Inverse Association between Coffee and Tea Consumption

Abstract

Coffee and tea are commonly consumed beverages. Inverse associations with mortality have been suggested for coffee and tea, but the relationships with cause-specific mortality are not well understood. We examined regular and decaffeinated coffee and tea in relation to mortality due to all causes, vascular, nonvascular, and cancer in the multi-ethnic, prospective, population-based Northern Manhattan Study. The study population included 2461 participants with diet data who were free of stroke, myocardial infarction, and cancer at baseline (mean age 68.30 ± 10.23 y, 36% men, 19% white, 23% black, 56% Hispanic). During a mean follow-up of 11 y, we examined the associations between coffee and tea consumption, assessed by food frequency questionnaire, and 863 deaths (342 vascular related and 444 nonvascular including 160 cancer deaths) using multivariable-adjusted Cox models. Coffee consumption was inversely associated with all-cause mortality [for each additional cup/d, HR = 0.93 (95% CI: 0.88, 0.99); P = 0.02]. Caffeinated coffee was inversely associated with all-cause mortality, driven by a strong protection among those who drank ≥4 cups/d. An inverse dose-response relationship between tea and all-cause mortality was suggested [for each additional cup/d, HR = 0.91 (95% CI: 0.84, 0.99); P = 0.01]. Coffee consumption ≥4/d was protective against nonvascular death [vs. <1/mo, HR = 0.57 (95% CI: 0.33, 0.97)] and tea consumption ≥2/d was protective against nonvascular death [HR = 0.63 (95% CI: 0.41, 0.95)] and cancer [HR = 0.33 (95% CI: 0.14, 0.80)]. There was a strong inverse association between coffee and vascular-related mortality among Hispanics only. Further study is needed, including investigation into the mechanisms and compounds in coffee and tea responsible for the inverse associations with mortality. The differential relationship between coffee and vascular death across race/ethnicity underscores the need for research in similar multi-ethnic cohorts including Hispanics.

Introduction

In 2000, The National Coffee Association reported that 54% of adults in the US drink coffee daily. Based on the antioxidants per serving and the per capita consumption, coffee is the primary source of antioxidants in the U.S. diet. An average cup of brewed coffee has 396 mg of polyphenols, including ∼100 mg of chlorogenic acid. Tea is the second most commonly consumed beverage in the world and also contains many polyphenols, including catechins and theaflavins. However, coffee and tea are also important sources of caffeine, and studies have shown some negative health consequences of caffeine consumption, particularly in relation to cardiovascular disease (CVD)6 risk, including raised blood pressure, arterial stiffness, circulating norepinephrine, and decreased endothelial-dependent vasodilation. These effects may only be acute and therefore not relevant for long-term cardiovascular health risk of coffee and tea consumption, but that remains unclear.

Elucidation of the health benefits of coffee and tea is important due to their popularity in the US and globally, yet the relationship between coffee and tea and mortality remains unclear. Several large studies have shown inverse associations between coffee consumption and all-cause mortality. However, the cause-specific mortality driving the associations is not well understood. High coffee consumption is associated with decreased mortality due to CVD, respiratory disease, stroke, diabetes, infections, and inflammatory diseases, but inconsistent results have been observed across studies. A potential J-shaped relationship for CVDs has also been suggested. The relationship with cancer mortality is particularly unclear, with a few studies failing to show an association  or showing differences in the association across gender.

The relationship between tea consumption and all-cause and cause-specific mortality has been less well studied. Whereas some studies have suggested a protective effect of tea on all-cause mortality, specifically CVD mortality, the findings have been inconsistent and much of the research has been conducted in Japan. The evidence for a relationship between tea consumption and cancer mortality is lacking and more research is needed.

This study’s goal was to examine the relationship of coffee and tea with all-cause mortality, mortality due to vascular causes, nonvascular causes, and cancer specifically in the prospective population-based Northern Manhattan Study (NOMAS). This study adds to the literature by examining the relationship of both coffee and tea with cause-specific mortality in a racially/ethnically diverse population of whites, blacks, and Hispanics living in the same community. Hispanics represent the fastest growing U.S. minority group and little is known about the potential effects of coffee and tea on mortality in a predominantly Hispanic population, where the risk of stroke and other causes of death are elevated.

Materials and Methods
Study population.

NOMAS is a prospective cohort study designed to determine the incidence and risk factors for vascular outcomes in a multi-ethnic urban population. Study details have been published.

Eligible participants had never been diagnosed with stroke, were >40 y old, and resided in Northern Manhattan for ≥3 mo in a household with a telephone. Participants were identified by random-digit dialing and recruited from the telephone sample to have an in-person interview and assessment. The enrollment response rate was 75% for a total of 3298 participants, with an average annual contact rate of 95%. Participants with a previous myocardial infarction (MI) (n = 201) or cancer diagnosis (n = 303) and those without information on coffee or tea consumption (n = 333) were excluded. The study was approved by the Columbia University and University of Miami institutional review boards. All participants provided informed consent.

Baseline evaluation (1993–2001).

Data were collected through interviews with trained research assistants in English or Spanish. Physical and neurological examinations were conducted by study neurologists. Race-ethnicity was based on self-identification through questions modeled after the U.S. census and conforming to standard definitions outlined by Directive 15. Standardized questions were adapted from the Behavioral Risk Factor Surveillance System by the CDC regarding hypertension, diabetes, smoking, and cardiac conditions. Measurement of blood pressure and fasting blood specimens for glucose and lipids and the definitions of hypertension, hypercholesterolemia, diabetes, moderate-heavy physical activity, and moderate alcohol use have been described.

Coffee and tea consumption.
At baseline, participants were administered a modified Block National Cancer Institute FFQ by trained bilingual research assistants. The questionnaire contained questions regarding the average consumption of decaffeinated coffee, regular (caffeinated) coffee, and tea (hot or iced). For this study, instances of intake referred to the consumption of approximately one medium cup. The possible responses were: never or <1/mo, 1–3/mo, 1/wk, 2–4/wk, 5–6/wk, 1/d, 2–3/d, 4–5/d, and ≥6/d.

Due to small sample sizes for each individual consumption category, and to make our analyses comparable to other studies, all coffee (regular and decaffeinated combined) and regular coffee were categorized as: <1/mo (reference), 1/mo to 4/wk, 5–7/wk, 2–3/d, and ≥4/d. Due to the lower frequency of tea and decaffeinated coffee consumption in our population, the top 3 exposure categories for these variables were combined in categorical analyses as ≥2/d. The coffee and tea exposures were also examined as pseudo-continuous variables approximated as cups/day, assigning the middle value for each questionnaire response category.

The consumption frequency of other beverage variables that were assessed in a similar manner included regular soft drinks, diet soft drinks, orange and grapefruit juice, other fruit juices or fortified drinks, Tang or Start breakfast drinks, whole milk, 2% milk, and skim or 1% milk. The consumption frequency of these other beverages was summed to create a continuous variable representing other non-water beverage consumption per day. The FFQ also assessed the frequency of consumption of commonly used additives to coffee and tea, including cream or half-and-half, milk, and nondairy creamer. The frequency of consumption of these additives was assessed in a similar manner and included individually as continuous covariates to examine the effects of coffee and tea consumption independent of their creamers.
Annual follow-up and definition of outcomes.

Participants were telephone screened annually to determine changes in vital status. Hospital surveillance of admission and discharge data, including ICD-9 codes, also provided morbidity and mortality data. Family members were reminded to notify us in the event of death.

The primary outcome was all-cause mortality. Secondary outcomes were death due to confirmed vascular causes (stroke, MI, heart failure, pulmonary embolus, cardiac arrhythmia), death due to nonvascular causes (accident, cancer, pneumonia, chronic obstructive pulmonary disease, other pulmonary disorders, and other nonvascular causes), and death due to cancer. Deaths were classified as vascular or nonvascular based on information obtained from the family and physicians and validated with medical records and death certificates. Follow-up procedures and outcome classifications were previously detailed.

Statistical analysis.

We constructed Cox proportional hazards models to examine the associations between coffee (any and regular only) and tea consumption and mortality, and HRs and 95% CIs were calculated. Decaffeinated coffee alone was examined in secondary exploratory analyses. Person-time of follow-up was accrued from baseline to the end of follow-up (March, 2012), death, or loss to follow-up, whichever came first. We used the following sequence of models: 1) adjusted for demographics (age, sex, race/ethnicity, education); 2) demographics, behavioral risk factors [smoking (never, former, current), moderate-heavy physical activity, moderate alcohol consumption], daily diet (total kilocalories, grams protein, total fat, saturated fat, carbohydrates), and BMI; and 3) demographics, behavioral risk factors, diet, BMI, previous cardiac disease, diabetes, hypertension, and hypercholesterolemia. In model 3, we mutually controlled for tea and coffee. We examined interactions of coffee and tea with age, sex, and race/ethnicity in relation to each of the mortality outcomes in model 3, and stratified analyses were conducted when effect modification was suggested (P-interaction < 0.05). We observed no interaction between coffee and tea in relation to any of the mortality outcomes. Because self-reported total daily kcal <500 or >4000 might indicate inaccurate reporting of diet, we conducted sensitivity analyses excluding these participants (n = 73). In an exploratory analysis, we also examined the combined effect of coffee and tea by adding the coffee and tea cups per day and examined this exposure as a continuous variable in relation to the mortality outcomes in model 3. The proportionality assumption of the hazards was first confirmed in the Cox models. This was done by examining interactions between the coffee and tea exposures of interest with the logarithm of follow-up time in relation to the mortality outcomes in model 3. No significant interactions were found.

We also ran a sensitivity analysis model 4 that included a variable representing the number of other non-water beverages (soda, juice, and milk) consumed per day and nondairy creamer, cream or half-and-half, and milk added to coffee and tea. In model 4, we controlled for smoking in continuous pack-years rather than as a categorical variable and alcohol as a continuous variable, representing the glasses of beer, wine, and liquor consumed per day, rather than as a dichotomous variable.

Lastly, we ran a sensitivity analysis simultaneously examining vascular and nonvascular death using competing risks regression rather than Cox models in model 3 and sensitivity analysis model 4.

In all analyses α = 0.05. All analyses were conducted using SAS version 9.3 (SAS Institute).

Results

Of the 3298 NOMAS participants, 2461 were included in this study. Supplemental Table 1 shows the distribution of regular coffee, decaffeinated coffee, and tea consumption in NOMAS. Table 1 shows the vascular risk factor profile of the study population overall and according to coffee consumption categories. During a mean follow-up of 11 y (range 0–18 y), 863 deaths accrued, 342 due to vascular causes (40 strokes, 48 MIs, 42 heart failure events, 8 pulmonary emboli, 165 cardiac arrhythmias, 36 other vascular causes) and 444 due to nonvascular causes (11 accidents, 160 cancer, 100 pneumonia or chronic obstructive pulmonary disease, 61 infections, 30 renal causes, 72 other nonvascular causes).

The relationship between coffee and tea and all-cause mortality. A dose-response inverse relationship was suggested between coffee and all-cause mortality with a 7% reduced mortality for each additional cup per day in model 3 (P < 0.05). Regular caffeinated coffee in particular was inversely associated with all-cause mortality, driven by a strong protection among those who drank ≥4 cups/d. An inverse dose-response relationship between tea consumption and all-cause mortality was also suggested, with 9% decreased mortality for each increased cup per day (P < 0.05). In sensitivity model 4, the association for all coffee was slightly attenuated but was somewhat stronger for tea. Decaffeinated coffee consumption was relatively rare, limiting statistical power to examine it separately, but exploratory analyses suggested a possible inverse trend with all-cause mortality [model 3: decaffeinated cups per day, HR = 0.88 (95% CI: 0.79, 0.98)]. When coffee and tea were combined, an inverse association was found, with an 8% decreased mortality for each cup per day [model 3, (95% CI: 0.88, 0.97)].

For vascular death, the associations for coffee and tea were slightly attenuated and no significant associations were observed, including when they were combined as a single variable [model 3 cups/d, HR = 0.95 (95% CI: 0.88, 1.02)]. We found a negative interaction between Hispanic ethnicity and all coffee and regular coffee in relation to vascular death (P-interaction < 0.05). There was no association between coffee consumption, any or regular only, and vascular-related mortality among whites or blacks. However, among Hispanics, there was a protective association between both any coffee and regular coffee consumption and vascular death [any coffee HR = 0.78 (95% CI: 0.65, 0.94)].

There was a significant protective association for nonvascular death observed among those who consumed ≥4 cups/d of coffee [model 3, HR = 0.48 (95% CI: 0.29, 0.81)]. The potential protective association for all coffee appeared to be driven by decaffeinated coffee. In an exploratory analysis of decaffeinated coffee assessed continuously, a strong inverse trend was observed [model 3, HR = 0.81 (95% CI: 0.68, 0.96)]. An inverse association was found for tea consumption, with a 13% reduction in nonvascular mortality with each additional tea cup per day in model 3 (P < 0.05), and this association was only strengthened in model 4. When coffee and tea were combined, a 9% decreased risk of nonvascular death was observed for each cup per day of coffee or tea [model 3, (95% CI: 0.85, 0.98)].

Although the strength of association between coffee consumption, assessed continuously, and cancer mortality was slightly more protective than that for all nonvascular death, the association was not significant. The association for regular coffee was also not significant. However, the power to conduct these analyses was limited (Supplemental Tables 3 and 4) and the effect estimates were not inconsistent with a potential protective association of high coffee consumption (e.g., ≥4 cups/d) with cancer mortality. There was an inverse relationship between tea consumption and cancer mortality such that cancer mortality decreased by 26% for each additional cup per day in model 3 (P = 0.02). This association appeared to be driven by a potentially decreased cancer mortality among those in the highest category of tea consumption (≥2/d). A 14% reduced risk of death due to cancer was found for each cup per day of coffee or tea in the combined exposure analysis (P < 0.05).

For all analyses, the associations remained consistent when we excluded those with improbably low (<500) or high (>4000) reported daily kilocalorie consumption (not shown). We observed no significant interaction between coffee or tea consumption and the demographic variables in relation to overall mortality, nonvascular mortality, or cancer mortality.

Table 7 shows the relationship between all coffee, regular coffee, decaffeinated coffee, and tea consumption in relation to vascular death and nonvascular death in models 3 and 4 using competing risk regression. Overall, the conclusions from these analyses were consistent with the results of the corresponding Cox models, although the effect estimates were attenuated. In these analyses, neither coffee nor tea was associated with vascular death in the full cohort. However, consumption of ≥4 cups/d of coffee was associated with a decreased risk of nonvascular death, as was consumption of ≥2 cups/d of tea in model 4. In fact, a protective dose-response relationship between tea consumption and risk of nonvascular death remained apparent.

Discussion

The majority of adults sampled in NOMAS drank coffee, often daily, and almost two-thirds drank tea. Due to the popularity of these beverages, a greater understanding of their health consequences is imperative. In this prospective cohort study, we observed a protective dose-response relationship between coffee and tea consumption and all-cause mortality.

For nonvascular death, an inverse association with coffee consumption was also suggested, driven by a protective effect observed among those in the highest exposure category (≥4/d). The inverse association between any coffee with nonvascular death was likely attributed to decaffeinated rather than caffeinated coffee, although further study in populations with greater consumption of decaffeinated coffee is needed to confirm these findings. Tea consumption also appeared to be potentially protective for nonvascular death, particularly cancer death. In fact, tea appeared to be more associated with nonvascular death and cancer than coffee. Despite effect estimates consistent with a potentially protective association between frequent coffee consumption (≥4 cups/d) and cancer mortality, no significant association was found in this power-limited analysis of cancer mortality.

Overall, our findings in a multi-ethnic, predominantly Hispanic cohort are consistent with other studies that have demonstrated inverse associations between coffee consumption and mortality. The results of several studies, including the Nurses’ Health Study and Health Professionals Follow-up Study, have suggested that death due to CVD may be driving the association for coffee rather than cancer or other nonvascular causes. However, our results did not support those conclusions, as those with the highest consumption of coffee in NOMAS had a decreased risk for nonvascular death and the effect estimates for coffee cups per day were similar for vascular and nonvascular death. Although studies have shown inverse associations between coffee consumption and morbidity and mortality due to overall CVD, an effect on stroke has also been suggested, yet evidence is limited and inconsistent.

To the best of our knowledge, our study is the first to show differences by race/ethnicity for the association between coffee and vascular mortality. We observed a strong inverse association between coffee and vascular-related mortality among Hispanics only, suggesting the need for further research in large, multi-ethnic cohorts including Hispanics. The Hispanic population in NOMAS was primarily of Caribbean descent. We previously showed differences in dietary patterns across race/ethnic groups in our cohort, and in the current study, we found that whites and Hispanics in NOMAS consumed coffee more frequently than blacks. However, our study was not designed to elucidate whether there may be relevant differences in the types of coffees consumed by Hispanics and non-Hispanics in our community-based cohort. Some of the effects of coffee are dependent on the genotypes of certain enzymes, such as the CYP1A2 enzyme that is the main metabolizer of caffeine. Both positive and negative associations of coffee are affected by the speed at which its constituents are metabolized and how long they remain in the body. It is possible that variability in genes involved in the metabolism of caffeine, antioxidants, and other components of coffee could be responsible for differences in associations across race/ethnic groups, but other future studies will have to investigate this possibility further. For example, there is some evidence to suggest that there are race/ethnic differences in CYP1A2 enzyme activity in liver microsomes.

Our findings of an inverse association between tea consumption and all-cause mortality are also consistent with the findings of a few previous studies, although the literature has been scant and inconclusive. In our study, the protective relationship between tea and overall mortality appeared to be driven by nonvascular causes, particularly cancer. However, the limited statistical power of our analyses of tea is important to note (Supplemental Tables 3 and 4) and no strong conclusions can be made regarding the relationship between tea consumption and vascular mortality. Other studies have shown tea to be protective against CVD mortality.

Evidence for a protective effect of coffee or tea on the risk of death from cancer is lacking, which may be attributed to the etiologic heterogeneity of cancer when all types are grouped together, yet our data suggested an inverse trend between tea consumption and cancer death. Unfortunately, we were not able to examine cancer types separately. A large Japanese study showed an inverse relationship between coffee and all-cause mortality in both men and women, whereas a weak inverse association with cancer mortality was observed only in women. In terms of cancer morbidity, the relationships for coffee or tea consumption are inconclusive. Protective associations have been suggested for particular cancer types in some studies, but the findings have been inconsistent. For example, in some, but not all, studies, coffee consumption was inversely associated with colon cancer incidence, aggressive prostate cancer, and bladder cancer. Although our findings were consistent with a possible protection against cancer mortality among those who drank ≥2 cups/d of tea, more research is required before any strong conclusions can be made. Much of the research on tea consumption in relation to cancer and other diseases has been conducted in Asian countries, where tea is consumed in higher volumes, and findings may not be generalizable across populations.

Epidemiologic studies like ours are not able to identify the underlying mechanisms or compounds in coffee/tea that may be responsible for the protective associations with mortality. However, the high antioxidant content of these beverages is hypothesized to play a role. The chlorogenic acid in coffee may decrease blood pressure by increasing NO. The antioxidants in coffee, such as phenolic acid, have been shown to increase the resistance of LDL to oxidation. Coffee consumption is also associated with lower plasma concentrations of certain markers of inflammation and endothelial dysfunction as well as uric acid. An inverse association between caffeine and coffee consumption and risk of diabetes, an important cause of mortality in the elderly, has been reported, likely due to the reduction in free radical generation from antioxidants derived from coffee consumption. In mice, both coffee and caffeine have been shown to improve glucose tolerance, insulin sensitivity, and hyperinsulinemia, findings that are at least partly a result of reduced inflammatory adipocytokine expression.

A role of caffeine consumption in mortality, particular vascular-related mortality, should also be considered, because coffee is a leading source of caffeine intake. As previously mentioned, caffeine has myriad health consequences, including raised blood pressure, arterial stiffness, circulating norepinephrine, and decreased endothelial-dependent vasodilation. Whereas acute consumption of caffeine can increase blood pressure, habitual use of caffeine does not have a linear association with incident hypertension over the long term. It may be that the antioxidant content or other beneficial components of coffee consumption simply outweigh a potential deleterious effect of caffeine. When we restricted our analysis to specifically examine regular coffee, the associations did not become appreciably stronger, and in fact when we performed exploratory analyses examining decaffeinated coffee only we saw that it was inversely associated with all-cause mortality and nonvascular-related mortality, with dose-response effect estimates slightly stronger than those for regular coffee. Previous studies with more power also showed protective effects for decaffeinated coffee in relation to overall mortality and CVD-related mortality. Therefore, the caffeine in regular coffee is most likely not responsible for the protective associations observed.

The examination of both coffee and tea, separately and combined, in relation to cause-specific mortality makes this study particularly innovative and the primary strength is the use of a racially/ethnically diverse population of adults living in the same community. This primarily Hispanic population represents one that has not been well studied in terms of the health consequences of coffee and tea consumption. However, the use of an older adult population from northern Manhattan with a unique race/ethnic composition limits the generalizability of our findings to other populations. Although we controlled for many potential confounders that are known risk factors for death, residual confounding by unmeasured and measured risk factors, including correlated dietary habits, is possible. Despite the use of a validated and reliable Block FFQ, there are some limitations to our coffee and tea exposure data. The analysis focuses on frequency of consumption and is not standardized for cup size. Though the majority of coffee and tea consumers reported that their normal dose was a “medium cup,” this may be interpreted differently across individuals. Misclassification due to self-reported beverage consumption is possible, although the study is prospective and we excluded those with improbably low or high self-reported total daily energy consumption in sensitivity analyses with the goal of minimizing misclassification bias. Although the FFQ is designed to measure average consumption over the previous year, dietary information was collected at one time (baseline) and we lacked information on duration of coffee and tea consumption as well as changes during follow-up. Although most Americans drink filtered coffee, NOMAS did not distinguish in the questionnaire whether individuals were drinking boiled unfiltered coffee or filtered coffee. Unfiltered coffee has been associated with an increased risk for CVD due to the diterpene oils found in unfiltered coffee that raise serum cholesterol concentrations. Filtered coffee has <0.1 mg/100 mL of the diterpene oils and unfiltered coffee can have 0.2–18.0 mg/100 mL. An additional limitation is that the FFQ did not specify the types of tea consumed (black, green, etc.). Different types of teas have varying amounts and types of polyphenols and other constituents. Reverse causality is also possible, if individuals with mortality-related diseases reduce or eliminate their coffee consumption. However, our cohort was stroke-free at baseline and we excluded individuals with an MI or cancer diagnosis prior to baseline. Lastly, some misclassification of the cause of death cannot be ruled out. In practice, it is difficult to tease out causes of death and vascular causes are often ascribed due to lack of definitive information. Also, if someone has a history of cancer and dies, they may get classified as a cancer death, when the actual cause is something else.

In conclusion, in our multi-ethnic population-based cohort of adults living in northern Manhattan with >10 y of follow-up, we found that coffee and tea consumption were inversely associated with mortality. Further study is needed, including investigation into the potential underlying mechanisms and compounds in coffee and tea that may be responsible for this inverse association, before public health recommendations are warranted.

Resource: http://www.ncbi.nlm.nih.gov
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A new way to production of Coffee Bean Extract

Abstract

Coffee is a critically important agricultural commodity for many tropical states and is a beverage enjoyed by millions of people worldwide. Recent concerns over the sustainability of coffee production have prompted investigations of the coffee microbiome as a tool to improve crop health and bean quality. This review synthesizes literature informing our knowledge of the coffee microbiome, with an emphasis on applications of fruit- and seed-associated microbes in coffee production and processing. A comprehensive inventory of microbial species cited in association with coffee fruits and seeds is presented as reference tool for researchers investigating coffee-microbe associations. It concludes with a discussion of the approaches and techniques that provide a path forward to improve our understanding of the coffee microbiome and its utility, as a whole and as individual components, to help ensure the future sustainability of coffee production.

INTRODUCTION

The cup of coffee we drink today is prepared from the roasted seeds of Coffea arabica and/or Coffea canephora var. robusta, which are produced in many tropical nations. Coffee exports topped 109 million bags, valued at over $20 billion in the 2011-to-2012 coffee year, and production continues to increase. Given its economic importance, an extensive body of scientific literature exists examining agronomic methods and the chemistry of the coffee beverage. The microbiology of coffee seeds and changes in microbial communities induced by coffee processing have also been investigated, though less extensively.

Increasing interest in improving coffee quality, protecting coffee production, and creating sustainable coffee markets has prompted recent investigations to understand and exploit the microbial communities associated with coffee at all stages of plant development and seed processing. This review highlights and synthesizes the literature relevant to our understanding of the coffee microbiome with a strong focus on seed- and fruit-associated microbes and their roles in coffee production and processing. This work also reviews how coffee microbes are currently being exploited and how more extensive use of microbes may improve coffee health, production practices, and beverage quality.

COFFEE PRODUCTION AND THE COFFEE MICROBIOME

In order to understand the nature of the coffee microbiome, one needs to understand the production and processing activities that could affect its structure. Coffee plants are predominantly propagated using seed stock germinated under conditions with various degrees of control in greenhouses or outdoor planters. Seedlings are colonized by seed endophytes, with some microbes adhering to the seed parchment and others present in the germination substrate. After 6 to 12 months, seedlings are transferred to field settings, where they are exposed to prevailing environmental conditions, opening the plant to additional microbial colonization from a variety of sources. Some modes of microbial immigration and emigration, such as splash dispersal and human vectoring, are strongly impacted by crop density and specific cultural practices that vary based on cultivar and growing region. Regardless of origin, coffee plants are actively pruned to increase flowering potential and maintain a manageable plant size. Pruning can vector microbes between plants, provide new entry points for colonization, and open new vegetative growth to colonization Another factor generally affecting microbial colonization is the plant variety. For example, while they are producers of high-quality seeds, some of the oldest coffee varieties are often susceptible to serious coffee diseases. Breeding programs are actively pursuing new cultivars with improved disease resistance and good crop quality. Coffee plants flower and fruit biennially, and fruit development is not synchronized. Fruits are generally harvested manually or are harvested mechanically with a mix of mature and immature fruits. Thus, human vectoring of microbial populations onto fruit that enter the processing stream is likely to occur during harvest. Following harvest, the coffee fruits, or cherries, are sorted and subjected to various postharvest processes to ready them for storage and shipment as green coffee. These processes remove fruit debris, allow microbial growth, and promote fermentation, which can change the physical, chemical, and biological properties of the seeds.

The different processing methods used in the industry likely impact the seed microbiome in very different ways. Dry, or natural, processing involves harvesting ripe coffee fruits and spreading them over a drying surface (e.g., cement patios or packed earth), where they are allowed to dry for 18 to 24 days. This extended environmental exposure likely results in a significant lot-to-lot variation in microbial load due to differences in temperature, humidity, and arthropod activity. Once the cherries are dry, the desiccated fruit tissue is removed to produce parchment coffee, which is stored and subsequently polished to remove the endocarp, making green coffee. Parchment and green coffee seeds are stored at 10 to 15% moisture, a level that allows only minimal microbial activity, prior to export and subsequent roasting. Wet processing, or washed coffee, produces more-dramatic but shorter-lived changes in the chemical and microbial environment of the seed. After the fruit exocarp and pulp are removed, the mucilage-covered seeds then ferment for 24 to 72 h in water at ambient temperatures. Microorganisms from the fruit, seed, handlers, water, and processing machinery can all seed the fermentation and have the potential to affect the coffee quality. After fermentation, the degraded mucilaginous pectin layer is removed from the seeds. The seeds are then rinsed and dried to form parchment coffee. A large body of literature exists that explores the impacts of these coffee processing methods on coffee quality, and it is generally accepted that wet-processed beans tend to produce a more acidic cup of coffee with less body.

MICROBIAL POPULATIONS INHABITING COFFEE FRUIT AND SEED: WHO IS THERE?

In an effort to better understand coffee fermentation, a number of studies have examined the microbial diversity of the coffee seeds during the use of the different coffee processing methods. Other studies have looked beyond the fermentation process and have monitored microbes throughout the processing chain, from endophytic communities of coffee seeds and fruit to epiphytic communities present during green coffee storage. By synthesizing the results of these studies here, we present a more complete picture of coffee seed-associated microbial communities that can be developed and exploited to improve coffee health and quality.

FILAMENTOUS FUNGI AND YEASTS

Fungi are known for their metabolic plasticity, variable metabolite production, and highly prevalent plant associations, and they comprise a substantial portion of the coffee microbiome. A total of 215 named species of fungi have been found in association with coffee fruit and seed tissues. These fungi represent 12 classes from three different phyla. The Basidiomycota (n = 17 species) and Mucoromycotina (n = 5 species) were cited more rarely than the Ascomycota (n = 193 species). Over 40% (n = 80) of the coffee-associated Ascomycota fungi belong to the Eurotiomycetes and consist almost exclusively of members of the genera Aspergillus and Penicillium. Of these, Aspergillus niger was the most commonly cited species.

The extensive knowledge of Eurotiomycetes is likely due, in part, to the emphasis given to unraveling the sources and levels of ochratoxin A (OTA) contamination in coffee. OTA contamination is linked to Aspergillus ochraceus and Aspergillus carbonarius, though Aspergillus niger and several Penicillium species have been implicated as well. Both Aspergillus and Penicillium spp., including those that are capable of producing OTA, were encountered very frequently and appear to be ubiquitous in coffee production from fruit to roasting.

The ascomycete yeasts, in the classes Saccharomycetes (n = 58) and Schizosaccharomycetes (n = 1), accounted for a third of coffee-associated Ascomycota species. The majority of these citations have come from studies examining the microbial community dynamics of coffee fermentation. Agate and Bhat  and Van Pee and Castellani showed that a number of yeasts from Saccharomycetes derived from wet fermentations. Avallone et al. and Masoud et al. confirmed that Saccharomyces, Pichia, and Candida spp. dominated the yeast communities in these wet fermentations through the use of culture-independent surveys. Silva et al. neatly showed that Debaryomyces and Pichia spp. dominate natural fermentation communities after the turnover of bacteria from earlier on in the fermentation process.

Sordariomycetes, dominated by Fusarium spp., representing 18% of the Ascomycota listed in Table 1, were noted primarily in studies focused on microbial community dynamics during natural coffee processing . Other Sordariomycetes, such as Beauveria bassiana, were found living endophytically in coffee seeds. The remaining 10% of coffee-associated Ascomycota were distributed among the Dothideomycetes (n = 14 species) and Leotiomycetes (n = 2 species), with a few species of uncertain taxonomic placement (incertae sedis, n = 2 species). Of these, the dothideomycete Cladosporium cladosporioides was cited as appearing in high abundance among species encountered in microbial succession studies and as an endophyte of coffee tissues.

Members of the other fungal taxa were generally encountered at low frequency. Some members of the Mucoromycotina were cited as spoilage organisms in a number of studies, though poor identification of many of these taxa hindered their inclusion in this review (Table 1). Of note, members of other fungal taxa, especially those from groups with complex systematics such as Fusarium spp., are often given genus-only identifications, which may lead to an underestimation of their importance in the coffee microbiome. The coffee pathogen Hemileia vastatrix (Basidiomycota) is also commonly encountered in the coffee literature due to its negative impact on coffee production. Most of the Basidiomycota and the rarely cited Ascomycota showed up in surveys of endophytic coffee fungi, which suggests a casual association with coffee fruits and seed.

BACTERIA

Bacteria play critical roles in food processing and fermentation and in plant and soil health and participate in important microbe-microbe interactions. Previous studies have noted 106 coffee-associated bacterial species, distributed among six classes belonging to four bacterial phyla (the Proteobacteria, Firmicutes, Actinobacteria, and Bacteroides). Proteobacteria was the most species-rich phylum (n = 58 species), followed by Firmicutes (n = 32) and Actinobacteria (n = 14). Bacteroides was represented by two species in a single class.

Gammaproteobacteria made up 85% of the total Proteobacteria from coffee; the Enterobacteriales family appears to be the most species rich (n = 37) . Enterobacter aerogenes and Pantoea agglomerans were the two most abundantly encountered Enterobacteriales species. Members of Enterobacteriales, along with members of Pseudomonadales, are common soil- and plant-associated Gram-negative bacteria. These groups are expected in humid, nutrient-rich environments like those found during coffee processing.

Bacillus spp. constituted 43% of the coffee-associated Firmicutes species. Of these, Bacillus cereus and B. subtilis were cited more commonly than any other bacterial species. Both species were found in both wet and dry fermentations and as endophytes. The members of Firmicutes also include Lactobacillales (46% of Firmicutes), which were found in high abundance during wet fermentations, especially Leuconostoc spp. The Actinomycetales (n = 15 species) were represented by 12 different genera. The high number of different genera and low citation frequency among the members of this group suggest that they are casually associated with coffee fruits and seeds. Alternatively, the Actinomycetales may be involved only casually in coffee fermentations, which have been the focus of the majority of studies on coffee seed microbiology. To date, no reports of Archeae on coffee have been made, a reflection on the lack of application of the appropriate profiling tools.

MICROBES FROM COFFEE FRUIT AND SEED: WHAT CAN WE DO WITH THEM?

The current understanding of the activities of the coffee seed microbiome is heavily based on investigations of microbial roles in coffee fermentation, microbial community composition and dynamics during fermentation, and microbial impacts on coffee quality. Given the wide variety of conditions that fruit and seeds are exposed to, it is likely that geography- and process-specific variations exist in seed microbiomes beyond what has been recorded to date. However, as the majority of plant microbial populations are chemoheterotrophs, the chemistry of the fruits and seeds, combined with the moisture regimens to which they are subjected, likely contributes to a common set of environmental parameters that select for a core microbiome. As the research community begins to move beyond descriptive surveys of microbial diversity, it is from this core of coffee-adapted microbes that the most functionally interesting and useful microbes will emerge. Microbial inoculants have long been used to alter, preserve, and improve food products. It is now recognized that diverse microorganisms can be utilized to improve a wide range of agricultural and industrial products and processes beyond fermentation. Here, we highlight several applications where microbes have been, or could be, introduced to help improve coffee production and quality .

IMPROVING COFFEE QUALITY WITH MICROBES

OTA is now a major factor in assessing coffee quality, and it is allowable in coffee imports to Europe at levels of <5 ppb. Currently, cultural controls are promoted as the best methods for controlling OTA contamination, though the application of fungicides is also cited as a possible preventative measure. Recently, interest has turned to trying to prevent OTA contamination through the use of biological agents to outcompete or inhibit the growth of Aspergillus spp. during processing. Djossou et al. examined the use of Lactobacillus plantarum, isolated from coffee pulp, as a biological antagonist against OTA-producing fungi. The authors were able to show an inhibitory effect on ochratoxigenic fungal growth; however, no mode of action was ascribed. In their work with yeasts isolated from coffee fermentation, Masoud et al. demonstrated an inhibitory effect on Aspergillus ochraceus growth and germination during cocultivation with coffee yeast strains. Further, they were able to identify several volatile compounds produced by the yeasts, including 2-phenyl ethyl acetate, which could replicate the growth inhibition when Aspergillus ochraceus cultures were exposed to the compound alone. Such mode-of-action studies will become important in developing in vivo assays and applications. Velmourougane et al. were able to demonstrate in vivo that application of a yeast treatment during processing resulted in a reduction of total ochretoxigenic mold incidence and OTA levels. Combined, these works present a pathway to establish biocontrol alternatives for control of unwanted microbes, namely, in vivo control efficacy studies coupled with in vitro mode of action studies. This pipeline can be improved on by introducing a more efficient, high-throughput screening strategy for candidate microbe recovery and identification.

MANAGING COFFEE WASTE

Coffee production and utilization generates byproducts in the form of coffee cherry pulp and hulls, wastewater, and spent coffee grounds. Only 6% of coffee fruit mass contributes to the final coffee product; the rest is considered a byproduct. Coffee pulp is one of the most abundant examples of agricultural waste, and, due to its high concentrations of caffeine and phenolic compounds, it is relatively useless for traditional value-added products (e.g., animal feed and supplements) and is often treated as an environmental hazard. There has been much research into ways to detoxify or repurpose coffee byproducts , many using microbial transformation. Several studies have explored the use of microbes isolated from coffee pulp for its remediation, focusing on caffeine detoxification. This would allow the creation of either a value-added product in the form of silage for animal feed or, at the very least, the detoxification of the byproducts so that they could be safely stored or composted.

The massive amount of waste created each year in the coffee production pipeline can also serve as a substrate for the manufacturing of other valuable products for industry, such as enzymes. Boccas et al. examined the use of coffee pulp as a substrate for the microbial production of pectinase. Others have looked at byproducts of roasting for enzyme production, including α-amylase and β-fructofuranosidase from silver skin and xylanase from spent coffee grounds. Others have examined the production of industrially useful small molecules from coffee byproducts. For example, while exploring the phenol- and pectin-degrading abilities of Gluconacetobacter hansenii isolated from wine, Rani and Appaiah found that when the bacterium was incubated with coffee cherry extract (another waste product), it was able to alter concentrations of free phenolic acids, such as vanillic acid and tannic acid. These compounds are important flavoring components used in the food industry. Given the highly distinctive environment that coffee waste presents, this material is a rich source of microbes able to produce or alter plant metabolites that may transform coffee pulp into a significant resource for many important industrial applications.

MICROBES TO CONTROL COFFEE DISEASES AND ENVIRONMENTAL STRESSES

Recent changes in the distribution and host range of coffee diseases and pests have made the control of coffee disease of keen interest. A number of efforts aimed at breeding resistant cultivars are being pursued, in addition to the targeted use of pesticides. Given the desire to support sustainable cropping systems and reduce the use of toxic pesticides, a number of investigations have been aimed at the use of biological agents to control coffee pests and disease. While some have examined epiphytic microbial communities, many have focused on endophytes from coffee tissues . Endophytes have been known for their ability to confer increased pest resistance, resistance to herbivory , and tolerance of environmental stress. With regard to disease resistance, both bacterial endophytes  and fungal endophytes have been explored as agents to promote resistance to the coffee leaf rust pathogen. Other comprehensive studies have been aimed at isolating endophytic bacteria and fungi to protect crops against the coffee berry borer Hypothenemus hampei.

The chemical profile of coffee plants makes them an interesting model for studies in chemical ecology and plant host defense. Looking at how compounds such as caffeine, a compound thought to reduce herbivory , respond to microbial pressure would have implications not only for coffee pest control but for microbially mediated host defense in other systems as well. For example, Chaves et al. demonstrated that an endophytic Aspergillus oryzae strain isolated from coffee leaves was able to induce the caffeine defense response though kojic acid production, which might protect plants from insect pests. Deciphering the exact roles that different endophytes play in stimulating plant defenses will help inform their utility as biocontrol agents.

LOOKING FORWARD: NEW APPROACHES TO STUDY THE COFFEE MICROBIOME

The studies reviewed above collectively indicate that diverse microbial populations impact coffee yield and quality. Here, we describe how new approaches, such as marker-assisted selection, could alter the efficiency and speed with which scientists could address some of the larger issues of coffee sustainability in a changing climate. These issues include identifying coffee-adapted microbial agents to help control coffee disease, increase tolerance to environmental stress, and improve coffee quality.

While a majority of plant-associated microbes are considered commensal, some can have positive impacts on plant health through the promotion of plant growth, disease resistance, and tolerance to environmental stresses. A large body of literature exists that describes the biocontrol- and plant growth-promoting properties of dozens of microbial agents and the mechanisms through which these benefits are conferred. The development of biocontrol agents has depended greatly on traditional isolation and screening procedures for discovery , the same procedures employed in most coffee microbiology studies to date. Sifting through hundreds to thousands of isolates to remove redundant genotypes and test their efficacy is very time-consuming and inefficient. Given the rapidity of coffee pest and pathogen spread in recent years , a quicker approach to bioprospecting for novel natural products or coffee-adapted biocontrol agents is needed. Benítez et al. employed community profiling to rapidly identify microbes statistically associated with disease suppression. Subsequently, the markers identified were used to direct the isolation and characterization of the potentially disease-suppressing microbes, resulting in the rapid recovery and characterization of two novel species of biocontrol bacteria . Together, these studies highlight how adjusting our approach to the use of a popular scientific tool, microbial community profiling, can result in the efficient generation of targets for disease control organisms using limited resources and time. The “next-generation” DNA sequencing technologies have at last become a competitive alternative for community analysis through massively parallel sequencing of community-derived DNA amplicons and direct metagenomic sequencing. These new approaches generate sequence data that can be directly employed to design selective isolation procedures for targeted microbe recovery. Such approaches are currently being used to identify microbial populations associated with increased millet growth in Senegal. The application of such marker-assisted selection and recovery to coffee-associated microbes will likely provide new inoculants of value to prevent disease, promote stress tolerance, and improve bean quality.

In order to address the issue of mechanism and timing for specific microbial activities, identifying targets is a critical first step, but it is only a part of the equation. To demonstrate a microbe's impact on a desired trait and, indeed, to exploit it during production, the microbe needs to be recovered in culture. It is agreed that the majority of the microbial diversity has yet to be cultured. This could be due to a number of factors, including improper isolation media or growth conditions. With this in mind, future coffee microbiome research efforts should focus on sampling and selection factors specific to coffee to increase the isolation of novel microbial targets, such as the yet-to-be studied Archeae. Park et al. present one such model for increasing the isolation efficacy for novel bacterial agents. Using marker-assisted selection and a balanced multifactorial sampling design, they were able to increase the efficiency of recovering novel bacterial genotypes with the targeted function by 5-fold. Such a sampling strategy can be used to select previously unstudied coffee microbes for further phenotypic screening.

Once in culture, microbial strains can be used in manipulative experiments to determine their impacts on many facets of coffee production and quality. The techniques and approaches to studying the coffee microbiome mentioned here will allow us to address more-specific questions concerning coffee microbiology. Are there other microbial endophytes that promote crop health and disease and pest resistance? Can microbes be used to improve coffee tolerance of a changing environment? To what extent are microbes associated with coffee seed defects, and how might they be employed to prevent them? Utilizing the aforementioned techniques, coffee researchers will advance our understanding of the coffee microbiome and help ensure a future for the world's largest-production agricultural commodity.

Resource: http://www.ncbi.nlm.nih.gov
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