Enzymatic Hydrolysis of Salmon Frame Proteins Using a Sequential Batch Operational Strategy: An Improvement in Water-Holding Capacity.

The meat industry uses phosphates to improve the water-holding capacity (WHC) of meat products, although excess phosphates can be harmful to human health. In this sense, protein hydrolysates offer an alternative with scientific evidence of improved WHCs. Salmon frames, a byproduct rich in protein, must be processed for recovery. Enzymatic technology allows these proteins to be extracted from muscle, and the sequential batch strategy significantly increases protein nitrogen extraction. This study focused on evaluating the WHC of protein hydrolysates from salmon frames obtained through double- and triple-sequential batches compared to conventional hydrolysis. Hydrolysis was carried out for 3 h at 55 °C with 13 mAU of subtilisin per gram of salmon frames. The WHC of each hydrolysate was measured as the cooking loss using concentrations that varied from 0 to 5% (w/w) in the meat matrix. Compared with those obtained through conventional hydrolysis, the hydrolysates obtained through the strategy of double- and triple-sequence batches demonstrated a 55% and 51% reduction in cooking loss, respectively, when they were applied from 1% by weight in the meat matrix. It is essential to highlight that all hydrolysates had a significantly lower cooking loss (p ≤ 0.05) than that of the positive control (sodium tripolyphosphate [STPP]) at its maximum allowable limit when applied at a concentration of 5% in the meat matrix. These results suggest that the sequential batch strategy represents a promising alternative for further improving the WHC of hydrolysates compared to conventional hydrolysis. It may serve as a viable substitute for polyphosphates.

Application of CO2-Laser Micro-Perforation Technology to Freeze-Drying Whole Strawberry (Fragaria ananassa Duch.): Effect on Primary Drying Time and Fruit Quality.

Freeze-drying (FD) processing preserves foods by combining the most effective traditional technologies. FD conserves the structure, shape, freshness, nutritional/bioactive value, color, and aroma at levels similar to or better than those of refrigerated and frozen foods while delivering the shelf-stable convenience of canned/hot-air-dehydrated foods. The mass transfer rate is the essential factor that can slow down the FD process, resulting in an excessive primary drying time and high energy consumption. The objective of this study was to reduce the FD processing time using CO2 laser technology to improve product competitiveness in the preservation of whole strawberries. The research process consisted of the selection and characterization of fresh strawberries, followed by preparation, pre-treatment, freeze-drying, a primary drying time assessment, and a quality comparison. Experiments were carried out using strawberries without micro-perforation and with five and eight micro-perforations. Quality parameters were determined for fresh, frozen/thawed, and freeze-dried/rehydrated strawberries. It was found that the primary drying time can be significantly reduced by 20% (95% CI) from 26.7 h for non-perforated fruits to 22.3 h when five micro-perforations are made on each strawberry. The quality parameters used to evaluate the strawberries did not show significant differences when comparing frozen/thawed fruits with freeze-dried/rehydrated fruits. The experiments conducted in this study showed that freeze-drying may efficiently compete with freezing technology when processing whole strawberries.

Effect of Adding Bovine Skin Gelatin Hydrolysates on Antioxidant Properties, Texture, and Color in Chicken Meat Processing.

(1) Background: Phosphates are used in the food industry to improve water retention and product quality. However, when consumed in excess, they can be harmful to health. Instead, bovine skin gelatin hydrolysates present health benefits such as being a rejuvenating agent, stimulating collagen production, and improving food quality, in addition to being a source of protein. The effect of the addition of bovine skin gelatin hydrolysates on the texture and color of thermally processed chicken meat (boiled type) and antioxidant activity was evaluated. (2) Methods: Hydrolysates were prepared with subtilisin with the degree of hydrolysis being 6.57 and 13.14%, which were obtained from our previous study. (3) Results: The hydrolysates improved the firmness of the meat matrix compared to the control. Additionally, the hydrolysate with a 13.14% degree of hydrolysis reached the same firmness (p > 0.05) as the commercial ingredient sodium tripolyphosphate at its maximum limit allowed in the food industry when it was applied at 5% (w/w meat) in the meat matrix, improving firmness over the control by 63%. Furthermore, both hydrolysates reached a similar color difference to sodium tripolyphosphate at its maximum allowed limit when applied at a concentration of 2% (w/w meat). Additionally, it was found that these hydrolysates obtained the same antioxidant activity as sodium tripolyphosphate, capturing free radicals at 10%. (4) Conclusion: The findings of this study suggest that bovine skin gelatin hydrolysates can be applied as an ingredient with functional properties, being an alternative to phosphates to improve the quality of meat products.

Modeling Tool for Studying the Influence of Operating Conditions on the Enzymatic Hydrolysis of Milk Proteins.

Systematic modeling of the enzymatic hydrolysis of milk proteins is needed to assist the study and production of partially hydrolyzed milk. The enzymatic hydrolysis of milk proteins was characterized and evaluated as a function of the temperature and protease concentration using Alcalase, Neutrase and Protamex. Modeling was based on the combination of two empirical models formed by a logarithmic and a polynomial equation to correlate the kinetic constants and the operating conditions. The logarithmic equation fitted with high accuracy to the experimental hydrolysis curves with the three proteases (R2 > 0.99). The kinetic constants were correlated with the operating conditions (R2 > 0.97) using polynomial equations. The temperature and protease concentration significantly affected the initial rate of hydrolysis, i.e., the kinetic constant a, while the kinetic constant b was not significantly affected. The values for the kinetic constant a were predicted according to the operating conditions and they were strongly correlated with the experimental data (R2 = 0.95). The model allowed for a high-quality prediction of the hydrolysis curves of milk proteins. This modeling tool can be used in future research to test the correlation between the degree of hydrolysis and the functional properties of milk hydrolysates.

Assessing the Enzymatic Hydrolysis of Salmon Frame Proteins through Different By-Product/Water Ratios and pH Regimes.

The enzymatic hydrolysis of fish by-product proteins is traditionally carried out by mixing ground by-products with water. In addition, pH control is used to avoid pH drops. Higher costs are involved due to the use of pH control systems and the consequent energy cost in the drying stage. This work aimed to evaluate the effect of these conditions on the hydrolysis of salmon frame (SF) proteins, including the SF hydrolysis without added water. SF hydrolysis by subtilisin at 50, 75, and 100% SF under different pH regimes were evaluated by released α-amino (α-NH) groups, total nitrogen, degree of hydrolysis, and estimated peptide chain length (PCL) at 55 °C. The concentration of released α-NH groups was higher in the conditions with less added water. However, the nitrogen recovery decreased from 50 to 24% at 50 and 100% SF, respectively. Changing the SF/water ratio had a more significant effect than changing the pH regime. Estimated PCL changed from 5-7 to 7-9 at 50 and 100% SF, respectively. The operating conditions affected the hydrolysis performance and the molecular characteristics of the hydrolysate.

Variable Retort Temperature Profiles (VRTPs) and Retortable Pouches as Tools to Minimize Furan Formation in Thermally Processed Food.

Furan and its derivates are present in a wide range of thermally processed foods and are of significant concern in jarred baby and toddler foods. Furan formation is attributed to chemical reactions between a variety of precursors and a high processing temperature. Also, some kinetic models to represent its formation in different food materials have been studied and could predict the furan formation under simulated operating conditions. Therefore, this review aims to analyze and visualize how thermally processed foods might be improved based on optimal control of processing temperature and package design (e.g., retort pouches) to diminish furan formation and maximize quality retention. Many strategies have been studied and applied to reduce furan levels. However, an interesting approach that has not been explored is the thermal process design based on optimum variable retort temperature profiles (VRTPs) and the use of retortable pouches considering the microstructural changes of food along the process. The target of process optimization would be developed by minimizing the microstructural damage of the food product. It could be possible to reduce the furan level and simultaneously preserve the nutritional value through process optimization.

Native Yeasts and Lactic Acid Bacteria Isolated from Spontaneous Fermentation of Seven Grape Cultivars from the Maule Region (Chile).

Grapes are a source of native yeasts and lactic acid bacteria (LAB); however, the microbial make up is dependent on the grape cultivar and the regional growth conditions. Therefore, the aim of this study was to characterize the yeast and LAB in seven grape cultivars cultivated in Chile. Grape juices were fermented at 25 °C for 7 days. Samples were collected to analyze sugar, organic acids, and ethanol. Microbial evolution was measured with culture-dependent and molecular approaches. Then, a native isolated Candida oleophila was selected for further sequential fermentations with Saccharomyces cerevisiae. The grape cultivars in the Maule showed a diversity of non-Saccharomyces yeasts, with a greater diversity observed at the beginning of the fermentation. However, species from the Hansenasporia, Metschnikowia, Torulaspora, Lachancea, and Candida genera were detected after 7 days, suggesting tolerance to environments rich in ethanol, capability may be associated to the terroir studied, which is characterized by torrid weather and antique and traditional vineyards. The alcoholic fermentation negatively impacted the LAB population, and after 7 days only Leuconostoc mesenteroides was isolated. In the sequential fermentations, C. oleophila was able to produce fermented grape juices with <1.5 g/L glucose, 12.5% (v/v) alcohol, and low concentrations of malic (<1.00 g/L) and succinic (2.05 g/L) acids, while acetic acid reached values >0.3 (g/L). To our knowledge this is the first time C. oleophila has been reported as a potential starter culture for wine production. However, more studies are necessary to fully characterize the potential of C. oleophila on wine attributes.

Effects of Enzymatic Hydrolysis on the Functional Properties, Antioxidant Activity and Protein Structure of Black Soldier Fly (Hermetia illucens) Protein.

The effects of chemical protein extraction, and enzymatic hydrolysis with Alcalase, papain and pepsin, on the functional properties, antioxidant activity, amino acid composition and protein structure of black soldier fly (H. illucens) larval protein were examined. Alcalase hydrolysates had the highest degree of hydrolysis (p < 0.05), with the highest hydrolysate and oil fraction yield (p < 0.05). Pepsin hydrolysates showed the lowest oil holding capacity (p < 0.05), whereas no significant differences were observed among other enzymes and protein concentrates (p > 0.05). The emulsifying stability and foam capacity were significantly lower in protein hydrolysates than protein concentrate (p < 0.05). The antioxidant activity of protein hydrolysates from protein concentrate and Alcalase was higher than that with papain and pepsin (p < 0.05), owing to the higher hydrophobic amino acid content. Raman spectroscopy indicated structural changes in protein α-helices and ß-sheets after enzymatic hydrolysis.

Prediction of the Limiting Flux and Its Correlation with the Reynolds Number during the Microfiltration of Skim Milk Using an Improved Model.

Limiting flux (JL) determination is a critical issue for membrane processing. This work presents a modified exponential model for JL calculation, based on a previously published version. Our research focused on skim milk microfiltrations. The processing variables studied were the crossflow velocity (CFV), membrane hydraulic diameter (dh), temperature, and concentration factor, totaling 62 experimental runs. Results showed that, by adding a new parameter called minimum transmembrane pressure, the modified model not only improved the fit of the experimental data compared to the former version (R2 > 97.00%), but also revealed the existence of a minimum transmembrane pressure required to obtain flux (J). This result is observed as a small shift to the right on J versus transmembrane pressure curves, and this shift increases with the flow velocity. This fact was reported in other investigations, but so far has gone uninvestigated. The JL predicted values were correlated with the Reynolds number (Re) for each dh tested. Results showed that for a same Re; JL increased as dh decreased; in a wide range of Re within the turbulent regime. Finally, from dimensionless correlations; a unique expression JL = f (Re, dh) was obtained; predicting satisfactorily JL (R2 = 84.11%) for the whole set of experiments.

Collagen as a source of bioactive peptides: a bioinformatics approach

BACKGROUND: Collagen is the most abundant protein in animals and can be obtained from residues of the food industry. Its hydrolysate has many desirable properties that make it suitable as an additive in foods and cosmetics, or as a component of scaffold materials to be used in biomedicine. RESULTS: We report here the characterization of type I collagen from five different sources, namely bovine, porcine, chicken, trout and salmon, as well as their hydrolysates by means of bioinformatics tools. As expected, the results showed that bovine and porcine collagen, as well as trout and salmon collagen, can be used interchangeably due to their high identity. This result is consistent with the evolution of proteins with highly identical sequences between related species. Also, 156 sequences were found as potential bioactive peptides, 126 from propeptide region and 30 from the central domain, according to the comparison with reported active sequences. CONCLUSIONS: Collagen analysis from a bioinformatic approach allowed us to classify collagen from 5 different animal sources, to establish its interchangeability as potential additive in diverse fields and also to determine the content of bioactive peptides from its in silico hydrolysis.

Enzyme Bioprospection of Marine-Derived Actinobacteria from the Chilean Coast and New Insight in the Mechanism of Keratin Degradation in Streptomyces sp. G11C.

Marine actinobacteria are viewed as a promising source of enzymes with potential technological applications. They contribute to the turnover of complex biopolymers, such as pectin, lignocellulose, chitin, and keratin, being able to secrete a wide variety of extracellular enzymes. Among these, keratinases are a valuable alternative for recycling keratin-rich waste, which is generated in large quantities by the poultry industry. In this work, we explored the biocatalytic potential of 75 marine-derived actinobacterial strains, focusing mainly on the search for keratinases. A major part of the strains secreted industrially important enzymes, such as proteases, lipases, cellulases, amylases, and keratinases. Among these, we identified two streptomycete strains that presented great potential for recycling keratin wastes-Streptomyces sp. CHA1 and Streptomyces sp. G11C. Substrate concentration, incubation temperature, and, to a lesser extent, inoculum size were found to be important parameters that influenced the production of keratinolytic enzymes in both strains. In addition, proteomic analysis of culture broths from Streptomyces sp. G11C on turkey feathers showed a high abundance and diversity of peptidases, belonging mainly to the serine and metallo-superfamilies. Two proteases from families S08 and M06 were highly expressed. These results contributed to elucidate the mechanism of keratin degradation mediated by streptomycetes.

Prevalence of retrovirus, hepatitis B and D infection in the Matsés ethnic group in Loreto, Peru. / Prevalencia de infección por los virus de la hepatitis B, D y por retrovirus en la etnia Matsés (Loreto, Perú).

Observational, cross-sectional, populational study to determine the prevalence of infection by hepatitis B virus (HBV), hepatitis D virus (HDV), human immunodeficiency virus (HIV) and human T-lymphotropic virus type 1 and 2 (HTLV-1/2) in the Matsés ethnic group, after immunization against HBV. ELISA and qPCR tests were used in 963 residents. The prevalence of HBsAg, Anti-HBc and Anti-HBs was 3.32%, 36.03% and 58.67% respectively. In 3.1% of the population the viral load was greater than 2000 IU/mL. In children under 10 years, the prevalence of HBsAg and anti-HBc was 0.0% and 2.6%, respectively, while protective antibodies were found in 94.4%. The prevalence of HIV and HTLV-1/2 infection was 1.5% and 0.6%, respectively. It is therefore concluded that there are low rates of HBV and HDV infection in the Matsés child population. Likewise, the presence of HIV and HTLV-1/2 infection is confirmed.

Para determinar la prevalencia de infección por los virus de la hepatitis B y D (VHB y VHD, respectivamente), VIH y HTLV-1/2 en la etnia matsés, después de la inmunización contra el VHB se realizó un estudio transversal y poblacional, utilizando pruebas de ELISA y qPCR en 963 pobladores. Las prevalencias de HBsAg, anti-HBc y anti-HBs fueron 3,3%, 36,0% y 58,7%, respectivamente. En el 3,1% de la población la carga viral fue mayor a 2000 UI/mL. En menores de 10 años, la prevalencia de HBsAg y anti-HBc fue 0,0% y 2,6%, respectivamente, mientras que en el 94,4% se encontraron anticuerpos protectores. La prevalencia de infección por el VIH y el HTLV-1/2 fue 1,5% y 0,6%, respectivamente. Se concluye que existen tasas bajas de infección por el VHB y el VHD en la población infantil de la etnia matsés. Asimismo, se confirma la presencia de infección por el VIH y el HTLV-1/2.

Bovine skin gelatin hydrolysates as potential substitutes for polyphosphates: The role of degree of hydrolysis and pH on water-holding capacity.

The effects of adding bovine skin gelatin hydrolysate obtained with subtilisin, on water-holding capacity (WHC), in a thermally processed chicken meat model, were investigated. Hydrolysates with different degrees of hydrolysis (DH) (6.57%, 13.14%, and 26.28%) were prepared. The results showed that all the tested hydrolysates improved water retention in the meat matrix. The hydrolysate with 26.28% DH showed similar behavior throughout the full range of concentrations [0% to 5% w/w] compared to that of the positive control (sodium tripolyphosphate [STPP]). In addition, the other hydrolysates [6.57% DH and 13.14% DH at 3% and 2.5% w/w concentrations, respectively] showed behaviors that coincided with that of STPP at its maximum limit allowed. A correlation was observed between the WHC and the pH of the meat samples treated with each hydrolysate or STPP. In addition, it was found that the WHC of the hydrolysates was due to increases in pH and the specific effects of the hydrolysate beyond the typical effects of pH and ionic strength in meat systems. The solubility of all hydrolysates was high (>90%). In conclusion, bovine skin gelatin hydrolysates could serve as an alternative to polyphosphates to improve water retention and the functional properties of thermally processed meat products. PRACTICAL APPLICATION: This study investigated the effects of adding bovine skin gelatin hydrolysate obtained with subtilisin on water-holding capacity (WHC) in a thermally processed chicken meat model. It was found that the hydrolysis of bovine skin gelatin with subtilisin can replace chemical products harmful to health, such as STPP, in terms of water-holding capacity. Therefore, bovine skin gelatin hydrolysate can be used as an ingredient in the formulation of thermally processed meat products.

Prevalencia de infección por los virus de la hepatitis B, D y por retrovirus en la etnia Matsés (Loreto, Perú) / Prevalence of retrovirus, hepatitis B and D infection in the Matsés ethnic group in Loreto, Peru

RESUMEN Para determinar la prevalencia de infección por los virus de la hepatitis B y D (VHB y VHD, respectivamente), VIH y HTLV-1/2 en la etnia matsés, después de la inmunización contra el VHB se realizó un estudio transversal y poblacional, utilizando pruebas de ELISA y qPCR en 963 pobladores. Las prevalencias de HBsAg, anti-HBc y anti-HBs fueron 3,3%, 36,0% y 58,7%, respectivamente. En el 3,1% de la población la carga viral fue mayor a 2000 UI/mL. En menores de 10 años, la prevalencia de HBsAg y anti-HBc fue 0,0% y 2,6%, respectivamente, mientras que en el 94,4% se encontraron anticuerpos protectores. La prevalencia de infección por el VIH y el HTLV-1/2 fue 1,5% y 0,6%, respectivamente. Se concluye que existen tasas bajas de infección por el VHB y el VHD en la población infantil de la etnia matsés. Asimismo, se confirma la presencia de infección por el VIH y el HTLV-1/2.

ABSTRACT Observational, cross-sectional, populational study to determine the prevalence of infection by hepatitis B virus (HBV), hepatitis D virus (HDV), human immunodeficiency virus (HIV) and human T-lymphotropic virus type 1 and 2 (HTLV-1/2) in the Matsés ethnic group, after immunization against HBV. ELISA and qPCR tests were used in 963 residents. The prevalence of HBsAg, Anti-HBc and Anti-HBs was 3.32%, 36.03% and 58.67% respectively. In 3.1% of the population the viral load was greater than 2000 IU/mL. In children under 10 years, the prevalence of HBsAg and anti-HBc was 0.0% and 2.6%, respectively, while protective antibodies were found in 94.4%. The prevalence of HIV and HTLV-1/2 infection was 1.5% and 0.6%, respectively. It is therefore concluded that there are low rates of HBV and HDV infection in the Matsés child population. Likewise, the presence of HIV and HTLV-1/2 infection is confirmed.

Recent advances and perspectives of ultrasound assisted membrane food processing.

Power ultrasound (US) transmits substantial amounts of small mechanical movements serving for particle detaching in membrane filtrations. This topic has been reviewed in recent years mainly focused on the mechanisms by which the flux is improved under specific processing conditions. US also been shown to improve food quality by changing physical properties and modifying the activity of enzymes and microorganisms. Surprisingly, limited information exists regarding on how the application of US results in terms of process and quality during membrane filtration of complex matrices such as liquid foods. This review highlights the recent advances in the use of US in membrane filtration processes focused in the manufacturing of foodstuffs and food ingredients, and perspectives of novel hybrid membrane-US systems that may be quite interesting for this field. The application of US in food membrane processing increases the flux, but the lack of standardization regarding to experimental conditions, make suitable comparisons impossible. In this sense, careful attention must be paid regarding to the ultrasonic intensity (UI), the membrane configuration and type of transducers and volume of the treated solution. Dairy products are the most studied application of US membrane food processing, but research has been mainly focused on flux enhancement; hitherto there have been no reports of how operational variables in these processes affect critical aspects such as quality and food safety. Also, studies performed at industrial scale and economical assessments are still missing. Application of US combined with membrane operations such as reverse osmosis (RO), forward osmosis (FO) and enzyme membrane bioreactors (EMBR) may result interesting for the production of value-added foods. In the perspective of the authors, the stagnation of the development of acoustic filtration systems in food is due more to a prejudice on this subject, rather than actual impedance due to the lack of technological development of transducers. This later has shown important advances in the last years making them suitable for tailor made applications, thus opening several research opportunities to the food engineering not yet explored.

Freeze-Drying of Blueberries: Effects of Carbon Dioxide (CO2) Laser Perforation as Skin Pretreatment to Improve Mass Transfer, Primary Drying Time, and Quality.

Freeze-dried berry fruits are generally consumed as they are, whole and without peeling or cutting, as the conservation of their original shape and appearance is often desired for the final product. However, usually, berries are naturally wrapped by an outer skin that imparts a barrier to vapor flow during freeze-drying, causing berry busting. Photo-sequence, experimental, and theoretical methodologies were applied to evaluate the application of CO2 laser microperforations to blueberry skin. Under the same set of freeze-drying conditions, blueberries with and without perforations were processed. The results showed that the primary drying time was significantly reduced from 17 ± 0.9 h for nontreated berries to 13 ± 2.0 h when nine microperforations per berry fruit were made. Concomitantly, the quality was also significantly improved, as the percentage of nonbusted blueberries at the end of the process increased from an average of 47% to 86%. From a phenomenological perspective, the analysis of the mass transfer resistance of nontreated fruits, in agreement with reported studies, showed a Type II curvature, with a sharp decrease at low time, followed by a linear increase. In contrast, blueberries with nine perforations depicted a Type III regime, with a saturation curvature toward the time axis. It was demonstrated that CO2-laser microperforation has high potential as a skin pretreatment for the freeze-drying of blueberries.

Calculation of statistic estimates of kinetic parameters from substrate uncompetitive inhibition equation using the median method.

We provide initial rate data from enzymatic reaction experiments and tis processing to estimate the kinetic parameters from the substrate uncompetitive inhibition equation using the median method published by Eisenthal and Cornish-Bowden (Cornish-Bowden and Eisenthal, 1974; Eisenthal and Cornish-Bowden, 1974). The method was denominated the direct linear plot and consists in the calculation of the median from a dataset of kinetic parameters Vmax and Km from the Michaelis-Menten equation. In this opportunity we present the procedure to applicate the direct linear plot to the substrate uncompetitive inhibition equation; a three-parameter equation. The median method is characterized for its robustness and its insensibility to outlier. The calculations are presented in an Excel datasheet and a computational algorithm was developed in the free software Python. The kinetic parameters of the substrate uncompetitive inhibition equation Vmax , Km and Ks were calculated using three experimental points from the dataset formed by 13 experimental points. All the 286 combinations were calculated. The dataset of kinetic parameters resulting from this combinatorial was used to calculate the median which corresponds to the statistic estimator of the real kinetic parameters. A comparative statistical analyses between the median method and the least squares was published in Valencia et al. [3].

Application of the median method to estimate the kinetic constants of the substrate uncompetitive inhibition equation.

In 1974, Eisenthal and Cornish-Bowden published the direct linear plot method, which used the median to estimate the Vmax and Km from a set of initial rates as a function of substrate concentrations. The robustness of this non-parametric method was clearly demonstrated by comparing it with the least-squares method. The authors commented that the method cannot readily be generalized to equations of more than two parameters. Unfortunately, this comment has been misread by other authors. Comments such as "this method cannot be extended directly to equations with more than two parameters" were found in some publications. In addition, recently, the most drastic comment was published: "this method cannot be applied for the analysis of substrate inhibition." Given all of these presumptions, we have been motivated to publish a demonstration of the contrary: the median method can be applied to more than two-parameter equations, using as an example, the substrate uncompetitive inhibition equation. A computer algorithm was written to evaluate the effect of simulated experimental error of the initial rates on the estimation of Vmax, Km and KS. The error was assigned to different points of the experimental design. Four different KS/Km ratios were analyzed with the values 10, 100, 1000 and 10,000. The results indicated that the least-squares method was slightly better than the median method in terms of accuracy and variance. However, the presence of outliers affected the estimation of kinetic constants using the least-squares method more severely than the median method. The estimation of KS using the median method to estimate 1/KS was much better than the direct estimation of KS, causing a negative effect of non-linearity of KS in the kinetic equation. Considering that the median method is free from the assumptions of the least-squares method and the arbitrary assumptions implicit in the linearization methods to estimate the kinetic constants Vmax, Km and KS from the substrate uncompetitive inhibition equation, the median method is highly superior to all published methods, including the non-linear regression by least squares. We concluded that the median method can be applied to the substrate uncompetitive inhibition equation and other equations with more than two parameters. In addition, as we can project, the median method is the most reliable and robust method for the estimation of kinetic parameters from enzyme kinetic models.