IS parameters increased while HNT parameter decreased in the milk-based model systems with respect to the water-based model systems. Under our experimental conditions, the balancing between the two effect resulted in lower levels of the freezing point depression and therefore in higher values of the initial freezing point for the milk-based model systems with respect to the water-based ones. Despite they have very close levels  of Mw/Ms, the WSUCR + WSORB samples showed an average value of freezing point depression (IFP at −1.58 °C) lower than that (IFP at −2.35 °C) of the MSUCR ones. Similar results were observed by comparing MSUCR + MDEXTR against WSORB samples for which Mw/Ms was close to 1.38: the sucrose equivalents were 51.85 and 55.99 g/100 g of water while the initial freezing point were −2.94 and −2.12 °C for MSUCR + MDEXTR and WSORB, respectively.An intermediate strength of the colligative solute-solvent interaction was observed in the ice cream mixtures. Such behavior was attributed to the main technological functions of stabilizers and emulsifiers from the multicomponent basis used in their formulations. Concerning the stabilizers, carrageenan, Tara-seed flour and carboxymethylcellulose are the hydrocolloids with the greatest water binding capacity and thickening ability, followed by inulin and maltodextrin. Such high molecular weight hydrocolloids have a great ability to interact with free water molecules through hydration  and gelling , thus occupying a large solution volume and entangling and interacting with each other and with milk proteins and partially-destabilized fat globules to form large hydrophilic colloids . The synCompelling evidence of the destabilizing effects of the emulsifying hydrocolloids, resulting in the increase of the interactions on the colloidal scale, is provided in the b that shThe mathematical description of the water crystallization in batch ice cream production was performed through a phenomenological modeling of the overall freezing occurring inside the SSHE that include the main freezing steps observed on a macroscopic scale, that is, the heterogeneous nucleation, ice crystal growth and air incorporation. From one hand, the improvement of the quality of ice cream produced in batch SSHE requires accurate and quantitative data about the composition of the starting liquid mixture and its freezing properties but also about the way in which structural and physical changes occurring under dynamic freezing conditions may affect melting and sensory properties of the final ice cream. From another hand, the time-temperature history experienced by the ice cream mixture inside the SSHE can be used as an accurate fingerprinting of the multiple events taking place on a wide range of scale size and that include heterogeneous nucleation, ice crystal growth, destabilizing of fat globules, cross-linking of hydrocolloids to form a viscoelastic network and air incorporation. In order to characterize the water crystallization kinetics that takes place inside the SSHE during ice cream production, the mass fraction of the initial water content was used as a kinetic descriptor of the temperatures experienced by the ice cream mixtures inside the SSHE. On the other hand, the direct measurement of the freezing point depression of the ice cream mixture under dynamic conditions is difficult to obtain instrumentally as a consequence of (i) the increased sample viscosity which is responsible of the slowdown of the rate of heat transfer and (ii) the dynamic scraping conditions causing the ice nuclei to be sheared, melted and mixed on the large ice crystals.The initial freezing point IFP and freezable water fraction n states . It is wn states . They reIn the present work, the freezable water fraction was based on the numerical analysis of the whole cooling curve throughout three preliminary steps that include (i) the individuation of the first supercooling event, (ii) the determination of IFP and (iii) the calibration of the whole cooling curve. Finally, a re-parameterized Gompertz’s function was used to describe the water crystallization kinetics by quantifying the time-dependent changes in As an example, a shows tIn the first step, derivative analysis of the cooling curve was performed to evaluate the time corresponding to the first supercooling event according to . In the The value of the function is expected to rapidly increase at the first supercooling  before the rate of the increasing can reach an asymptotical level during freezing a. The quexit, which was assumed to include all the equilibrium freezing temperatures experienced by the ice cream mixture inside the freezer. To this aim, the whole cooling curve was previously calibrated by algebraically subtracting the difference calculated between the experimental value of the initial freezing point IFP and the theoretical value IFPt. The calibration of the experimental temperature profile was necessary to compensate the overestimation of the temperature levels measured by the temperature probe due to the specific location inside the SSHE, that is, at intermediate position between the colder zone (freezing surface) and warmer zone (shaft rotor) and the effects exerted by other experimental factors that include the time-of-response of the temperature probe and the solute-solute and solute-solvent interactions. In the present work, the freezable water fraction t method  as the mDuring freezing, the concentration of the colligative solutes increases in the unfrozen serum phase, thus causing the progressive reduction of the equilibrium freezing temperature.In the present work, the temperature dependence of the freezable water fraction was established by coupling the The assumption that the bulk temperatures of the ice cream mixture correspond to its equilibrium freezing points is supported by several basic studies focused on sugar solutions which proved that the bulk temperature inside the SSHE is very close to the thermodynamic equilibrium temperatures (freezing point) since the secondary nucleation rate of ice is fast ,28. The eff range can be considered an induction step of the whole water crystallization process. In this induction step, cooperative phenomena take place on a microscopic scale, which include heat and mass transfer, supercooling, crystallization and melting events, before the ice started to growth significantly at the effective freezing temperature point IFPeff. After this equilibrium temperature, the growth of the ice crystals dominates on the heterogeneous nucleation: the growth rate of WF(t) reaches a maximum level before to decelerate and level off to an upper asymptotic level  towards the Twhipp point, where the air incorporation dominates on the ice formation until the Texit point.From a macroscopic point of view, the water crystallization kinetics occurring in the batch SSHE follows a sigmoidal trend as described by the time-dependent changes in the freezable water fraction during cooling. As can be inferred from b, the dyA re-parameterized Gompertz’s function was proposed as a phenomenological model to mathematically describe the time-changes in the freezable water fraction The three model parameters were estimated by performing a non-linear fitting procedure of the re-parameterized Gompertz’s function. The lack-of-fit was evaluated by calculating the square relative mean error :eff), (2) at its maximum rate (GRtime and GRT) and (3) at its maximum deceleration (tTwhipp and Twhipp).First-, second- and third-order derivative analysis of the modified Gompertz’s function was performed to determine the time-temperature onsets corresponding to the water crystallization kinetics (1) at its maximum acceleration  beyond the set point of desired stiffness, due to the fine-modulation of the stiffness target by the electronic system of the SSHE: the smaller crystals melt causing the increase of the bigger ones  , Tiffness , which was able to cause the overrun loss and more likely melting of the smaller ice crystals and growth of the larger ones.It is widely known that the greatest number of ice crystals formed in the mechanical freezer are obtained with the highest level of the initial freezing point of the mix, thus resulting in a smoother texture for the ice cream. The initial freezing point also determines the amount of water melts at room temperature. In general, the higher is the initial freezing point, the lowest is the melting rate at room temperature. However, in this study, an opposite behavior was observed for LSC ice cream mixtures for which a prolonged time of freezing was required to incorporate the air and obtain a given overall stiffness at the exit of the SSHE. This unexpected behavior was attributed to an excess of the mechanical energy provided by the SSHE scraping blades during the prolonged freezing time (as indicated by higher levels of tTResults from PCA corroborates the idea that some freezing properties obtained from the modeling of the cooling curve can be used for classification purposes of the ice cream mixtures.rate and Twe were also considered. The stepwise analysis retained all temperature- and time-domain freezing properties in the predictive models, while all composition properties were discarded from the models, except for the fat concentration that affected the Flavor Intensity attribute. As can be inferred from data, the predictive models were satisfactorily fitted to the calibration data as suggested by the multiple R2. The cross-validation of the predictive models provided acceptable results for most ice cream quality indices with exception of those referred to overrun, cool sensation, smoothness and sweetness, whose multiple R2 were lower than 0.500.To represent the structural changes taking place during the chilling step of cooling and those occurring during the asymptotic ice formation, CLA preliminary comparative study of the freezing behavior of water and milk-based model systems and ice cream mixtures was useful to individuate and understand the main freezing steps of the water crystallization kinetics and to highlight the strength of the interactions occurring between the colligative solutes and free water molecules in the SSHE at the conditions required for the batch ice cream production. During the cooling of the ice cream mixtures, more than one supercooling events occurred with lower temperature fluctuations than in the two model systems. This is a consequence of the presence of stabilizers and emulsifiers, which are responsible respectively for the increase of the bulk viscosity and for the extent of the destabilization of the milk fat globules. The whole cooling curve was numerically analyzed and the derived parameters used as descriptors of the main freezing processes underlying the conversion of the colloidal structure of the ice cream mixture into the soft-solid ice cream. in-situ starting from the experimental cooling curve registered inside the SSHE and it was identified as the point corresponding to the highest temperature reached after the first supercooling event. A re-parameterized Gompertz’s function was advantageously used to mathematically describe the water crystallization kinetics together with some key kinetic properties. Time-temperature onsets discriminating heterogeneous nucleation, ice growth and air incorporation were objectively determined by performing the first and second-order derivative analysis of the re-parameterized Gompertz’s model. Composition and freezing properties were treated as technological properties of the ice cream mixtures and were used as input variables in the multivariate analysis to classify the ice cream mixtures as a function of their ability to produce high-quality ice creams. Statistical models were developed and validated to predict the final overrun, ice cream melting rate and some sensory attributes such as appearance, cool sensation, sweetness, meltdown, flavor intensity and flavor persistence.For the first time, the initial freezing point was numerically individuated The proposed methodology represents a powerful and science-based approach overcoming the drawbacks of the usual methods based on trial-and-error assessment for setting ice cream formulations and freezing time. The numerical analysis of the experimental time-temperature profiles acquired under dynamic freezing conditions could provide an affordable decision-tool to opportunely select the starting ice cream mixture formulations able to obtain the desired quality properties in the final product. It also represents the first step to build robust predictive models of the ice cream quality indices to be transferred at the industrial level with the perspective to develop a computer-aided tool able to support the managing of the freezing time during batch ice cream production. Obviously, the successive step is to collect a large data set of cooling data accounting for a greater number of ice cream ingredients and their combinations.


Thus, lateral inhibition is conditional on activity in the principal neurons connected to a granule cell and tightly intertwined with recurrent inhibition. This notion allows us to infer that lateral inhibition between principal neurons occurs “on demand,” i.e., selectively on coactive mitral and tufted cells, and thus can provide directed, dynamically switched lateral inhibition in a sensory system with 1000 input channels organized in glomerular columns. The mechanistic underpinnings of this hypothesis concur with findings from odor discrimination behavior in mice with synaptic proteins deleted in granule cells. In summary, our hypothesis explains the unusual microcircuit of the granule cell reciprocal spine as a means of olfactory combinatorial coding.The role of granule cells in olfactory processing is surrounded by several enigmatic observations, such as the purpose of reciprocal spines and the mechanisms for GABA release, the apparently low firing activity and recurrent inhibitory drive of granule cells, the missing proof for functional reciprocal connectivity, and the apparently negligible contribution to lateral inhibition. Here, we summarize recent results with regard to both the mechanisms of GABA release and the behavioral relevance of granule cell activity during odor discrimination. We outline a novel hypothesis that has the potential to resolve most of these enigmas and allows further predictions on the function of granule cells in odor processing. Briefly, recent findings imply that GABA release from the reciprocal spine requires a local spine action potential and the cooperative action of NMDA receptors and high voltage-activated Ca The role of granule cells in olfactory processing is surrounded by several enigmatic findings  in more detail, odorant molecules bind to olfactory receptor neurons in the nasal epithelium. The axons of these receptor neurons terminate in glomeruli of the olfactory bulb (OB) where they interact with local interneurons and the apical dendritic tufts of the bulbar projection neurons, the mitral and tufted cells (MTC), which in turn send their axons mostly to olfactory cortical areas  recurrent inhibition, which refers to interneuron feedback onto an exciting MTC, and (2) lateral inhibition, which refers to interneuron feedforward onto MTCs other than the exciting MTC. GCs, the most numerous neuron population of the OB, participate in both recurrent and lateral inhibition of MTCs at the level of the EPL, where they receive glutamatergic synaptic input from MTC lateral dendrites which they provide with reciprocal GABAergic output. Axonless GCs feature basal dendrites and an apical dendrite, both spinous, with the apical dendrite extending from the granule cell layer (GCL) into the EPL and contacting MTC lateral dendrites via large spines that house the reciprocal synapses , but did not provide data for an involvement of voltage-gated Na+ (NaV) channels as in “classical” axonal release  in MTCs, the recurrent rIPSPs were strongly reduced. These results demonstrated that an AP was required for GABA release at the reciprocal synapse, just like neurotransmitter release from regular axonal presynaptic terminals  that in turn activate HVACCs. This mechanism relies on the tight electrical isolation of the spine head by the high spine neck resistance, boosting the AMPAR-mediated EPSP  a rapid relief of Mg2+ block  during the repolarization phase of the AP, the driving force for Ca2+ entry through HVACCs will strongly increase SPINE will closely resemble that of the spine spike  unitary release upon single MTC input; (2) the same spine was already activated within the NMDAR memory time window and the subsequent glutamate release generates recurrent inhibition as described above, e.g., during a theta burst of an MTC; (3) the GC fires an action potential that enters all spines , but GABA is only released from those spines that were activated during the NMDAR memory time window, thereby generating lateral inhibition. In essence, short MTC bursts will facilitate recurrent inhibition and lateral inhibition will mostly affect MTCs that are currently active or were active very recently.r_GABA from spiking GCs remains in the absence of glutamate, which is then amplified by the large number of stimulated GCs. Alternatively, the stimulation activates other, yet unknown inhibitory inputs to MCs, e.g., from deep short-axon cells or axons of EPL interneurons  The involved GCs have to be excited sufficiently to generate a non-local spike  or global spike. Evidence for global GC spiking upon uniglomerular activation in vitro has been observed by several groups, including our own  Moreover, because of the necessary activation of the NMDARs in the GABAergic active zone, there will be only little GC-mediated lateral inhibition upon activation of a single column because there is no glutamate provided at the lateral spines  to enhance recurrent inhibition. Non-topographical lateral interactions in the bulb have been proposed already previously by others based on functional evidence and simulations  by Arevian et al. ; one “seActivity dependence is also reflected in the labeling of glomerular columns spanning all layers of the bulb by markers of neuronal activity  of the apical dendrite or involve local dendritic spikes and will therefore not be visible to recordings from the GC soma. Furthermore, this spiking activity may also remain undetected in field potential recordings, because the limited space within which currents will flow might not suffice to generate sources and sinks far enough apart to be detected. In our view, global spikes mostly serve to enhance (but not trigger) a GC’s output across coactive columns.GCs do not prevent MC spiking but influence MC spiking frequency and therewith could contribute to the synchronization of coactive columns. In this scenario, recurrent inhibition is inherently linked to lateral inhibition.According to the model proposed here, lateral inhibition does exist, but predominantly between coactive columns and always in combination with recurrent inhibition, making it easy to miss experimentally.V1.2 subunit in mouse GCs, consistently affected odor discrimination time, such that manipulations that both should reduce GABA release  result in decreased recurrent inhibition and a slowed odor discrimination. Conversely, the GluA2 KO manipulation should increase GABA release  and indeed increased recurrent inhibition and accelerated odor discrimination. Since according to our hypothesis lateral inhibition is tightly related to recurrent inhibition, it is likely to be affected in a similar manner. Hence, our framework is further closing the loop from molecules to synapses, networks, and behavior in olfactory bulb function.Manipulations of essential components of the reciprocal microcircuit, i.e., deletion of the glutamate receptor subunits GluA2 or GluN1 and knockdown of the sodium channel Na