During processing, a wide range of indigenous microbiota, including yeasts, bacteria and to a lesser extent filamentous fungi, grow and perform metabolic activities on the beans, a process called coffee fermentation, which has been shown to have a strong effect on it. The main function of fermentation is to remove the outer fruit layers, leaving two dried seeds. A wide range of endogenous isolates with pectinolytic activities have been identified, including bacteria such as Klebsiella, Erwinia, Bacillus and Lactobacillus, and yeasts such as Pichia, Saccharomyces , Candida . Leaving cherries untreated for more than 8 h after harvest may initiate uncontrolled fermentation and alter the quality of the final products. In addition, several endogenous metabolic activities have been detected in the endosperm during the drying process, causing significant changes in its chemical composition, including the type and amount of reducing sugars and amino acids, which are considered important precursors of coffee aroma and color during roasting. During fermentation, remaining mucilage is used as substrates for microbial growth, which produces secondary metabolites including organic acids, alcohols, esters, ketones and aldehydes that can affect final product quality.
For example, studies have shown that the resulting green beans have high levels of desirable microbial metabolites such as ethyl acetate, acetaldehyde, glycerol, and ethanol, which are associated with fruity notes and high sensory scores of flavor, aroma, body, acidity, and uniformity after roasting.
The mechanical mucilage process is reported to reduce the weight loss of the fruit reported by fermentation and prevents off-flavors that can occur during uncontrolled washed fermentation. Honey process coffee is another specialty coffee made in countries such as Costa Rica and El Salvador. Its production is relatively similar to the semi-dry method, where the mechanical pulping process is adjusted to retain a certain amount of pulp material around the seeds and then subjected to a natural dry process of 12 to 30 days.
The resulting beans are called black, red, yellow or white honey beans, depending on the amount of pulp material remaining attached to the beans, which ranges from 80% to 20%, respectively. Such a process has been reported to alter the concentration and profile of certain sugars and amino acids in the coffee beans. For example, coffees produced by the hock process reported higher concentrations of galactinol and glutamic acid compared to natural coffees, which are believed to modulate flavor development during the roasting process. Coffee beans produced by the natural dry process contained higher levels of reducing sugars compared to the washed process, while washed process contained higher levels of aspartic and glutamic acid. Washed process coffees were also reported to have floral, caramel and fruity notes, while dry processed beans were reported to have buttery and nutty notes. The main chemical changes that occur during the roasting process include loss of water content and protein denaturation, pyrolysis and caramelization. At this stage, various aromatic compounds are developed, including esters, phenolic, carbonyl compounds and polycyclic compounds, which give coffee its characteristic taste and aroma.
Diversity of yeast community in coffee fermentation:
Yeasts are detected in almost all types of coffee fermentation. In the wet process, yeast species such as Saccharomyces, Schizosaccharomyces, Candida, Hanseniaspora, Pichia, Debaryomyces, Cryptococcus and Rhodotorula species have been frequently isolated from different parts of the world. Recently, a wide variety of yeast species such as Pichia guilliermondii, P. anomala, P. ofunaensis, Debaryomyces hansenii and Starmerella were detected in Natural process, with Arxula adeninivorans being the most prevalent during fermentation. Metabolic activities of yeasts such as fermentation of pulp sugars in coffee beans produce secondary metabolites such as alcohols, acids, esters, aldehydes and ketones. These metabolites are believed to pass into the coffee beans and affect the coffee quality. In addition, the ability of yeasts to degrade the stickiness of coffee beans and to suppress mycotoxin-producing fungi has also been reported.
Diversity of bacterial community in coffee fermentation:
The dominant LAB species included Lactiplantibacillus plantarum, Levilactobacillus brevis, Lactobacillus sp, Leuconostoc mesenteroides dextranicum, Leuconostoc sp, Leuconostoc citreum, Leuconostoc pseudomesenteroi, while lower populations included Lactococcus lactis, Streptococcus faecalis and Weissella sp.
LAB are well known for producing organic acids such as lactic, acetic, butanoic, formic and glutamic acids and others. Many of these acids have been detected in coffee bean fermentations. The presence of such metabolites contributes to the fermentation process and affects the quality of the coffee. Low levels of acetic acid have a desirable clean and sweet sensory taste in the final product, but when present in high concentrations (above 2 mg/ml) it causes an undesirable acetic acid taste.
Recently, high concentrations of alcohol, aldehyde and ester compounds were also detected in beans fermented with the washed process, which improved the sensory quality of the final product. The authors speculated that these metabolites were produced by heterofermentative LAB, such as lactobacillus, leuconostoc and Lactococcus, which were present in high populations during fermentation. A wide variety of bacteria other than LAB have been detected during different coffee fermentation processes. Washed process was the first to report coliform bacteria resembling Aerobacter and Escherichia in addition to Bacillus species, which are believed to contribute to the mucilage degradation process during fermentation.
Fungal diversity in coffee fermentation:
While a large number of fungal species are isolated from coffee fermentation, mostly in the Natural and Honey processes, their levels are generally low in the washed process. The main reason for the low number of molds compared to yeast and bacteria is thought to be the short fermentation time and the underwater environment of the wet process.
Mycotoxins such as ochratoxin A as well as poor sensory quality are the main concerns with filamentous fungi in coffee fermentation. Such problems have been reported occasionally, especially in cases where large numbers of coffee berries are over-ripened and floating or in contact with the ground. Therefore, it is important to harvest at the right time to minimize the percentage of floaters and prevent ground contamination to ensure that the level of fungal contamination is kept low.
While it might be intuitive to think that the role of molds in coffee fermentation is mostly negative, some studies have shown that this is not always the case.
To date, no experimental or industrial methods have been used to accelerate mucilage degradation.
coffee fermentation in the environment
There is no report of an attempt to add organic acids. In contrast, Oumer & Abate (2017) added pectinase extraction of Bacillus subtilis to wet coffee fermentation and observed complete mucilage degradation in 24 h compared to the 36 h required to complete the process in the untreated control. Therefore, bacteria may play a role in mucilage degradation during wet coffee fermentation by extracellular enzymatic activity, acidification, or a combination of both.
Silva et al. (2008) reported high propionic acid production by B. megaterium when fermentation lasted longer than 14 days in the dry process. Long fermentations may also result in the accumulation of short-chain fatty acids and their esters (cyclohexanoic acid ethyl ester, 3-methyl butanoic acid ethyl ester and 2-methyl butanoic acid ethyl ester), which are undesirable for coffee aroma.
While basic flavors such as sweetness, acidity and bitterness have a strong influence on the overall sensory impression of coffee beverages, it is the complex aromas of volatile compounds in coffee that determine the subtle sensory characteristics and qualities of coffee. Various factors such as plant genetics, geographical origin, environmental conditions, pre- and post-harvest processing can affect the composition and concentration of volatile compounds in coffee beans. Volatile compounds found in green and roasted coffee beans mainly consist of pyrazines, furans, esters, ketones, aldehydes, acids, phenols and nitrogen-containing compounds. These compounds are formed mainly during roasting through complex reactions such as Maillard reactions, caramelization, pyrolysis and other thermal processes involving green coffee bean components. Proteins, amino acids, lipids, polysaccharides, simple sugars, phenolic and chlorogenic compounds are the main aroma precursors found in green beans. During thermal processing, mainly roasting, sugars and polysaccharides are responsible for the formation of caramelization products. Phenols are obtained from a series of thermal reactions involving strong phenolic volatiles such as guaiacol and vinyl guaiacol, chlorogenic acids and phenolics. During the thermal processing of green coffee beans, free amino acids interact with reducing sugars via Maillard reactions to form important coffee aromatic compounds such as pyrazines and pyridines, which are responsible for the roasted aroma and nutty sensory properties of coffee beverages. Pyrazines and pyrroles are also produced by another route during roasting, via the pyrolysis of hydroxyl amino acids such as serine and threonine. Trigonelline is an alkaloid compound found in green coffee beans that undergoes degradation during thermal processing to produce pyridine and pyrrole compounds. Therefore, the level and concentration of aroma precursors present in green coffee beans have a significant effect on the aromas of roasted beans and final coffee beverages.
Fermented coffee is generally considered to have superior quality characteristics to unfermented coffee; however, both incomplete and excessive fermentation can produce coffee with undesirable aromas (2015). Fermentation can affect coffee aroma in several ways. First, the production of microbial metabolites that can migrate into the coffee beans. Second, microbial activity during fermentation largely determines the residual amount of free sugars and amino acids in the beans, which can affect the Maillard reaction and the volatile compounds produced therein during subsequent drying and roasting. In addition, fermentation can affect the intrinsic metabolic activity of the coffee bean through changes in the chemical composition of the bean. This can directly affect the flavor of the bean and, more importantly, indirectly affect coffee quality through changes in the compounds involved in the Maillard reaction, i.e., aroma precursors. Finally, fermentation can prevent the growth of undesirable microorganisms, such as filamentous fungi, which can produce metabolites that have a negative effect on coffee aroma. Recently, several studies have investigated the metabolic activities of endogenous microorganisms identified during coffee fermentation and their ability to produce secondary metabolites that can migrate into the beans and affect coffee quality (2015).
In a 2019 study, the initial microbiota of fermentation mostly belonged to LAB, AAB, enterobacteria, and yeasts, but LAB became the dominant species during fermentations, while the population of other microbial groups decreased continuously. The authors believed that most of these metabolic and sensory differences were due to LAB activities. The authors reported that the sensory quality of coffee produced from fermented beans without yeast was lower. It is well known that yeasts produce aromatic metabolites in other fermented products through alcoholic fermentation of sugars and produce primary ethanol and carbon dioxide in addition to secondary metabolites including higher alcohols, esters, aldehydes, sulfur, and nitrogen compounds. Similarly, LAB can convert sugars to lactic acid (homofermentative) and produce other metabolites such as ethanol, esters, diacetyl, and lactic acid (heterofermentative).
Traditionally, the fermentation process relies on the natural microbiota occurring in the coffee substrate. Early attempts attempted to accelerate the fermentation process by applying reverse slop techniques using residual water from previous coffee fermentation tanks as starters for a new process (1965). Subsequently, the identified starter cultures were first introduced into coffee fermentation (1966) using Saccharomyces spp. S. marxianus, S. bayanus and S. cerevisiae var. ellipsoideus, which were found to accelerate the fermentation process and prevent the negative effects of prolonged fermentation. The authors also observed signs of external discoloration in the inoculated beans compared to the natural process, which led the authors to suggest the use of enzyme extracts of yeasts in processing to avoid changes in the color and appearance of the beans.
Following the initial studies on inoculated fermentation, trials were conducted using microbial strains, particularly yeast strains, aiming to improve the flavor, aroma and other sensory qualities of coffee in dry, wet and semi-dry processes. Higher levels of alcohols, furans, aldehydes and esters, which impart caramel, herbal and fruity flavor notes, were found in beans obtained from inoculated fermentation. A higher concentration of acetic, malic and citric acids was also detected in the inoculated batch, which may contribute to the acidic taste of the coffee beverage, but this did not adversely affect its superior overall sensory quality compared to the uninoculated batch (2014). Beans produced with inoculated fermentations had higher concentrations of lactic, acetic and succinic acids, as well as higher levels of volatile compounds. Propionic, butyric, oxalic and tartaric acids were absent, indicating that yeasts suppressed the production of these undesirable metabolites. In general, coffee obtained from fermentations inoculated with selected yeasts was observed to have a caramel, acidic and bitter taste that was not detected in the control. S. cerevisiae has also been reported to improve coffee quality by increasing the level of organic acids (i.e. citric, acetic and succinic acids), sugar (mainly sucrose) and volatile compounds (2017).
In a 2021 study, four yeast species, Meyerozyma caribbica, Saccharomyces cerevisiae, Candida parapsilosis, and Torulaspora delbrueckii, were individually inoculated into natural whole or pulped beans for 40 hours. Higher levels of volatile compounds were detected in the natural process compared to pulped beans. Sensory evaluation found a higher acidic flavor in the inoculations compared to spontaneously fermented beans. Additionally, fruity and caramel aroma notes were more pronounced in M. caribbica-inoculated beans, while citric fruit and caramel flavor with a long aftertaste were perceived in S. cerevisiae-inoculated beans.
In a 2017 study, a new approach to modulate coffee flavor and aroma using microbial species not found among native coffee isolates yielded promising results. In this approach, the yeast Yarrowia lipolytica was used to control the solid-state fermentation of green coffee beans at 6 log CFU/g for 3 days under sterile conditions. In green beans, total alcohol and phenol levels increased, while volatile acids and aldehyde levels decreased. In addition, the levels of non-volatile compounds, including most reducing sugars, organic acids, total free amino acids, and phenolic acids, decreased. After roasting, high levels of 4-vinyl guaiacol, 4-vinyl phenol, alpha-butyrolactone, and sulfur compounds were produced, while similar concentrations of pyrazine were observed in both treatments. The authors believed that thermal hydrolysis of proteins and polysaccharides occurring during roasting may have compensated for the initial low levels of reducing sugars and amino acids and did not affect the occurrence of Maillard reactions and the production of such volatiles. A 2016 study showed that L. plantarum produced high concentrations of lactic acid, ethyl acetate, ethyl isobutyrate, acetaldehyde and ethyl propionate among selected isolates in fermentation with LAB. Such metabolites have fruity aromas and improve the sensory quality of the final products. In addition, batches inoculated with bacteria halved the fermentation time from 24 to 12 h for the control, which is believed to be due to acidification caused by the inoculated bacteria.
In a 2019 study, another member of LAB, Lacticaceaeibacillus rhamnosus, was inoculated into wet coffee fermentation at 7.5 log CFU/ml with or without glucose supplementation for 3 days. High levels of lactic acid (5.2-fold increase) and significant increases in diacetyl and acetoin levels were detected in the inoculation series. Glucose supplementation increased LAB growth and overall aroma of the coffee beans, as well as improving the color of the roasted beans.
The most important contribution of LAB to coffee quality is the inhibition of potential mycotoxin production, such as ochratoxin, by filamentous fungi during the primary processing of coffee beans.
A 2022 study also used non-lactic acid bacteria such as Klebsiella sp, B. subtilis, Pantoea dispersa, and Arthrobacter koreensis as starter cultures and showed evidence of improving sensory scores with each strain causing specific modulations.
Filamentous fungi as starter cultures; In 2010, we tried to explore the positive role of filamentous fungi by inoculating Aspergillus niger into the wet coffee fermentation process. It was found that the mold greatly accelerated the fermentation process by pectinolytic enzymes, which were completed in one hour compared to 24 hours in natural spontaneous fermentation. The coffee aroma was also improved due to the higher concentration of reducing sugars. The authors attributed these changes to enzymatic degradation activities that produced peptides, amino acids and sugars that migrated to the coffee bean endosperm. These findings were later confirmed by adding pectinase produced by Aspergillus niger to the fermentation. Filamentous fungi such as Rhizopus oligosporus are well known for their potent capacity for extracellular enzymes including lipolytic and proteolytic enzymes as well as for the degradation of polysaccharides. Several trials have been reported recently for bioreactor applications in coffee industries. A 2021 study incorporated new technology into the coffee fermentation process called self-induced anaerobic fermentation (SIAF), which claims to improve coffee quality. Fermentation is carried out using closed bioreactors, which gradually lead to the production of CO2, driven by microbial metabolic activities that support the growth of endogenous yeast and lactic acid bacteria, which increase the level of microbial metabolites compared to the traditional fermentation process. A comparative study conducted in 2022 showed superior quality of coffee produced by SIAF over traditional fermentation using both whole and dehulled beans. The study showed high initial microbiota diversity, which then selectively increased LAB and yeast populations under the SIAB bioreactor condition. The authors reported fruity, caramelized sugar, chocolate and herbaceous notes with SIFA coffee, while the traditional process showed lower sensory notes. Such changes were associated with the anaerobiosis environment occurring in bioreactors, which altered bean chemical compounds including lactic acid, chlorogenic glycerol, trigonelline, and sucrose compared to the traditional fermentation process. In a related study conducted in 2021, bioreactor and starter culture technology were combined and applied to improve the coffee fermentation process. Yeasts: Saccharomyces cerevisiae, Candida parapsilosis, and Torulaspora delbruecki were inoculated separately and in selective combinations in closed polypropylene bioreactors and compared with spontaneous fermentations. The authors showed that co-inoculations of C. parapsilosis T. delbruecki produced coffees with the highest sensory scores, while other yeast inoculation treatments resulted in lower scores compared to spontaneously fermented coffee. In the 2021 study, similar to the above study, yeast inoculations were performed in polyethylene bioreactors and found the best performance of S. cerevisiae and T. delbrueckii in the doughy and natural dry methods, respectively.
In summary of this publication, Coffee berries undergo three different primary processes, wet, dry and semi-dry, where spontaneous fermentation is dependent on endogenous microorganisms occurring in the beans. A complex microbial community consisting of bacteria, yeast and, to a lesser extent, filamentous fungi is involved in coffee fermentation. Their main role is to degrade the mucilage layer. The mechanism of pulp and mucilage liquefaction or degradation that occurs during wet coffee fermentation is controversial and it is not clear whether it is due to yeasts, bacteria, acidification or endogenous enzymes, further studies are needed. During fermentation, the endogenous microbiota utilizes the nutrients present in the beans, grows and produces a wide range of non-volatile and volatile metabolites.
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