Effect of selective fermentation on nutritional parameters and techno-functional characteristics of fermented millet-based probiotic dairy product.

The primary goal of this study was to assess the effect of selective fermentation on the nutritional and techno-functional characteristics of fermented millet-skim milk-based product. The product was made with HHB-311 biofortified pearl millet (PM) flour, skim milk powder, and isolated cultures (either alone or in combination) of Limosilactobacillus fermentum MS005 (LF) and Lactobacillus rhamnosus GG 347 (LGG). To optimize fermentation time, time intervals 8, 16, and 24 h were explored, while the temperature was kept 37 °C. Results of protein digestibility showed that LF (16 h) and LGG (24 h) fermented samples had significantly higher (P < 0.05) protein digestibility of 90.75 ± 1.6% and 93.76 ± 3.4%, respectively, than that of control (62.60 ± 2.6%). Further, 16 h fermentation with LF showed enhanced iron (39%) and zinc (14%) bioavailability. The results suggested that LF with 16 h fermentation is most suitable for making millet-based fermented products with superior techno-functional attributes and micronutrient bioavailability.

Effect of Blueberry Pomace Addition on Quality Attributes of Buttermilk-Based Fermented Drinks during Cold Storage.

The fruit and beverage industry faces challenges related to waste management and environmental pollution due to rapid industrial expansion. Fruit industry waste, such as blueberry pomace, holds the promise of enhancing gut health and providing valuable antioxidants. Concurrently, buttermilk, a prominent dairy product, offers nutritional and technological benefits but remains underutilized. This study aimed to evaluate the incorporation of blueberry pomace (0%, 2%, 4%, 6%, 8%, and 10%) into buttermilk at varying levels and assess its impact on the physicochemical, antioxidant, microbiological, and sensory characteristics of the buttermilk. Buttermilk samples were supplemented with different concentrations of blueberry pomace and subjected to analysis over a two-week storage period (4 ± 1 °C). The addition of blueberry pomace led to alterations in the pH, dry matter, water holding capacity, color parameters, total phenolic content, and antioxidant activity. Microbiological analysis revealed the absence of Enterobacteriaceae, yeast, or molds. Sensory evaluation indicated significant differences among samples, with the highest scores observed for the buttermilk supplemented with 2% and 4% blueberry pomace. Incorporating blueberry pomace improved the overall acceptability and sensory properties. This research highlights the potential of fruit industry by-products to enhance the functionality and health benefits of dairy products, which is a promising way to effectively utilize waste.

Protein hydrolysates prepared by Alcalase using ultrasound and microwave pretreated almond meal and their characterization.

The study aimed to optimize ultrasonic (US: 40 kHz/200 W for 10, 20, 30, 40, and 50 min), and microwave (MW: 160 W for 45, 90, 125, 180, and 225 s) pretreatment conditions on protein extraction yield and degree of protein hydrolysis (DH) from almond de-oiled meal, an industrial by-product. First order model was used to describe the kinetics of almond protein hydrolysates obtained with Alcalase. The highest DH, 10.95% was recorded for the US-50 min and 8.87% for MW-45 s; while it was 5.76% for the untreated/control sample. At these optimized pretreatment conditions, a 1.16- and 1.18-fold increment in protein recovery was observed for the US and MW pretreatments, respectively in comparison to the conventional alkaline extraction. The molecular weight distribution recorded for pretreated samples disclosed a significant reduction in the band thickness in comparison with control. Both the pretreatments resulted in a significant increase (P < 0.05) in the antioxidant activity, and TCA solubility index when compared with the control. Results evinced that US and/or MW pretreatments before enzymatic hydrolysis can be a promising approach for the valorization of almond meal for its subsequent use as an ingredient for functional foods/nutraceuticals which otherwise fetches low value as an animal feed.

High-pressure microfluidisation positively impacts structural properties and improves functional characteristics of almond proteins obtained from almond meal.

Almond protein isolate (API) obtained from almond meal was processed using dynamic high-pressure microfluidisation (0, 40, 80, 120, and 160 MPa pressure; single pass). Microfluidisation caused significant reductions in the particle size and increased absolute zeta potential. SDS-PAGE analysis indicated reduction in band intensity and the complete disappearance of bands beyond 80 MPa. Structural analysis (by circular dichroism, UV-Vis, and intrinsic-fluorescence spectra) of the API revealed disaggregation (up to 80 MPa) and then re-aggregation beyond 80 MPa. Significant increments in protein digestibility (1.16-fold) and the protein digestibility corrected amino acid score (PDCAAS; 1.15-fold) were observed for the API (80 MPa) than control. Furthermore, significant improvements (P < 0.05) in the functional properties were observed, viz., the antioxidant activity, protein solubility, and emulsifying properties. Overall, the results revealed that moderate microfluidisation treatment (80 MPa) is an effective and sustainable technique for enhancing physico-chemical and functional attributes of API, thus potentially enabling its functional food/nutraceuticals application.

A comparative study on comprehensive nutritional profiling of indigenous non-bio-fortified and bio-fortified varieties and bio-fortified hybrids of pearl millets.

Seven indigenous pearl millet varieties, including non-bio-fortified (HC-10 & HC-20) and bio-fortified (Dhanashakti) and bio-fortified hybrids, viz., AHB-1200, HHB-299, HHB-311, and RHB-233, were studied in the present work. There was not any significant difference observed in the crucial anti-nutrients content, i.e., phytate (24.88-32.56 mg/g), tannin (3.07-4.35 mg/g), and oxalate (0.33-0.43 mg/g). Phytochemical content and antioxidant activity showed significantly high (p < 0.05) TPC and FRAP, TFC, and DPPH radical scavenging activity in the HHB 299 and Dhanashakti, respectively. Quantitative analysis of polyphenols by HPLC (first report on these varieties) revealed that HHB-299 has the highest amount of gallic acid. Fatty acid profiling by GC-FID showed that Dhanashakti, AHB-1200, and HHB-299 have rich monounsaturated fatty acid (MUFA) and polyunsaturated fatty acids (PUFA). Mineral analysis by ICP-OES showed high iron (87.79 and 84.26 mg/kg) and zinc (55.05 and 52.43 mg/kg) content in the HHB-311 and Dhanashakti, respectively. Results of the present study would help facilitate the formulation of various processed functional food products (RTC/RTE) that are currently not reported/unavailable. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05452-x.

Antioxidant potential and amino acid profile of ultrafiltration derived peptide fractions of spent hen meat protein hydrolysate.

Spent hen meat is considered as a category of waste generated by the poultry sector which can lead to serious environmental concerns if not disposed and utilized properly. In this work, spent hen meat was hydrolysed by 2% Flavourzyme (6.5 pH, 55 °C) followed by ultrafiltration to produce three peptide fractions with molecular weights > 10 kDa, 5-10 kDa and < 5 kDa. These fractions were evaluated for antioxidant potential, SDS PAGE and amino acid profile. The SDS PAGE profile demonstrated bands in the low molecular weight (< 10 kDa) region. Peptide fractions of < 5 kDa exhibited highest antioxidant activity and, essential as well as hydrophobic amino acid composition than whole hydrolysate and other peptide fractions. Incorporation of the identified hydrolysate fraction in food could improve its shelf stability while serving as a preventive component against human degenerative diseases.

Formulation and characterization of nano-curcumin fortified milk cream powder through microfluidization and spray drying.

In the present investigation, novel nano-curcumin enriched milk cream powder (CP) was formulated using microfluidization (at 100 MPa/2 passes) followed by spray drying (at three different temperatures: 150, 170 and 190 °C) with sodium caseinate (N) and gum arabic (G) as encapsulating materials. The effect of processing and encapsulating materials on the powder functionalities, particle size, encapsulation efficiency, morphology, fluorescence properties, bioaccessibility and cytotoxicity were studied. Results showed that NCP (spray dried at 190 °C) had significantly higher yield (68 %), encapsulation efficiency (EE) (93 %), and lower particle size (724 nm) than that of GCP. Fluorescence spectra of the powders revealed characteristic 'blue shift' phenomenon indicating better encapsulation and protection of the nano-curcumin corroborating EE results. SEM images showed distinctive features of NCP and GCP; wherein, NCP had shrivelled and irregular surface as compared to the GCP which exhibited round shape and smooth surface. TEM results confirmed that curcumin particles were in the nano-scale (50-250 nm) for both NCP and GCP. In vitro simulated digestion showed significantly high (88.48 %) curcumin bioaccessibility of NCP plus remarkable inhibition of HepG2 cells; whereas, no cytotoxicity was observed in Caco-2 cells by MTT assay. Formulation's applicability was shown by reconstituting powders as a 'cream spread' wherein high sensory acceptability observed. Sodium caseinate was found to be an excellent delivery vehicle for the fortification of nano-curcumin in the cream powder. To our knowledge, this is the first report of formulating a novel nano-curcumin fortified cream powder which has tremendous potential as a functional food ingredient.

Application of Aloe vera Gel Coating Enriched with Cinnamon and Rosehip Oils to Maintain Quality and Extend Shelf Life of Pomegranate Arils.

A completely randomized design was applied on pomegranate arils for several post-harvest treatments before the packaging in polypropylene boxes for 15 days at (5 ± 1 °C, 95 ± 2% RH): control (untreated), Aloe vera gel (10% or 20%), 10% Aloe vera + rosehip oil (0.25% or 0.50%), 20% Aloe vera + rosehip oil (0.25% or 0.50%), 10% Aloe vera + cinnamon oil (0.25% or 0.50%), and 20% Aloe vera + cinnamon oil (0.25% or 0.50%). Aloe vera in combination with cinnamon oil resulted in an enhanced shelf life (15 d) compared to the uncoated arils (control). The Aloe vera + cinnamon oil coating led to the retention of total phenolics, anthocyanin, ascorbic acid, and antioxidant activity in context to the quality attributes. Moreover, this coating showed minimal change in the color, total soluble solids, titratable acidity, firmness, delayed ethylene production, respiration rate, and physiological weight loss. Also, A. vera + cinnamon oil coatings significantly (p < 0.05) inhibited the total counts of mesophilic aerobics, coliforms, and yeast and mold. Organoleptic attributes, including color, flavor, aroma, texture, and purchase acceptability were higher for the arils that were treated with 10% A. vera + 0.25% cinnamon oil; thus, this highly economical and easily available coating material can be formulated and used commercially to extend the shelf life and enhance the profit of the producers and/or processors.

Critical overview of biorefinery approaches for valorization of protein rich tree nut oil industry by-product.

This review explores reutilization opportunities of protein-rich bio-waste derived from the major tree nuts (almonds, walnuts, and cashew nuts) oil processing industries through biorefinery strategies. The mechanically pressed out oil cakes of these nuts have high protein (45-55%), carbohydrate (30-35%), and fiber that could be utilized to produce bioactive peptides, biofuels, and dietary fiber, respectively; all of which can fetch substantially greater value than its current utilization as a cattle feed. Specific attention has been given to the production, characterization, and application of nut-based de-oiled cake hydrolysates for therapeutic benefits including antioxidant, antihypertensive and neuroprotective properties. The often-neglected safety/toxicological evaluation of the hydrolysates/peptide sequences has also been described. Based on the available data, it is concluded that enzymatic hydrolysis is a preferred method than microbial fermentation for the value addition of de-oiled tree nut cakes. Further, critical insights on the existing literature as well as potential research ideas have also been proposed.

Health-Promoting and Therapeutic Attributes of Milk-Derived Bioactive Peptides.

Milk-derived bioactive peptides (BAPs) possess several potential attributes in terms of therapeutic capacity and their nutritional value. BAPs from milk proteins can be liberated by bacterial fermentation, in vitro enzymatic hydrolysis, food processing, and gastrointestinal digestion. Previous evidence suggested that milk protein-derived BAPs have numerous health-beneficial characteristics, including anti-cancerous activity, anti-microbial activity, anti-oxidative, anti-hypertensive, lipid-lowering, anti-diabetic, and anti-osteogenic. In this literature overview, we briefly discussed the production of milk protein-derived BAPs and their mechanisms of action. Milk protein-derived BAPs are gaining much interest worldwide due to their immense potential as health-promoting agents. These BAPs are now used to formulate products sold in the market, which reflects their safety as natural compounds. However, enhanced commercialization of milk protein-derived BAPs depends on knowledge of their particular functions/attributes and safety confirmation using human intervention trials. We have summarized the therapeutic potentials of these BAPs based on data from in vivo and in vitro studies.

Bioactive peptides from meat: Current status on production, biological activity, safety, and regulatory framework.

By-products of the meat processing industry which are often discarded as waste are excellent protein substrates for producing bioactive peptides through enzymatic hydrolysis. These peptides have tremendous potential for the development of functional food products but there is scanty information about the regulations on bioactive peptides or products in various parts of the world. This review focuses on the diverse bioactive peptides identified from different meat and meat by-products, their bioactivity and challenges associated in their production as well as factors limiting their effective commercialization. Furthermore, this report provides additional information on the possible toxic peptides formed during production of the bioactive peptides, which enables delineation of associated safety and risk. The regulatory framework in place for bioactive peptide-based foods in different jurisdictions and the future research directions are also discussed. Uniform quality, high cost, poor sensory acceptance, lack of toxicological studies and clinical evidence, paltry stability, and lack of bioavailability data are some of the key challenges hindering commercial advancement of bioactive peptide-based functional foods. Absorption, distribution, metabolism and excretion (ADME) studies in rodents, in vitro genotoxicity, and immunogenicity data could be considered as absolute pre-requisites to ensure safety of bioactive peptides. In the absence of ADME and genotoxicity data, long term usage to evaluate safety is highly warranted. Differences in legislations among countries pose challenge in the international trade of bioactive peptides-based functional foods. Harmonization of regulations could be a way out and hence further research in this area is encouraged.

Polyphenols of Edible Macroalgae: Estimation of In Vitro Bio-Accessibility and Cytotoxicity, Quantification by LC-MS/MS and Potential Utilization as an Antimicrobial and Functional Food Ingredient.

Macroalgae are a rich source of polyphenols, and their ingestion promotes various health benefits. However, information on factors contributing to health benefits such as antioxidants, antimicrobial properties, bioaccessibility, and cytotoxicity is less explored and often unavailable. Therefore, this study aims to investigate the above-mentioned parameters for the brown and green macroalgae Sargassum wightii and Ulva rigida, respectively, collected from the southeast coast of India. S. wightii exhibited higher antioxidant activity and moderate antimicrobial activity against major food pathogens in an agar well diffusion assay and in the broth microdilution method (MIC50 being <0.5 mg/mL for all microorganisms tested). Both macroalgae extracts exhibited significantly high bioaccessibility of polyphenols. To evaluate the safety of the extracts, in vitro cytotoxicity by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was carried out on the primary cells: mouse splenic lymphocytes. An almost complete decline in the cell viability was seen at considerably high concentration (50 mg/mL), expressing the reasonably high safety of the extracts. The extracts of both macroalgae were quantified for polyphenols, wherein fucoxanthin (9.27 ± 2.28 mg/kg DW) and phloroglucinol (17.96 ± 2.80 mg/kg DW) were found to be greater in the S. wightii apart from other phenolics, like gallic acid, quercetin, vanillin, and ferulic acid. The results signify the tremendous scope for the value addition of S. wightii through extraction and purification of polyphenols for its potential exploitation in functional foods and nutraceuticals or as an antimicrobial ingredient in active or smart packaging.

Persistent organic pollutants in foods, their interplay with gut microbiota and resultant toxicity.

Persistent Organic Pollutants (POPs) have become immensely prevalent in the environment as a result of their unique chemical properties (persistent, semi-volatile and bioaccumulative nature). Their occurrence in the soil, water and subsequently in food has become a matter of concern. With food being one of the major sources of exposure, the detrimental impact of these chemicals on the gut microbiome is inevitable. The gut microbiome is considered as an important integrant for human health. It participates in various physiological, biochemical and immunological activities; thus, affects the metabolism and physiology of the host. A myriad of studies have corroborated an association between POP-induced gut microbial dysbiosis and prevalence of disorders. For instance, ingestion of polychlorinated biphenyls, polybrominated diphenyl ethers or organochlorine pesticides influenced bile acid metabolism via alteration of bile salt hydrolase activity of Lactobacillus, Clostridium or Bacteroides genus. At the same time, some chemicals such as DDE have the potential to elevate Proteobacteria and Firmicutes/Bacteriodetes ratio influencing their metabolic activity leading to enhanced short-chain fatty acid synthesis, ensuing obesity or a pre-diabetic state. This review highlights the impact of POPs exposure on the gut microbiota composition and metabolic activity, along with an account of its corresponding consequences on the host physiology. The critical role of gut microbiota in impeding the POPs excretion out of the body resulting in their prolonged exposure and consequently, enhanced degree of toxicity is also emphasized.

Nanofluid research advances: Preparation, characteristics and applications in food processing.

There has been growing interest and substantial improvement in thermal processes for enhancing the heat transfer rate in food industry applications. The replacement of conventional heat transfer fluids with nanofluids is now being considered as a novel and emerging solution to the heat transfer problem of the food processing sector. This review covers state-of-the-art methods for production and application of these nanofluids with emphasis on the decontamination of liquid foods. The review also discusses the influence of processing conditions such as temperature and nanoparticle concentration on the thermal and viscous characteristics of the developed nanofluids. Further, the effect of these developed nanofluids on the quality attributes of food materials has also been reviewed and analyzed. Based on the current technological status, certain knowledge gaps in nanofluid research have been identified, including controlled (shape and size) and systematic experimental studies, stability of nanofluids with increasing thermal cycles, increasing the compatibility of base fluid to nanomaterials, and toxicity and environmental impact assessment.

Strategies and advances in the pretreatment of microalgal biomass.

Modification of structural components, especially the cell wall, through adequate pretreatment strategies is critical to the bioconversion efficiency of algal biomass to biorefinery products. Over the years, several physical, physicochemical, chemical and green pretreatment methods have been developed to achieve maximum productivity of desirable by-products to sustain a circular bioeconomy. The effectiveness of the pretreatment methods is however, species specific due to diversity in the innate nature of the microalgal cell wall. This review provides a comprehensive overview of the most notable and promising pretreatment strategies for several microalgae species. Methods including the application of stress, ultrasound, electromagnetic fields, pressure, heat as well as chemical solvents (ionic liquids, supercritical fluids, deep eutectic solvents etc.) have been detailed and analyzed. Enzyme and hydrolytic microorganism based green pretreatment methods have also been reviewed. Metabolic engineering of microorganisms for product specificity and lower inhibitors can be a future breakthrough in microalgal pretreatment.

Nanoencapsulated curcumin emulsion utilizing milk cream as a potential vehicle by microfluidization: Bioaccessibility, cytotoxicity and physico-functional properties.

Curcumin loaded milk cream emulsion was microfluidized at different pressures (50-200 MPa) and passes (1-4) using a full-factorial experimental design. Ultrasonicated and microfluidized emulsion was evaluated for particle size, morphological characteristics, antioxidant activity, rheological properties, bioaccessibility and cytotoxicity. Significant reduction was observed in the average particle size (358.2 nm) after microfluidization at 100 MPa/2nd pass. Transmission electron micrographs of the control (homogenized) and microfluidized (100 MPa/2nd pass) samples showed uniform distribution of fat globules in the microfluidized sample with partially dissolved curcumin particles (50-150 nm). Encapsulation efficiency of microfluidized emulsion was found to be significantly higher (97.88%) after processing as compared to control (91.21%). Two-fold (100%) increase in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and 25% increase in ferric-reducing antioxidant power (FRAP) was observed for microfluidized emulsions over control. Infrared spectrums of the emulsion exhibited shift in high intensity peaks indicating bond cleavage after microfluidization. After characterization, emulsions were subjected to in vitro digestion (oral, gastric and intestinal phase) to evaluate its bioaccessibility which was found to be remarkably increased by 30% after microfluidization. For assessing processing induced safety of the formulation, in vitro cytotoxicity of the microfluidized nanocurcumin emulsion was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on HepG2 cells, wherein high % of cell viability (>93%) was seen even at a dose as high as 900 µg/mL revealing no toxic effect of the processing technique (microfluidization). This study highlights the efficacy of microfluidization as a technique and that of milk cream as an inexpensive, yet potential vehicle for generating stable and bio-accessible nano-curcumin emulsion.

Comparative functional and spectroscopic analysis of spent hen meat hydrolysate by individual and combined treatment of microbial proteases.

Simultaneous (Sm) and sequential (Sq) use of microbial proteases for the hydrolysis of spent hen/chicken meat from antioxidant potential perspective is relatively unexplored and requires attention. In this work, meat was hydrolyzed using Flavourzyme (Fz) and Alcalase (Ac), each at 1, 2, and 3% for 6 h as well as using both enzymes (at 2% each) in Sm and Sq treatment. Maximum attained %DPPH-RSA (Fz:68.25; Ac:77.18; Sm:59.82; and Sq:65.97) and FRAP (mM TEAC/g) values (Fz:3.77; Ac:2.56; Sm:2.54; and Sq:3.37) were measured as a function of hydrolysis time. The highest (23.38%) and lowest (10.68%) degree of hydrolysis (DH) was obtained with 3% Ac and 1% Fz, respectively. FTIR spectroscopy clearly revealed changes in the secondary structure of proteins. SDS PAGE profiling of hydrolysates showed that Fz produces low molecular weight peptides (2-75 kDa) as compared to Ac or its combination with Ac. As per the results of this study, Sq enzyme treatment is recommended for preparing spent hen meat hydrolysate with higher functional attributes for possible use as functional food/nutraceutical.

p53 Gene Mutation in Blood of Zebu Cattle with Squamous Cell Carcinoma of the Horn.

Horn cancer is one of the most important diseases in Zebu castrated male cattle. The objective of this study was to investigate the presence of p53 gene mutation in the blood of affected cattle and its value for early diagnosis and prognosis. The study was conducted on blood samples from 20 affected cattle and six healthy control cattle from Western India. Plasma samples were evaluated for the presence of p53 gene mutation using the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique and the results were correlated with the stage of cancer. Five of the 20 cases had stage I neoplasms, nine stage II and six stage III, based on histopathological examination. PCR-SSCP analysis revealed an aberrant pattern of DNA migration on polyacrylamide gel electrophoresis of DNA extracts from blood samples of six animals with stage II and stage III cancer. No mutation was identified in blood from cattle with stage I cancer or from healthy control cattle. These results suggest that PCR-SSCP detection of p53 gene mutation in blood has potential diagnostic and prognostic value, and indicate the need for further large-scale investigation.

Effect of Indian brown seaweed Sargassum wightii as a functional ingredient on the phytochemical content and antioxidant activity of coffee beverage.

Seaweed is a novel food source that is packed with bioactive compounds but is rarely used in its raw/cooked form. An efficient nutrient delivery medium is required to make the potential health benefits of seaweeds accessible to the general population while maintaining its palatability. In this study, coffee infused with different seaweed concentrations (1%, 3%, 5%) were prepared and their physico-chemical, phenolics, flavonoids, antioxidants, rheology, thermal and spectral characteristics were observed. Increase in seaweed concentration resulted in increased acidity and decreased total soluble solids of the beverage with no distinct color change. Rheological measurements showed flow behavior index in the range of 1.09-1.34 indicating dilatant tendency of the seaweed-coffee infusions which gradually decreased towards a Newtonian nature with increase in seaweed concentration. Higher detection of flavonoids and ferric reducing antioxidant power was possible with increase in seaweed concentration from 1 to 5%. However, no significant changes in total phenols and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity was observed. Sensory evaluation of the coffee drinks was done using fuzzy logic which showed highest sensory acceptability for the infusion with 1% seaweed concentration. Thermograms showed changes in flavor profile on increasing seaweed concentration which was later confirmed using Fourier transform-infrared spectroscopy. Results of the study highlight that coffee can be successfully used to mask the off-flavor of seaweed while disseminating its health benefits to the general population through an effective and extensively utilized food medium.

Review of toxicological assessment of d-limonene, a food and cosmetics additive.

R-(+)-limonene (d-limonene) is a commonly used flavor additive in food, beverages and fragrances for its pleasant lemon-like odor. Considering its increasing applications, it's necessary to understand toxicological effects and risk associated with its use. R-(+)-limonene is rapidly absorbed in experimental animals and human beings following oral administration. In humans, it gets distributed to liver, kidney, and blood resulting in the formation of metabolites like perillic acid, dihydroperillic acid, limonene-1,8-diol and limonene-1,2 diol. Important toxic effects primarily reported in rodents are severe hyaline droplet nephrotoxicity (only in male rats due to specific protein α2u-globulin; however, this effect isn't valid for humans), hepatotoxicity and neurotoxicity. R-(+)-limonene does not show genotoxic, immunotoxic and carcinogenic effects. Substantial data is available about limonene's stability after treatment with thermal and non-thermal food processing techniques; however, information about toxicity of metabolites formed and their safe scientific limits is not available. In addition, toxicity of limonene degradation products formed during storage of citrus juices isn't known. Based on all available toxicological considerations, R-(+)-limonene can be categorized as low toxic additive. More detailed studies are required to better understand interaction of limonene with modern food processing techniques as well as degradation products generated and toxicity arising from such products.