Microbial ecology and starter culture technology in coffee processing

Coffee has been for decades the most commercialized food product and most widely consumed beverage in the world, with over 600 billion cups served per year. Before coffee cherries can be traded and processed into a final industrial product, they have to undergo postharvest processing on farms, which have a direct impact on the cost and quality of a coffee. Three different methods can be used for transforming the coffee cherries into beans, known as wet, dry, and semi-dry methods. In all these processing methods, a spontaneous fermentation is carried out in order to eliminate any mucilage still stuck to the beans and helps improve beverage flavor by microbial metabolites. The microorganisms responsible for the fermentation (e.g., yeasts and lactic acid bacteria) can play a number of roles, such as degradation of mucilage (pectinolytic activity), inhibition of mycotoxin-producing fungi growth, and production of flavor-active components. The use of starter cultures (mainly yeast strains) has emerged in recent years as a promising alternative to control the fermentation process and to promote quality development of coffee product. However, scarce information is still available about the effects of controlled starter cultures in coffee fermentation performance and bean quality, making it impossible to use this technology in actual field conditions. A broader knowledge about the ecology, biochemistry, and molecular biology could facilitate the understanding and application of starter cultures for coffee fermentation process. This review provides a comprehensive coverage of these issues, while pointing out new directions for exploiting starter cultures in coffee processing.     

Fermentation Characteristics of Lactobacilli in Okra (Hibiscus esculentus) Juice

Okra juice was fermented with nine strains of homofermentative and ten strains of heterofermentative lactobacilli as well as one commercial culture to study the metabolic activities of these cultures in an effort to select organisms for use in okra fermentation. During a 24 hr fermentation one Lactobacillus cellobiosus strain converted 29.7% of the mucilage, utilized 85.7% of the reducing sugars and 90.3% of the sucrose decreasing the pH value to 3.65. During the same period seven other strains converted from 20.2–26.8% of the mucilage, 78–98% of the reducing sugars and 75–80% of the sucrose giving a pH value between 3.45 and 4.23. All strains showed considerable variation in the ability to produce lactic acid, acetic acid and ethanol, and they all possessed low levels of lactate dehydrogenase activity.     

Involvement of pectolytic micro-organisms in coffee fermentation

During the fermentation of Coffea arabica L., the most frequently found pectolytic bacteria were Erwinia herbicola and Klebsiella pneumoniae . These micro-organisms produce pectatelyase which is unable to depolymerize esterified pectins of mucilage without previous de-esterification. Furthermore, the optimal activities are observed at pH 8.5 whereas fermentation conditions are acidic (5.3–3.5). The major lactic acid bacteria, Leuconostoc mesenteroides , do not produce pectolytic enzymes. Only a Lactobacillus brevis strain, rarely isolated with a low frequency, shows a polygalacturonase activity compatible with fermentation conditions. Mucilage decomposition seems to be correlated to acidification and not to enzymatic pectolysis. Inoculation with pectolytic micro-organisms allows microbiological control of the fermentation but does not speed up the process. It would be preferable to use lactic acid bacteria so that the pH remained as close as possible to natural fermentation, where acidification is important. This practice would standardize the coffee fermentation microflora and therefore control the end product quality.     

苹果酒酿造中的苹果酸-乳酸发酵

在苹果酒酿造中进行苹果酸-乳酸发酵,乳酸菌通过分解苹果酸,产生乳酸,引起其他有机酸的变化,使苹果酒的口感、质量得以改善。pH、温度、SO_2、酒度通过影响乳酸菌的活动而影响苹果酸-乳酸发酵的进行。保证苹果酒苹果酸-乳酸发酵进行的条件为:温度16～18℃,总SO_2含量<70mg/L,pH<3.70,酒精体积分数低于13％。     

The occurrence and prevention of ethanol fermentation in high‐dry‐matter grass silage

Ethanol is a common, usually minor fermentation product in ensiled forages, the major product being lactic acid. Occasionally, high levels of ethanol are found in silages. The aim of this study was to determine the incidence of high‐dry‐matter (DM) grass silages containing ethanol as the main fermentation product (ethanol silages), to describe the fermentation process in such silages and to determine the effect of grass maceration prior to wilting and addition of a bacterial inoculant containing Lactobacillus plantarum and Enterococcus faecium strains on fermentation. Twenty‐one laboratory silages produced between 1993 and 1995, 21 farm silages produced between 1980 and 1989 and 36 farm silages produced in 1995 (all produced without additive) were examined for pH and chemical composition. Dry matter (DM) loss during ensilage was determined for the laboratory silages only. Four laboratory silages were identified as ethanol silages. Mean concentrations of ethanol, lactic acid and acetic acid were 48.1, 15.5 and 6.0 g kg⁻¹ DM respectively. In the silages that contained lactic acid as the main fermentation product (lactic acid silages) these values were 7.7, 45.5 and 15.1 g kg⁻¹ DM. Mean DM loss and pH were 62.8 g kg⁻¹ DM and 5.32 respectively for ethanol silages and 24.4 g kg⁻¹ DM and 4.69 for lactic acid silages. There was no difference between ethanol silages and lactic acid silages in the mean concentration of ammonia‐N (94 g kg⁻¹ total N), and butyric acid was not detected (<0.2 g kg⁻¹ DM), indicating that both types of silages were well preserved. Analysis of the composition of the grass at ensiling showed a positive correlation between the concentration of soluble carbohydrates and the development into ethanol silage. Analysis of the farm silages indicated that 29% of the silages produced between 1980 and 1989 and 14% of those produced in 1995 were ethanol silages. Maceration prior to wilting and addition of silage inoculant improved lactic acid fermentation and prevented high ethanol levels. The micro‐organisms responsible for ethanol fermentation as well as the implications of feeding ethanol silages to livestock remain to be resolved. © 2000 Society of Chemical Industry     

Selective production of lactic acid in continuous anaerobic acidogenesis by extremely low pH operation

The selective production of lactic acid by anaerobic acidogenesis with low pH control was examined using a chemostat culture. By decreasing culture pH to 3.5 in a chemostat culture containing mixed microbial populations for anaerobic acidogenesis, heterolactic fermentation became dominant, resulting in the selective production of lactic acid and ethanol. This phenomenon was reversible between the acidic and neutral conditions, and was not affected by the dilution rate. The extremely low pH operation was effective for selective lactic acid production in anaerobic acidogenesis.     

DairyBeef: maximizing quality and profits--a consistent food safety message

To respond to meat safety and quality issues in dairy market cattle, a collaborative project team for 7 western states was established to develop educational resources providing a consistent meat safety and quality message to dairy producers, farm advisors, and veterinarians. The team produced an educational website and CD-ROM course that included videos, narrated slide sets, and on-farm tools. The objectives of this course were: 1) to help producers and their advisors understand market cattle food safety and quality issues, 2) help maintain markets for these cows, and 3) help producers identify ways to improve the quality of dairy cattle going to slaughter. DairyBeef. Maximizing Quality & Profits consists of 6 sections, including 4 core segments. Successful completion of quizzes following each core segment is required for participants to receive a certificate of completion. A formative evaluation of the program revealed the necessity for minor content and technological changes with the web-based course. All evaluators considered the materials relevant to dairy producers. After editing, course availability was enabled in February, 2003. Between February and May, 2003, 21 individuals received certificates of completion.     

Metagenomics and metabolomic profiles of Coffea canephora processed by honey/pulped natural technique

Post-harvest processing of coffee is the dominant factor affecting the metabolite composition and quality of the final brew. This study proposes a novel coffee processing method based on an established semi-dry process. Honey/pulped natural coffee (HC) process combines fermentation and drying excluding the washing stage thus conserving water, unlike wet method. Robusta coffee (Coffee canephora) mucilage was monitored for the evolution of bacterial and fungal diversity along with metabolites produced. Lactic acid bacteria (LAB) (48%) and the major fungal community of Wallemia (31%), a selective marker of Robusta coffee fermentation, were predominant throughout the processing. The process modulates volatiles without significantly affecting the total polyphenols (4.66%), chlorogenic acid (2.07%), caffeine (1.66%), trigonelline (0.53%) and theophylline (0.16%). HC contained compounds like 2-methoxy-4-vinyl phenol, 2-furancarboxaldehyde, 5-methyl-, acetic acid, pyrazine, methyl-, 2-propanone, 1-hydroxy-, and furfural that promotes sweet, caramelly, nutty, pungent and hazelnut taste to coffee.     

奶啤生产中乳酸菌对酵母菌发酵作用的研究

利用乳酸菌和酵母菌的共生作用,对牛乳进行了混合菌种的发酵研究。分析了共生作用对产品pH值、滴定酸度、乙醇含量和α-氨基酸态氮的影响。研究结果表明,乳酸菌的加入不仅可以提高酵母菌的生长速度,而且可以提高产品的风味,奶啤的酒精含量相对啤酒较低,是酒精度较低的健康型饮料。     

凝固型咖啡酸奶的研制

以全脂乳粉、脱脂乳粉、咖啡为主要原料,添加蔗糖,以安琪酵母8型菌为发酵剂,酸奶感官品质评分和酸奶黏度的综合评分为考察指标,采用正交试验方法对凝固型咖啡酸奶配方进行优化,得到最佳的发酵咖啡酸奶的工艺与配方。结果表明,在发酵温度为42 ℃条件下发酵6 h,脂肪含量3.4%,咖啡添加量1.0%,蔗糖添加量6%,8菌型发酵剂添加量0.1%,发酵后在4 ℃条件下冷藏24 h,获得的发酵酸奶的感官品质评分为89分,黏度为5 640 mPa·s,产品组织状态均匀,口感细腻,略带有咖啡香味,感官评价最佳。最佳发酵条件下的酸度为88 °T,pH值为4.24,乳酸菌活菌数为1.25×108 CFU/mL,符合GB 19302—2010对酸奶品质的规定。     

乳酸菌UV_3-9包埋固定化技术发酵产共轭亚油酸

采用包埋法将乳酸菌UV3-9固定化处理后,对该固定化乳酸菌小球进行了发酵生产共轭亚油酸(CLA)的研究。结果显示,固定化小球的最佳直径为2～3 mm,固定化小球在氯化钙和硼酸混合液中需浸泡过夜;固定后的小球发酵产CLA最佳条件为:发酵温度37℃,初始pH 6.5,发酵时间28 h,底物LA添加量为0.3%,CLA最大产量达到92.061μg/mL。该固定化乳酸菌小球已完成了7批次的共轭亚油酸发酵,其产量均在80.476μg/mL以上。而且固定化UV3-9的CLA发酵产量是游离UV3-9的1.28倍。     

葡萄酒中的乳酸菌

介绍了存在于葡萄酒中的乳酸菌及其在葡萄酒酿造中的作用。影响乳酸菌作用的主要因素包括pH、温度、酒精浓度、SO2、酵母菌、碳水化合物以及游离脂肪酸等。介绍了苹果酸－乳酸发酵的人工控制。     

Ripening and seasonal changes in microbial groups and in physico-chemical properties of the ewes’ cheese Pecorino del Poro

Three batches of Pecorino del Poro, ewes’ cheese made from raw milk, were examined throughout a 28-day ripening time at three different seasons. High logarithmic counts per gram of cheese for mesophilic coccal-shaped lactic acid bacteria (6.70–12.45), mesophilic lactobacilli (4.82–11.73), thermophilic coccal-shaped lactic acid bacteria (2.30–9.90), and thermophilic lactobacilli (2.95–8.15) were found. Coccal-shaped lactic acid bacteria were the dominant microorganisms throughout ripening. The microorganisms used as an indicator of hygiene during manufacture of the cheeses, coliforms and Escherichia coli, were considerably lower, as were enterococci and yeasts. Coliforms and E. coli decreased sharply throughout ripening. Physico-chemical parameters such as pH (5.07–7.03), dry matter (46.34–72.79%), ether extract (31.35–51.84% of dry matter), crude protein (29.93–44.73% of dry matter), and chloride content (2.36–4.11% of dry matter) were also determined. Probably, the use of selected autochthonous mesophilic lactococci as a starter would control or suppress the growth of undesirable microorganisms. The results obtained suggest the need for improvements in milking and dairy conditions.     

传统发酵蔬菜中耐乙醇型乳酸菌的研究进展

发酵蔬菜一般都是由乳酸菌等多种微生物发酵而成,发酵过程中会产生有机酸、乙醇、氨基酸等物质。乙醇和有机酸形成酯类物质,赋予发酵蔬菜特有的香气。但是发酵蔬菜发酵过程中如果乙醇浓度过高,则会抑制乳酸菌发挥作用。因此,该文主要总结传统发酵蔬菜中乳酸菌的筛选方法、乳酸菌培养基优化、乳酸菌的乙醇耐受性、乳酸菌的诱变、乳酸菌的复配及应用等方面的研究进展,为人工接种乳酸菌发酵传统蔬菜产业化生产提供依据。     

Characterization of Cucumber Fermentation Spoilage Bacteria by Enrichment Culture and 16S rDNA Cloning

Commercial cucumber fermentations are typically carried out in 40000 L fermentation tanks. A secondary fermentation can occur after sugars are consumed that results in the formation of acetic, propionic, and butyric acids, concomitantly with the loss of lactic acid and an increase in pH. Spoilage fermentations can result in significant economic loss for industrial producers. The microbiota that result in spoilage remain incompletely defined. Previous studies have implicated yeasts, lactic acid bacteria, enterobacteriaceae, and Clostridia as having a role in spoilage fermentations. We report that Propionibacterium and Pectinatus isolates from cucumber fermentation spoilage converted lactic acid to propionic acid, increasing pH. The analysis of 16S rDNA cloning libraries confirmed and expanded the knowledge gained from previous studies using classical microbiological methods. Our data show that Gram‐negative anaerobic bacteria supersede Gram‐positive Fermincutes species after the pH rises from around 3.2 to pH 5, and propionic and butyric acids are produced. Characterization of the spoilage microbiota is an important first step in efforts to prevent cucumber fermentation spoilage. Practical Application An understanding of the microorganisms that cause commercial cucumber fermentation spoilage may aid in developing methods to prevent the spoilage from occurring.     

发酵过程果酒成分对苹果酸乳酸发酵的影响

苹果酸-乳酸发酵（MLF）是果酒（葡萄酒、苹果酒）酿造中非常重要的二次发酵过程。本研究采用人工模拟果酒，研究果酒成分对MLF的影响。结果表明，葡萄糖抑制乳酸菌的MLF，果糖却有促进作用，而且果糖可解除葡萄糖对酿酒酒球菌MLF的抑制作用，果酒中葡萄糖和果糖的浓度及其比例是预测和控制MLF的重要参数；高浓度乙醇、低pH抑制乳酸菌的MLF，其抑制作用的大小与使用的菌种有关；酒中酚类物质（没食子酸和阿魏酸）对酿酒酒球菌的MLF几乎无任何显著影响；酒精发酵完成后果酒中营养的缺乏会抑制乳酸菌的MLF。     

Comparison of the bacterial species diversity of spontaneous cocoa bean fermentations carried out at selected farms in Ivory Coast and Brazil

To compare the spontaneous cocoa bean fermentation process carried out in different cocoa-producing regions, heap and box (one Ivorian farm) and box (two Brazilian farms) fermentations were carried out. All fermentations were studied through a multiphasic approach. In general, the temperature inside the fermenting mass increased throughout all fermentations and reached end-values of 42-48 °C. The main end-products of pulp carbohydrate catabolism were ethanol, lactic acid, acetic acid, and/or mannitol. In the case of the fermentations on the selected Ivorian farm, the species diversity of lactic acid bacteria (LAB) and acetic acid bacteria (AAB) was restricted. Lactobacillus fermentum and Leuconostoc pseudomesenteroides were the predominant LAB species, due to their ethanol and acid tolerance and citrate consumption. The levels of mannitol, ascribed to growth of L. fermentum, were fermentation-dependent. Also, enterobacterial species, such as Erwinia soli and Pantoea sp., were among the predominating microbiota during the early stages of both heap and box fermentations in Ivory Coast, which could be responsible for gluconic acid production. Consumption of gluconic acid at the initial phases of the Ivorian fermentations could be due to yeast growth. A wider microbial species diversity throughout the fermentation process was seen in the case of the box fermentations on the selected Brazilian farms, which differed, amongst other factors, regarding pod/bean selection on these farms as compared to fermentations on the selected Ivorian farm. This microbiota included Lactobacillus plantarum, Lactobacillus durianis, L. fermentum, Lactobacillus mali, Lactobacillus nagelii, L. pseudomesenteroides, and Pediococcus acidilactici, as well as Bacillus subtilis that was present at late fermentation, when the temperature inside the fermenting mass reached values higher than 50 °C. Moreover, AAB seemed to dominate the Brazilian box fermentations studied, explaining higher acetic acid concentrations in the pulp and the beans. To conclude, it turned out that the species diversity and community dynamics, influenced by local operational practices, in particular pod/bean selection, impact the quality of fermented cocoa beans.     

植物乳杆菌的筛选及其在泡菜中的应用

实验筛选了1株产酸较好的菌株,鉴定为植物乳杆菌(Laobacillusplantarum),将其应用在制作泡菜中,进行小批量自然发酵和人工发酵。结果表明,自然发酵的最佳周期为48h,人工接种发酵的最佳周期为36h。人工接种发酵从发酵周期、乳酸产量、感官评定等各方面都明显优于自然发酵,既适合家庭制作,又适于工业化大批量生产。     

葵仁乳酸发酵饮料的研究

以向日葵籽仁为原料，先经粉碎、浸泡、磨浆等工艺制成葵仁乳，再经乳酸菌发酵，生产出风味及状态良好的葵仁乳酸饮料，采用正交试验法确定了最佳发酵工艺条件，并测定了发酵过程中pH及总酸的变化。     

Conducting starter culture-controlled fermentations of coffee beans during on-farm wet processing: Growth,metabolic analyses and sensorial effects

In this study, the potential use of Pichia fermentans YC5.2 as a starter culture to conduct controlled coffee bean fermentations during on-farm wet processing was investigated. Inoculated fermentations were conducted with or without the addition of 2% (w/v) sucrose, and the resultant microbial growth and metabolism, bean chemistry and beverage quality were compared with spontaneous (control) fermentation. In both inoculated treatments, P. fermentans prevailed over indigenous microbiota and a restricted microbial composition was observed at the end of fermentation process. The inoculation also increased the production of specific volatile aroma compounds (e.g., ethanol, acetaldehyde, ethyl acetate and isoamyl acetate) and decreased the production of lactic acid during the fermentation process. Sucrose supplementation did not significantly interfere with the growth and frequency of P. fermentans YC5.2 inoculum but maintained high levels of wild bacteria population and lactic acid production similar to the spontaneous process. In roasted beans, the content of sugars and organic acids were statistically (p < 0.05) similar for all the treatments. However, the inoculated fermentations were shown to influence the volatile fraction of roasted coffee beans by increasing the concentration of yeast-derived metabolites compared to control. Sensory analysis of coffee beverages demonstrated that the use of the YC5.2 strain was favorable for the production of high-quality coffees with distinctive characteristics, e.g., intense perception of ‘vanilla’ taste and ‘floral’ aromas. In conclusion, the use of P. fermentans YC5.2 in coffee processing was shown to be a viable alternative to control the fermentation step and to ensure consistent quality of finished products.