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Somatic embryogenesis (SE) is a clear example of cellular totipotency. The SE of the genus Coffea has become a model for in vitro propagation for woody species and for the large-scale production of disease-free plants that provide an advantage for modern agriculture. Temporary immersion systems (TIS) are in high demand for the propagation of plants. The success of this type of bioreactor is based on the alternating cycles of immersion of the plant material in the culture medium, usually a few minutes, and the permanence outside the medium of the tissues for several hours. Some bioreactors are very efficient for propagating one species but not another. The efficiency of bioreactors depends on the species, the tissue used to propagate, the species' nutritional needs, the amount of ethylene produced by the tissue, and many more. In this protocol, we show how we produce C. canephora plants that are being taken to the field.

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Since the term proteomics was coined by Marc Wilkins in 1994, there has been an explosion in the number of articles reporting the use of the proteomics technique. As the layers of biological organization and their regulation increase, the complexity of living beings increases. Thus, we go from the genome to tissues, cells, cellular compartments, and phenotypes and the complexity of the tools used to study this complexity also increases. Unlike the genome study, in the case of the proteome, we have a more complex panorama. We have a spatial and temporal proteome. Proteomics helps to answer complex biological questions since proteins' function depends on their molecular structure, subcellular localization, and posttranslational modifications. In this protocol, we describe a methodology to extract proteins using different methods, separating proteins by electrophoresis in double-dimensional gels and analyzing the gels using specialized software that allows obtaining information on the number and abundance of the proteins from the gels.

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Omic tools have changed the way of doing research in experimental biology. The somatic embryogenesis (SE) study has not been immune to this benefit. The transcriptomic tools have been used to compare the genes expressed during the induction of SE with the genes expressed in zygotic embryogenesis or to compare the development of the different stages embryos go through. It has also been used to compare the expression of genes during the development of calli from which SE is induced, as well as many other applications. The protocol described here is employed in our laboratory to extract RNA and generate several transcriptomes for the study of SE on Coffea canephora.

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For the first time, the dual effect of coffee cherry sanitization methods to control the microbial load in processing and the influence of fermentation time on coffee quality parameters was evaluated. Two assays were carried out by wet processing: I) Sanitization of the coffee cherry (ST1: Unclassified processed cherries; ST2: Classified and sanitized cherries with drinking water; ST3: Classified and sanitized cherries with a chemical agents and II) Fermentation times (FT1: 12 h; FT2: 24 h; FT3: 48 h; FT4: 72 h and FT5: 96 h). pH, temperature, and dissolved oxygen were monitored during fermentation. Counts of Lactic Acid Bacteria - LAB, mesophiles, and yeasts were carried out on the coffee mass before and after fermentation. Caffeine and chlorogenic acid contents were determined by HPLC-DAD and the sensory profile by methodology for specialty coffees (SCA). The main findings showed that: sanitization with Timsen® did not significantly influence the evolution of pH during fermentation (p > 0.05), but it can reduce to a small extent the action of LAB at the end of the process. It was observed that the temperature of the coffee mass tends to balance with the ambient temperature, with significant effects (p < 0.05) of sanitization (ST2 and ST3) on the stability of this variable during fermentation. Timsen® as a disinfectant affected microbial populations and improved the sensory profile in the cup. In prolonged coffee fermentations (FT3, FT4 and FT5), the pH of the coffee mass tended to stabilize after 36 h, regardless of the process time. Likewise, a correlation was evident between a higher microbial load correlated with better sensory profiles in FT4 and FT5. Neither the sanitization process nor the fermentation time significantly affected the caffeine and chlorogenic acid contents of the coffee, both in its green and roasted states. Consequently, the sanitization of cherry coffee with Timsen® and prolonged fermentation times favor the safety and coffee final quality in the cup.

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Coffee (Coffea arabica) cultivation is vital to the global economic, social and cultural life of farmers. However, senescent and disease-susceptible plantations affect coffee productivity. Therefore, it is crucial to improve biotechnological strategies such as micropropagation to increase the number of plants for replanting. In this study, the dark condition (T1) and different light qualities (T0-white light 400-700 nm; T2-red light 660 nm and T3-blue light 460 nm) were evaluated to optimize the in vitro propagation of 4 and 9 month-old coffee seeds. The results showed that red light had the highest percentage, an outstanding germination rate index, which may suggest that in the case of coffee seeds could be involved phytochromes that promote germination in a red light quality. In summary, the ideal conditions for in vitro micropropagation of coffee are under white and red light condition.

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The maturation of Arabica coffee fruits is influenced by both endogenous and external factors. The stage of fruit maturation affects the chemical composition of the beans, which in turn impacts the quality of the coffee beverage. During maturation, the fruit peel changes colour from green to red (cherry), signalling the optimal harvest time and suggesting high fruit quality. However, the degree of redness can vary, indicating different levels of maturity. This study aimed to explore the variation in soluble sugar accumulation in relation to the redness of coffee fruit tissues. We classified ripe fruits into six ripeness categories based on the intensity of the red colour of the epicarp, measured using a colourimeter. We analysed total soluble sugar, sucrose, and starch in three parts: coat (exocarp + mesocarp), coat juice (obtained by squeezing the coat), and beans. Our findings reveal that the variation in sugar in the endosperm does not correspond to changes in the coat, suggesting separate regulation of sugar accumulation, particularly sucrose, which is crucial for coffee quality. Our data indicate that there is no transfer of sucrose and reducing sugars from the red coat to the bean.

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Specialty coffee beans are those produced, processed, and characterized following the highest quality standards, toward delivering a superior final product. Environmental, climatic, genetic, and processing factors greatly influence the green beans' chemical profile, which reflects on the quality and pricing. The present study focuses on the assessment of eight major health-beneficial bioactive compounds in green coffee beans aiming to underscore the influence of the geographical origin and post-harvesting processing on the quality of the final beverage. For that, we examined the non-volatile chemical profile of specialty Coffea arabica beans from Minas Gerais state, Brazil. It included samples from Cerrado (Savannah), and Matas de Minas and Sul de Minas (Atlantic Forest) regions, produced by two post-harvesting processing practices. Trigonelline, theobromine, theophylline, chlorogenic acid derivatives, caffeine, caffeic acid, ferulic acid, and p-coumaric acid were quantified in the green beans by high-performance liquid chromatography with diode array detection. Additionally, all samples were roasted and subjected to sensory analysis for coffee grading. Principal component analysis suggested that Cerrado samples tended to set apart from the other geographical locations. Those samples also exhibited higher levels of trigonelline as confirmed by two-way ANOVA analysis. Samples subjected to de-pulping processing showed improved chemical composition and sensory score. Those pulped coffees displayed 5.8% more chlorogenic acid derivatives, with an enhancement of 1.5% in the sensory score compared to unprocessed counterparts. Multivariate logistic regression analysis pointed out altitude, ferulic acid, p-coumaric acid, sweetness, and acidity as predictors distinguishing specialty coffee beans obtained by the two post-harvest processing. These findings demonstrate the influence of regional growth conditions and post-harvest treatments on the chemical and sensory quality of coffee. In summary, the present study underscores the value of integrating target metabolite analysis with statistical tools to augment the characterization of specialty coffee beans, offering novel insights for quality assessment with a focus on their bioactive compounds.

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Consumers are increasingly looking for healthy foods without the addition of synthetic additives. The aim of this study was to evaluate the efficiency of coffee extracts as a natural antioxidant in fresh pork sausage. Firstly, the conditions for obtaining coffee green extracts were optimized (Central Composite Rotatable Design 23, variables: extraction time, ethanol-water ratio, and sample-solvent ratio) in an ultrasound bath (70 °C). The response variables were the bioactive compounds levels and antioxidant activity. Valid models were obtained (p ≤ 0.05, R2 > 0.751), with higher bioactive content and antioxidant activity in the central point region. Extracts of Robusta and Arabica coffee green (RG and AG) and medium roast (RR and AR) obtained, and central point (10 min, an ethanol concentration of 30%, and a sample-solvent ratio of 10 g/100 mL) and optimized (14.2 min, 34.2%, and 5.8 g/100 mL) parameters were characterized. The RG presented a significantly (p ≤ 0.05) higher content of caffeine (3114.8 ± 50.0 and 3148.1 ± 13.5 mg/100 g) and 5-CQA (6417.1 ± 22.0 and 6706.4 ± 23.5 mg/100 g) in both extraction conditions, respectively. The RG and RR coffee presented the highest antioxidant activity. Two concentrations of RG and RR coffee extracts were tested in fresh pork sausage. The Robusta coffee extract presented the highest antioxidant activity in both roasted and green states. However, when applied to a meat product, the extract prepared with RG coffee showed better results, with efficiency in replacing synthetic antioxidants (content of malonaldehyde/kg of sample below 0.696 ± 0.059 in 20 days of storage), without altering the sensory attributes of the product (average scores above 7.16 ± 1.43 for all attributes evaluated). Therefore, the RG coffee extract was a suitable alternative as a natural antioxidant applied to fresh pork sausage.

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Cancer is the second leading cause of death worldwide, despite the many treatments available, cancer patients face side effects that reduce their quality of life. Therefore, there is a need to develop novel strategies to increase the efficacy of treatments. In this study, gold nanoparticles obtained by green synthesis with Coffea arabica green bean extract were loaded with Doxorubicin, (a highly effective but non-specific drug) by direct interaction and using commercial organic ligands that allow colloidal dispersion at physiological and tumor pH. Conjugation of these components resulted in stable nanohybrids at physiological pH and a tumor pH release dependent, with a particle size less than 40 nm despite having the ligands and Doxorubicin loaded on their surface, which gave them greater specificity and cytotoxicity in H69 tumor cells.