Immunomodulatory and Antioxidant Properties of Ipomoea batatas Flour and Extracts Obtained by Green Extraction.

Sweet potato (SP), Ipomoea batatas Lam, belongs to the Convolvulaceae family. It produces edible storage roots. Currently, orange varieties contribute to improving food systems and managing vitamin A deficiency. Processing of this food crop into flour allows better conservation. However, nutrition health data regarding SP flour obtained by green extraction remains scarce. In this study, we therefore explored its phytochemistry and its associated bioactivity potential for human health. We analyzed the nutritional composition of orange flesh sweet potato (OFSP) flour and assessed the antioxidant (free radical scavenging) and immunomodulatory (on inflammatory murine macrophages) properties of the extract. More specifically, we measured the impact of OFSP flour extract on mediators such as Nitric Oxide (NO) and the production of pro-inflammatory cytokines such as Interleukin-6 (IL-6), Tumor Necrosis Factor alpha (TNF-alpha), Monocyte Chemoattractant Protein-1 (MCP-1), and Prostaglandin-E2 (PGE-2). Our results indicated significant fiber, mineral, beta-carotene, and polyphenols content in the extracts, and antioxidant and immunomodulatory bioactivities were also demonstrated with a concentration-dependent inhibition of cytokine production. Taken together, our results suggest that Ipomoea batatas flour could, in addition to being a good source of energy and beta-carotene provitamin A, constitute a food of interest for the prophylaxis of metabolic diseases associated with an underlying low-grade inflammatory state.

Use of an experimental design to optimise the saponification reaction and the quantification of vitamins A1 and A2 in whole fish.

In ASEAN countries, small freshwater fish species contribute to the nutritional needs of people with few livelihoods by providing them with significant amounts of protein, fat, vitamins and minerals. Some species are eaten whole (with their organs, skin, bones, head and eyes). To estimate the vitamin A content of these foods, conventional saponification has been applied but has not been able to fully release the retinol. Our objective was to optimise the conditions of vitamin A saponification in whole fish to have a reliable estimate of their contribution to intakes. The effects of temperature and saponification time on the retinol quantification of whole fish were evaluated using a two-factor experimental design. Reaction time had a significant effect on the saponification of standard retinyl palmitate and whole fish (p≤0.05). For whole fish, the best conditions for the saponification were to heat the samples to 80 °C for 43 minutes. Under these conditions, the retinol is well liberated from the matrix and protected from degradation and isomerisation reactions. The time-temperature couple used is more intense than that recommended for quantifying vitamin A in milk or enriched margarines. The protective effect of the food matrix against the release of retinol is evident. Vitamin A2 alcohol (3,4-didehydroretinol) was detected in five species and the overall vitamin A contents ranged from 9.6 to 737.5 µg RE/100 g in species frequently consumed in Cambodia. The two species of small fish consumed whole were the ones that contained significantly more vitamin A among the ten tested (p≤0.05). Highlights: Vitamin A2 alcohol was quantified in five fish species. The official saponification partially released retinol in whole fish. The optimised reaction required heating the sample to 80 °C for 43 min.

Antifungal and antimycotoxic activities of 3 essential oils against 3 mycotoxinogenic fungi.

Fungal toxins can have various adverse health effects, including carcinogenic, teratogenic or hepatotoxic impacts. In addition, fungal alteration has also a negative impact on agricultural plant production. The use of chemical fungicides to control mycotoxin contamination is increasingly controversial and regulated. More environmentally friendly methods are therefore being explored. Essential oils, as compounds extracted from plants, are liquids whose specific aromatic compounds give each essential oil its own unique characteristics. Due to their rich chemical composition, essential oils (EOs) have many interesting properties, including antifungal activities. The objective of the present study was to analyze volatile chemical composition of EOs (Cymbopogon schoenanthus, Cymbopogon nardus and Eucalyptus camaldulensis) by GC/MS and to investigate their effects on the growth, sporulation and mycotoxin production of Aspergillus flavus, Aspergillus carbonarius and Fusarium verticillioides (aflatoxin B1, ochratoxin A and fumonisin B1, respectively). In addition, EOs influence on aflatoxin B1 (AFB1) and fumonisin B1 (FB1) biosynthesis pathways was explored using real-time qRT-PCR. The results obtained in vitro, by direct contact with the EOs and by diffusion of their volatile compounds, showed that the essential oils had inhibitory effects on the growth and the production of mycotoxins of the 3 fungal strains and modified the expression of some toxin synthesis genes. We conclude that the recorded effects were dependent on the combined effects of the EOs type, the fungal strains and the doses studied.

Effect of Elevated Carbon Dioxide Exposure on Nutrition-Health Properties of Micro-Tom Tomatoes.

(1) Background: The anthropogenically induced rise in atmospheric carbon dioxide (CO2) and associated climate change are considered a potential threat to human nutrition. Indeed, an elevated CO2 concentration was associated with significant alterations in macronutrient and micronutrient content in various dietary crops. (2) Method: In order to explore the impact of elevated CO2 on the nutritional-health properties of tomato, we used the dwarf tomato variety Micro-Tom plant model. Micro-Toms were grown in culture chambers under 400 ppm (ambient) or 900 ppm (elevated) carbon dioxide. Macronutrients, carotenoids, and mineral contents were analyzed. Biological anti-oxidant and anti-inflammatory bioactivities were assessed in vitro on activated macrophages. (3) Results: Micro-Tom exposure to 900 ppm carbon dioxide was associated with an increased carbohydrate content whereas protein, minerals, and total carotenoids content were decreased. These modifications of composition were associated with an altered bioactivity profile. Indeed, antioxidant anti-inflammatory potential were altered by 900 ppm CO2 exposure. (4) Conclusions: Taken together, our results suggest that (i) the Micro-Tom is a laboratory model of interest to study elevated CO2 effects on crops and (ii) exposure to 900 ppm CO2 led to the decrease of nutritional potential and an increase of health beneficial properties of tomatoes for human health.

Flash vacuum expansion: an alternative with potential for Ataulfo and Manila mango processing.

Mangoes of the Manila and Ataulfo varieties are characterized by having a pleasant flavor and an attractive color of both the peel and pulp. However, due to their post-harvest physiology, they are highly perishable and very susceptible to physical damage, which greatly limits marketing these fruits. Therefore, it is necessary to evaluate technologies that preserve their organoleptic and nutritional properties. There is also an increasing demand of products that are as natural as possible where only physical processes are used in their preparation to reduce the use of chemical compounds. A technology that satisfies these demands is the flash vacuum-expansion (FVE) process. In Ataulfo mango, the FVE process increased soluble solids by approximately 9% and total phenolic content from 100.9 to 122.8 mg GAE/100 g of puree, which led to an increase in antioxidant capacity of the puree, as well as slightly improving color stability. However, further optimization of this method of processing mango is required.

In Vitro Tests for a Rapid Evaluation of Antidiabetic Potential of Plant Species Containing Caffeic Acid Derivatives: A Validation by Two Well-Known Antidiabetic Plants, Ocimum gratissimum L. Leaf and Musanga cecropioides R. Br. ex Tedlie (Mu) Stem Bark.

Plant bioactive extracts represent a major resource for identifying drugs and adjuvant therapy for type 2 diabetes. To promote early screening of plants' antidiabetic potential, we designed a four in vitro tests strategy to anticipate in vivo bioactivity. Two antidiabetic plants were studied: Ocimum gratissimum L. (Oc) leaf extract and Musanga cecropoides R. Br. ex Tedlie (Mu) stem bark extract. Chemical compositions were analyzed by LCMS and HPLC. Antidiabetic properties were measured based on (1) INS-1 cells for insulin secretion, (2) L6 myoblast cells for insulin sensitization (Glut-4 translocation), (3) L6 myoblast cells for protection against hydrogen peroxide (H2O2) oxidative stress (cell mortality), and (4) liver microsomial fraction for glucose-6-phosphastase activity (G6P). Oc extract increased insulin secretion and insulin sensitivity, whereas it decreased oxidative stress-induced cell mortality and G6P activity. Mu extract decreased insulin secretion and had no effect on insulin sensitivity or G6P activity, but it increased oxidative stress-induced cell mortality. Results were compared with NCRAE, an antidiabetic plant extract used as reference, previously characterized and reported with increased insulin secretion and insulin sensitivity, protection against oxidative stress, and decreased G6P activity. The proposed set of four in vitro tests combined with chemical analysis provided insight into the interest in rapid early screening of plant extract antidiabetic potential to anticipate pharmaco-toxicological in vivo effects.

Oxidative status of a yogurt-like fermented maize product containing phytosterols.

This work describes the formulation of a functional yogurt-like product based on fermented maize with added phytosterols and its oxidative stability during cold storage. The technological challenge was to stabilize 3.5% esterified phytosterols (between 2 and 3 g of free sterols) in a low-fat emulsion and to preserve the obtained product throughout processing and storage. The natural bioactive compounds: lutein, zeaxanthin, ß-cryptoxanthin, ß-carotene and γ-tocopherol were detected in the yogurt, and remained stable during 12 days of refrigeration. Higher content of C18:1 n-9 and C18:3 n-3 (six and ninefold, respectively) were obtained in samples with phytosterols. This was desirable from a nutritional point of view, but at the same time it induced lipid oxidation that was 1.4-fold higher in the product with phytosterols than in the controls. The use of a multivariate approach served to find descriptors which were related to treatments, and to explain their behavior over time.

Does drying preserve the nutritional quality of small freshwater fish without excessive concentrations of heavy metals?

The potential of drying to preserve the nutritional quality of a small freshwater fish Henicorhynchus siamensis was assessed. Drying time to reach moisture content and water activity of 10 g/100g and 0.65 ranged from 55 h at 50 °C to 20 h at 80 °C, respectively. Dried fish powder is rich in macronutrients (protein, lipid and ash) and essential minerals (calcium, phosphorus, iron and zinc) due to water removal and despite lipid loss. It is still rich in polyunsaturated fatty acids although docosahexaenoic acid was reduced except at 60°C. Vitamin A was rapidly degraded and manganese was concentrated at high level. However, mean score for the nutritional adequacy of the 15 nutrients (SAIN) and score of nutrients to limit (LIM) show that the fish powder can be used as a food ingredient for example in the formulation of fish snack or instant soup. With the abundance of Henicorhynchus siamensis, dried fish powder from this species could contribute to food security in Cambodia, especially vulnerable people in rural areas.

Nutrition, Healthcare Benefits and Phytochemical Properties of Cassava (Manihot esculenta) Leaves Sourced from Three Countries (Reunion, Guinea, and Costa Rica).

(1) Background: Manihot esculenta, cassava, is an essential food crop for human consumption in many parts of the world. Besides the wide use of its roots, cassava leaves have been used locally as green vegetables and for medicinal purposes. However, nutritional health data regarding cassava leaves is limited, therefore we investigated its composition and associated potential bioactivity interest for human health. (2) Methods: Cassava leaf bioactivity investigations focused on antioxidant properties (free radical scavenging) in association with immunomodulatory activities on inflammatory murine macrophages to measure the impact of cassava extract on the production of pro-inflammatory cytokines such as Interleukin-6, Tumor Necrosis Factor alpha, Monocyte Chemoattractant Protein-1, Prostaglandin-E2 and mediators such as nitric oxide. (3) Results: Antioxidant and immunomodulatory bioactivities were significant, with a concentration-dependent inhibition of cytokines production by inflammatory macrophages; (4) Conclusions: Taken together, our results tend to suggest that Manihot esculenta leaves might be underrated regarding the potential nutrition-health interest of this vegetal matrix for both human nutrition and prophylaxis of metabolic disease with underlying low grade inflammation status.

Differential Nutrition-Health Properties of Ocimum basilicum Leaf and Stem Extracts.

(1) Background: Ocimum basilicum L. is an aromatic medicinal plant of the Lamiaceae family known as sweet basil. It is used in traditional medicine for its beneficial effects on gastrointestinal disorders, inflammation, immune system, pyrexia or cancer among others. Ocimum basilicum (OB) leaf extracts contain many phytochemicals bearing the plant health effects but no reports is available on the potential bioactivity of stem extracts. Our investigation aimed at assessing the differential biological activity between basil leaf and stem to promote this co-product valorization. (2) Method: For this purpose we explored phytochemical composition of both parts of the plant. Antioxidant activity was evaluated through total polyphenol content measure, DPPH and ORAC tests. Anti-inflammatory markers on stimulated macrophages, including NO (nitric oxide), TNFa (tumor necrosis factor alpha), IL-6 (interleukin 6), MCP1 (monocyte attractant protein 1) and PGE-2 (prostaglandin E2), were evaluated. In addition, we investigated OB effects on jejunum smooth muscle contractility. (3) Results: OB extracts from leaves and stems demonstrated a different biological activity profile at the level of both antioxidant, anti-inflammatory and smooth muscle relaxation effects. (4) Conclusion: Taken together our results suggest that Ocimum basilicum extracts from co-product stems, in addition to leaves, may be of interest at the nutrition-health level with specific therapeutic potential.

Impact of storage on the functional characteristics of a fermented cereal product with probiotic potential, containing fruits and phytosterols.

The aim of this work was to study the changes in the functional characteristics of a fermented maize product containing fruits, and enriched with phytosterols. Functional characteristics (natural antioxidants and phytosterols content, in vitro antioxidant capacity and probiotic viability), lipid oxidation, and physicochemical parameters were investigated during 4 weeks of storage at 4 °C. The differences between one formulation elaborated with semi-skimmed powdered milk (Basic Product) and another with whey protein isolate (WPI Product) were evaluated. The content of polyphenols, carotenoids and tocopherols remained unchanged during the storage of both formulations. These compounds increased the antioxidant capacity in both products compared to the control formulation (without fruits), which was displayed along the whole storage period. The doses of phytosterols and the probiotic potential were maintained to the end of the storage period for both formulations. Basic and WPI products represent novel foods with desirable functional characteristics preserved during commercial storage.

Carotenoid absorption in rats fed with vacuum-fried papaya chips depends on processed food microstructure associated with saturated and unsaturated oils.

Many studies indicate that food matrix microstructure and type of dietary oil or fat play a key role in carotenoid absorption. Therefore, this work was designed to highlight the relationship between processed food microstructure and carotenoid absorption. This study aimed to evaluate the consumption of a carotenoid-rich fruit snack on lipid profile, glycemia and especially on carotenoid absorption/bioconversion in Wistar rats. Animals were fed with mixtures based on vacuum-fried papaya chips with either soy oil (PC-S) or palm oil (PC-P) during 7 days, receiving 0.29 mg lycopene/kg/day and 0.35 mg total carotenoids/kg/day. Lycopene and retinoids were analyzed in plasma and liver of rats by HPLC-DAD. Results showed that the consumption of mixtures based on papaya chips did not affect the lipid profile or glycemia in rat plasma, regardless the type of oil. Wide-field and confocal microscopy analyses of food matrix helped to understand why lycopene accumulation in the liver was higher (p < 0.05) in rats fed with PC-P (0.442 µg/g liver) than in those fed with PC-S (0.291 µg/g liver). A better dissolution of crystalloid lycopene was found in PC-P. Conversely, a higher bioconversion of provitamin A carotenoids was observed for soy products. The effect of type of oil was underlined by epifluorescence microscopy of papaya mixtures showing homogeneous and small lipid droplets for soy products. These results showed that PC-S could be recommanded as a healthy snack, being a source of provitamin A carotenoids and bioavailable lycopene in a diversified diet.

A kinetic study of carotenoid degradation during storage of papaya chips obtained by vacuum frying with saturated and unsaturated oils.

The aim of the study was to evaluate the degradation kinetics of carotenoids (CTs) in vacuum-fried papaya (Carica papaya L.) chips (PCs) during storage at four temperatures (15, 25, 35 and 45 °C) for 52 and 94 days for the two highest and lowest temperatures, respectively. Three treatments were applied to obtain the chips: chips with soy oil (24% lipids) and chips with palm oil (24% and 29% lipids). All the chips were packaged under air or nitrogen conditions. The CTs analyzed by HPLC-DAD were per order of content all-E-lycopene (LYC), Z-lycopene (Z-LYC), all-E-ß-carotene (BC), all-E-ß-cryptoxanthin (BCX) and Z-ß-carotene (Z-BC). The all-E-forms represented 80% of carotenoids in PCs. No significant carotenoid degradation was observed in the PCs packaged under nitrogen conditions during storage. For chips stored under air conditions, a second-order kinetic model best fitted the experimental data. Rate constants for LYC degradation were the lowest, while BCX and BC presented similar rate constants 4-23-fold higher depending on lipid composition. All Z-isomers degraded faster than all-E-forms, but Z-BC degraded only 2-4-fold faster than Z-LYC. All CTs followed Arrhenius temperature-dependency pattern and LYC showed the lowest activation energies (5-21 kJ/mol). A higher lipid content in the chips with palm oil enhanced the carotenoid retention in PCs. Moreover, a greater retention (p < 0.05) of CTs was observed in PCs with soy oil. The use of soy oil instead of palm oil increased the theoretical half-life (at 25 °C) by 2.2, 1.3 and 5.9-fold for BCX, BC and LYC, respectively. Packaging under nitrogen conditions and lipid composition may be considered to optimize the shelf life and carotenoid retention in PCs during storage.

Pectin structure and particle size modify carotenoid bioaccessibility and uptake by Caco-2 cells in citrus juices vs. concentrates.

Citrus pectin is known to influence carotenoid bioaccessibility and absorption in humans, but limited attention has been given to the influence of pectin structure related to the particle size from differentially processed citrus food matrices. In this context, this study aims to investigate the nutritional health benefits of an innovative Citrus clementina concentrate, which is a new citrus-based food made by cross-flow microfiltration. This concentrated citrus-based food was selectively enriched 8-fold in ß-cryptoxanthin (43-55 µg g-1) and ß-carotene (6-9 µg g-1) as well as 6-fold in pectin (376-462 mg per 100 g). The bioaccessibility of pro-vitamin A carotenoids from commercial and fresh clementina juices versus their concentrates was assessed, including the intestinal carotenoid uptake by Caco-2 cells. Differences in particles size and pectin status resulted in a 7-fold increase in the bioaccessibility of carotenoids in industrial products versus fresh products while limiting their cellular uptake in correlation with larger micelle sizes (10.6 nm and 6.82 nm for industrial and fresh concentrates, respectively). Overall, the highest carotenoid bioaccessibility from industrial concentrate was sufficient to offset the lower carotenoid intestinal uptake related to micelle size. This study highlights that the structure of pectins, more specifically their degree of methoxylation, favors carotenoid bioaccessibility but impairs the intestinal absorption of carotenoids from citrus concentrates.

Bioaccessibility of Biofortified Sweet Potato Carotenoids in Baby Food: Impact of Manufacturing Process.

Orange-fleshed sweet potato (OFSP), a biofortified crop rich in ß-carotene, can be used as a component of baby food recipes in order to tackle vitamin A deficiency in children <6 years old. In this work, the impact of formulation (addition of pumpkin, oil, and egg yolk) and industrial heat processing (pasteurization, sterilization) on carotenoid content and bioaccessibility was evaluated in an OFSP-based baby puree. A commercial OFSP baby food product from Brazil and a homemade OFSP puree were used as references. The losses of all-trans-ß-carotene ranged from 16 to 21% (pasteurization, homemade) to 32% (sterilization). Because of higher particle sizes and despite a higher content in carotenoids, the homemade puree had a lower bioaccessibility (i.e., micellar transfer using in vitro digestion: 0.50%) compared with the sterilized and commercial purees (5.3-6.2%). Taking into account bioaccessibility and applying a 50% conversion to retinol, a 115 g baby portion of the sterilized OFSP-puree formulated with 2% oil provided 31.4% of the daily vitamin A requirement (RDA) for children under 6 years. In comparison, 115 g of homemade OFSP-puree provided only 3.5% of the RDA. Addition of pumpkin to OFSP did not improve the percentage of RDA. Interestingly, the incorporation of an emulsifier (egg yolk powder) before cooking could improve the percentage of provision by a factor of 2.7. These results showed that reaching a balance between formulation and processing is determinant to maximize carotenoid bioaccessibility of carotenoids from OFSP-based baby food.

Assessment of cocoa (Theobroma cacao L.) butter content and composition throughout fermentations.

Cocoa fermentation is a crucial step for the development of cocoa aroma and precursors of high quality cocoa and by-products. This bioprocess has been studied for years to understand cocoa chemical changes but some matters concerning changes in fat content remain that are investigated in this work. Changes in the quantity (free and total fat), extractability and composition of cocoa butter were assessed in samples from Madagascar, the Dominican Republic and Ecuador. Increases in free fat content were highlighted in samples from each origin thanks to the use of the 'soxtec' solvent method, which preserves the integrity of the butter. A 4.71% increase in free fat was measured in the Ecuadorian samples fermented for 144 h. Conversely, total fat content remained stable throughout fermentation. Protein and polyphenol contents decreases were linked to fat content augmentation by a strong negative interaction. Triglyceride and total and linked fatty acid kinetics (0 to 6 days) of the butter remained statistically stable during fermentation, as did unsaponifiable matter. The origin of fermentation had a predominant and significant impact on composition, revealed by PCA. This work underlines and explains the importance of fermentation process in improving yield of fat that can be extracted while preserving the composition of this cocoa butter. This study highlights an interaction in cocoa unfermented or partially fermented beans. This phenomenon causes butter content retention but is slowly broken after 72 h fermentation. Therefore, fermentation appears to be also necessary to enhance the cocoa butter content extracted from the nibs.

Culinary practices mimicking a polysaccharide-rich recipe enhance the bioaccessibility of fat-soluble micronutrients.

This study was carried out to assess the impact of heat processing of a complex emulsion on the behavior of fat soluble micronutrients (FSM) in a traditional Tunisian dish. A simplified recipe involved, dried mucilage-rich jute leaves, tomato paste and olive oil, followed by a cooking treatment (150min). Hydrothermal pattern and viscosity were monitored along with the changes of FSM content and the bioaccessibility (called micellarization, using an in vitro digestion model). Partitioning of carotenoids differed according to their lipophilicity: lycopene, ß-carotene and lutein diffused to the oil phase (100%, 70% and 10% respectively). In contrast with the poor carotenes/tocopherol bioaccessibility (0.9-1%), the highest micellarization was observed for lutein (57%) and it increased with heating time and viscosity change. Domestic culinary cooking practices probably increase the bioavailability of carotenes mainly by their diffusion to the oil phase, facilitating their in vivo transfer into micelles.

Changes in antioxidant activity during the ripening of jujube (Ziziphus mauritiana Lamk).

Phenolic compounds from jujube fruits and related antioxidant activities were investigated during the ripening stages. Three different antioxidant assays, including ORAC, FRAP and DPPH, were monitored on crude jujube extract (CJE). Jujube fruits were additionally fractionated into three selective fractions F1, F2, and F3. However, only the FRAP assay gave the relative antioxidant activity for the three fractions. Furthermore, HPLC-ESI-MSMS (Q-Tof) and GC-MS were used to identify the compounds in each purified fraction. Using FRAP, F1 mainly composed of lipids, exhibited the lowest antioxidant activity (≈0.080±0.015mmol trolox/100g, p<0.05). F2, rich in flavanols and flavonols, displayed 50-fold higher activity (4.27±0.11mmol trolox/100g). Remarkably, F3 with an elevated content of condensed tannins (polymeric proanthodelphinidins), exhibited the highest antioxidant activity (25.4±0.35mmol trolox/100g). The presented results showed that the phenolic profiles of the fruits were influenced by their developmental stage. Furthermore, during ripening, the antioxidant activity may be more impacted by the flavanols and condensed tannins. The purified condensed tannins of jujube fruits may be used as natural antioxidant extracts.

Near infra-red characterization of changes in flavan-3-ol derivatives in cocoa (Theobroma cacao L.) as a function of fermentation temperature.

Flavan-3-ols were successfully extracted from cocoa by the Fast-Prep device and analyzed by HPLC-DAD, and their identifications were confirmed by injection of authentic standards. (-)-Epicatechin was the most abundant component with an average of 9.4 mg/g dried cocoa powder. More than 700 cocoa samples were used to calibrate the NIRS. An efficient calibration model was developed to accurately determine any flavan-3-ol compound of ground dried cocoa beans (SEP = 2.33 mg/g in the case of total flavan-3-ols). This performance enabled NIRS to be used as an efficient and easy-to-use tool for estimating the level of targeted compounds. The analysis of the PLS loadings of the model and pure epicatechin spectra gave proof that NIRS was calibrated on an indirect strong correlation resulting in the changes in flavan-3-ols during fermentation and their interaction with some major components, such as proteins. Total flavan-3-ol concentration fell from an average of 33.3 mg/g for unfermented samples to an average of 6.2 mg/g at the end of fermentation. Changes in flavan-3-ol content were dependent upon the origin and highly correlated to the fermentation level expressed as the sum of temperatures (average R(2) = 0.74), a good marker of the fermentation process and of the heterogeneity of the batch.