Breeding Strategy To Generate Robust Yeast Starter Cultures for Cocoa Pulp Fermentations

Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at cocoa farms is allowed to ferment the pulp surrounding cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, therefore, we aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produce high-quality chocolate. Starting from specifically selected Saccharomyces cerevisiae strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate showed that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production.     

Functional yeast starter cultures for cocoa fermentation

The quest to develop a performant starter culture mixture to be applied in cocoa fermentation processes started in the 20th century, aiming at achieving high-quality, reproducible chocolates with improved organoleptic properties. Since then, different yeasts have been proposed as candidate starter cultures, as this microbial group plays a key role during fermentation of the cocoa pulp-bean mass. Yeast starter culture-initiated fermentation trials have been performed worldwide through the equatorial zone and the effects of yeast inoculation have been analysed as a function of the cocoa variety (Forastero, Trinitario and hybrids) and fermentation method (farm-, small- and micro-scale) through the application of physicochemical, microbiological and chemical techniques. A thorough screening of candidate yeast starter culture strains is sometimes done to obtain the best performing strains to steer the cocoa fermentation process and/or to enhance specific features, such as pectinolysis, ethanol production, citrate assimilation and flavour production. Besides their effects during cocoa fermentation, a significant influence of the starter culture mixture applied is often found on the cocoa liquors and/or chocolates produced thereof. Thus, starter culture-initiated cocoa fermentation processes constitute a suitable strategy to elaborate improved flavourful chocolate products.     

Fermentation for future food systems: Precision fermentation can complement the scope and applications of traditional fermentation

Modern biotechnology holds great potential for expanding the scope of fermentation to create novel foods and improve the sustainability of food production.

The growing human population and global warming pose an impending threat for global food security (Linder, 2019). This has prompted a critical re‐examination of the food supply chain from producers to consumers in order to increase the overall efficiency of food production, storage and transport. Much research in plant science consequently aims to increase production with new, high‐yield crop, fruit and vegetable varieties better adapted to changing climatic conditions. Yet, there is also much room for improving food safety by minimising food losses and recycling waste, valorising by‐products, improving nutritional value and increasing storage time. This is where fermentation comes in as a cost‐efficient, versatile and proven technology that extends the shelf life of food products and enhances their nutritional content. Moreover, there is enormous potential in fermentation to further increase efficiency and product range and even create new food products from non‐food biomass.

… there is enormous potential in fermentation to further increase efficiency and product range and even create new food products from non‐food biomass.

In a broader sense, fermentation can be defined as the cultivation of microorganisms such as bacteria, yeasts and fungi to break down complex molecules into simpler ones, notably organic acids, alcohols or esters. In a practical sense, it is one of the oldest food processing technologies to increase storage life along with cooking, smoking or air‐drying: fermentation was already fully industrialised for producing beer and bread millennia ago in ancient Mesopotamia and Egypt. It is also an elegant and simple technology as these microorganisms do most of the work without much human involvement.Louis Pasteur’s discovery that microorganisms cause fermentation laid the basis for further improvement of the technology from traditional spontaneous fermentation to the use of defined starter cultures. Fermentation is now widely used to produce alcoholic beverages, bread and pastry, dairy products, pickled vegetables, soy sauce and so on. More recent advances based on genomics and synthetic biology include precision and biomass fermentation to produce specific compounds for the food and chemical industry or medicinal use. This is not the limit though: when combined with genomics, fermentation has even greater potential for creating novel foods and other products.     

Functional bacterial cultures for dairy applications: Towards improving safety,quality, nutritional and health benefit aspects

Traditionally, fermentation was used to preserve the shelf life of food. Currently, in addition to favouring food preservation, well standardized and controlled industrial processes are also aimed at improving the functional characteristics of the final product. In this regard, starter cultures have become an essential cornerstone of food production. The selection of robust microorganisms, well adapted to the food environment, has been followed by the development of microbial consortia that provide some functional characteristics, beyond their acidifying capacity, achieving safer, high-quality foods with improved nutritional and health-promoting properties. In addition to starters, adjunct cultures and probiotics, which normally do not have a relevant role in fermentation, are added to the food in order to provide some beneficial characteristics. This review focuses on highlighting the functional characteristics of food starters, as well as adjunct and probiotic cultures (mainly lactic acid bacteria and bifidobacteria), with a specific focus on the synthesis of metabolites for preservation and safety aspects (e.g. bacteriocins), organoleptic properties (e.g. exopolysaccharides), nutritional (e.g. vitamins) and health improvement (e.g. neuroactive molecules). Literature reporting the application of these functional cultures in the manufacture of foods, mainly those related to dairy production, such as cheeses and fermented milks, has also been updated.     

Effect of lactic starter cultures on the organic acid composition of milk and cheese during ripening—analysis by HPLC

The major function of lactic starter cultures in cheese making is to produce lactic and other organic acids from the carbohydrates present in milk. The activity of six starter cultures consisting of two Lactococcus lactis ssp. lactis , two Lactococcus lactis ssp. lactis biovar. diacetylactis and two Leuconostoc strains, was tested by monitoring the evolution of the organic acid composition of milk by a modified HPLC method. In addition, their performance as cheese starters was also tested. The HPLC method developed proved to be a precise tool to monitor the organic acid content. Thus, it can be used to follow the fermentation ability of starter cultures, providing information about the type of fermentation. The use of any of the six starters assayed is suggested for manufacturing Afuega'l Pitu cheese.     

Application of rRNA-targeted oligonucleotide probes in biotechnology

Ribosomal RNA-targeted oligonucleotide probes have become valuable tools for the detection of microorganisms involved in important biotechnological processes. Microorganisms which are of major importance for processes such as wastewater treatment, microbial leaching or methane production can be detected and quantified in situ within a complex microbial community. For certain processes, such as nitrification or biological phosphate removal, new microorganisms have become the focus of interest and have led to an improved understanding of these bioremediation techniques. Hybridization techniques have become fast and reliable alternatives to conventional cultivation techniques in the food industry as a control method for starter cultures for fermentation processes or product control. Recent analytical tools such as flow cytometry and digital image processing have improved the efficiency of these techniques. This review is intended to present a summary of methodological aspects of rRNA-based hybridization techniques and their application in biotechnology.     

微生物发酵对木薯渣营养成分的影响

[背景]将木薯渣作为饲料资源进行开发和利用能够减轻环境污染,实现资源就地转化,已成为国内外研究热点.微生物发酵可降低木薯渣的粗纤维含量,改善适口性,提高饲料转化率.[目的]筛选对木薯渣发酵效果较好的微生物发酵剂及其发酵时长.[方法]试验选用A(芽孢菌+乳酸菌+酿酒酵母菌)、B(戊糖片球菌+酿酒酵母菌)和C(植物乳杆菌+...     

Method for the validation of intraspecific crosses of Saccharomyces cerevisiae strains by minisatellite analysis

The crossing of Saccharomyces strains by spore conjugation is one of the ways to obtain new starter cultures for the fermentation industry. One of the major difficulties of this practice is the identification of the newly formed hybrids. In this work we describe an effective molecular method for the validation of Saccharomyces intraspecific crosses. The method described is based in the hypothesis that hybrids constructed by spore conjugation contain the sum of the genomes of both parental strains. As a consequence, the conjugation of spores of two yeasts showing different genomic fingerprinting profiles will result in a hybrid culture that will show the sum of both profiles. We demonstrated that the detection of polymorphism in two genes containing minisatellite-like sequences, either SED1 or AGA1, is suitable for this purpose. Using this strategy we were able to validate 15 crosses out of 162 hybridization attempts.     

Identification of pOENI-1 and Related Plasmids in Oenococcus oeni Strains Performing the Malolactic Fermentation in Wine

Plasmids in lactic acid bacteria occasionally confer adaptive advantages improving the growth and behaviour of their host cells. They are often associated to starter cultures used in the food industry and could be a signature of their superiority. Oenococcus oeni is the main lactic acid bacteria species encountered in wine. It performs the malolactic fermentation that occurs in most wines after alcoholic fermentation and contributes to their quality and stability. Industrial O. oeni starters may be used to better control malolactic fermentation. Starters are selected empirically by virtue of their fermentation kinetics and capacity to survive in wine. This study was initiated with the aim to determine whether O. oeni contains plasmids of technological interest. Screening of 11 starters and 33 laboratory strains revealed two closely related plasmids, named pOENI-1 (18.3-kb) and pOENI-1v2 (21.9-kb). Sequence analyses indicate that they use the theta mode of replication, carry genes of maintenance and replication and two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tauE) and a NADH:flavin oxidoreductase of the old yellow enzyme family (oye). Interestingly, pOENI-1 and pOENI-1v2 were detected only in four strains, but this included three industrial starters. PCR screenings also revealed that tauE is present in six of the 11 starters, being probably inserted in the chromosome of some strains. Microvinification assays performed using strains with and without plasmids did not disclose significant differences of survival in wine or fermentation kinetics. However, analyses of 95 wines at different phases of winemaking showed that strains carrying the plasmids or the genes tauE and oye were predominant during spontaneous malolactic fermentation. Taken together, the results revealed a family of related plasmids associated with industrial starters and indigenous strains performing spontaneous malolactic fermentation that possibly contribute to the technological performance of strains in wine.     

Design of a low-cost culture medium based in whey permeate for biomass production of enological Lactobacillus plantarum strains

The production of malolactic starter cultures requires the obtention of suitably large biomass at low-cost. In this work it was possible to obtain a good amount of biomass, at laboratory scale, of two enological strains of Lb. plantarum, by formulating a culture medium based on whey permeate (WP), a by-product of the cheese industry usually disposed as waste, when this was supplemented with yeast extract (Y), salts (S) and Tween 80 (T) (WPYST). Bacteria grown in WPYST medium exhibited good tolerance to stress conditions of synthetic wine (pH 3.5, ethanol 13% vol/vol). However, when WPYST was added with 8% vol/vol ethanol, cultures inoculated in synthetic wine, showed a lower viability and capacity to consume L-malic acid than when they were cultured in WPYST without ethanol. Subsequently, strains grown in WPYST were inoculated in sterile wine samples (final stage of alcoholic fermentation) of the red varietals Merlot and Pinot noir, and incubated at laboratory scale. Cultures from WPYST, inoculated in Pinot noir wine, showed a better performance than bacteria grown in MRS broth, and exhibited a consumption of L-malic acid higher than 90%. However, cultures from WPYST or from MRS broth, inoculated in sterile Merlot wine, showed a lower survival. This study allowed the formulation of a low-cost culture medium, based on a by-product of the food industry, which showed to be adequate for the growth of two enological strains of Lb. plantarum, suggesting their potentiality for application in the elaboration of malolactic starter cultures.     

Fermentation at non-conventional conditions in food- and bio-sciences by the application of advanced processing technologies

The interest in improving the yield and productivity values of relevant microbial fermentations is an increasingly important issue for the scientific community. Therefore, several strategies have been tested for the stimulation of microbial growth and manipulation of their metabolic behavior. One promising approach involves the performance of fermentative processes during non-conventional conditions, which includes high pressure (HP), electric fields (EF) and ultrasound (US). These advanced technologies are usually applied for microbial inactivation in the context of food processing. However, the approach described in this study focuses on the use of these technologies at sub-lethal levels, since the aim is microbial growth and fermentation under these stress conditions. During these sub-lethal conditions, microbial strains develop specific genetic, physiologic and metabolic stress responses, possibly leading to fermentation products and processes with novel characteristics. In some cases, these modifications can represent considerable improvements, such as increased yields, productivities and fermentation rates, lower accumulation of by-products and/or production of different compounds. Although several studies report the successful application of these technologies during the fermentation processes, information on this subject is still scarce and poorly understood. For that reason, the present review paper intends to assemble and discuss the main findings reported in the literature to date, and aims to stimulate interest and encourage further developments in this field.     

Criteria for lactic acid bacteria screening to enhance silage quality

The main challenge of ensiling is conserving the feed through a fermentative process that results in high nutritional and microbiological quality while minimizing fermentative losses. This challenge is of growing interest to farmers, industry and research and involves the use of additives to improve the fermentation process and preserve the ensiled material. Most studies involved microbial additives; lactic acid bacteria (LAB) have been the focus of much research and have been widely used. Currently, LABs are used in modern and sustainable agriculture because of their considerable potential for enhancing human and animal health. Although the number of studies evaluating LABs in silages has increased, the potential use of these micro-organisms in association with silage has not been adequately studied. Fermentation processes using the same strain produce very different results depending on the unique characteristics of the substrate, so the choice of silage inoculant for different starting substrates is of extreme importance to maximize the nutritional quality of the final product. This review describes the current scenario of the bioprospecting and selection process for choosing the best LAB strain as an inoculant for ensiling. In addition, we analyse developments in the fermentation process and strategies and methods that will assist future studies on the selection of new strains of LAB as a starter culture or inoculant.     

Bacteriophage-resistance systems in dairy starter strains: molecular analysis to application

Starter inhibition by bacteriophage infection in dairy fermentations can limit the usage of specific bacterial strains used in the manufacture of Cheddar, Mozzarella and other cheeses and can result in substantial economic losses. A variety of practical measures to alleviate the problem of phage infection have been adopted over the years but has invariably resulted in a very limited number of strains which can withstand intensive usage in industry. The application of genetic techniques to improve the phage-resistance of starter cultures for dairy fermentations has been intensively studied for the last 20 years to a point where this approach now has significant potential to alleviate the problem. This paper highlights the recent findings and developments that have been described in the literature that will have an impact on improvement of the phage-resistance of starter cultures.     

Phenotypic typing, technological properties and safety aspects of Lactococcus garvieae strains from dairy environments

AIMS: To characterize Lactococcus garvieae strains of dairy origin and to determine their technological properties and safety for their possible use in starter culture preparation. METHODS AND RESULTS: Forty-seven L. garvieae isolates, recovered from two artisanal Italian cheeses were studied, in comparison with 12 fish isolates and the type strain of the species. Phenotypic typing revealed that the strains could be differentiated on the basis of their ecological niche of origin in lactose positive strains (all isolated from dairy sources) and lactose negative strains (all isolated from fish). Furthermore, the strains exhibited a high degree of physiological variability, showing the presence of 26 different biotypes. The strains possessed moderate acidifying and proteolytic activities and did not produce bacteriocins. A safety investigation revealed that all strains were sensitive to vancomycin and moderately resistant to kanamycin; some biotypes were tetracycline resistant. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some isolates. CONCLUSIONS: The prevalence of L. garvieae in some artisanal Italian cheeses can be linked to the typicity of the products. Although in a few cases an antimicrobial resistance or a presence of virulence determinants may imply a potential hygienic risk, most of the strains showed positive properties for their possible adjunction in a starter culture preparation, to preserve the natural bacterial population responsible for the typical sensorial characteristics of the traditional raw milk cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: L. garvieae strains can be considered an important part of the microbial population associated with the natural fermentation of artisanal Italian cheeses. A deepened characterization of the strains may aid in understanding the functional and ecological significance of their presence in dairy products and in selecting new strains for the dairy industry.     

传统与未来的碰撞：食品发酵工程技术与应用进展

随着生物技术的飞速发展,作为食品生物工程的主要组成部分,食品发酵工程技术不断升级,在传统发酵食品的菌种、发酵过程、产品品质得到改善的同时,生物制造的功能食品组分、未来食品等新型产品也应运而生。首先概述了由生物技术和信息技术的进步带来的食品发酵研究手段与生产方式的多层面变革,并重点阐释了利用食品合成生物学设计构建细胞工厂的思路和方法,以及食品生物工程在微生物分析、过程工程和分离工程方面的智能化进程。其次,介绍了现代食品生物工程技术在改善传统发酵食品品质及安全性、生产功能食品组分、添加剂和酶制剂、创制未来食品和开发新型益生食品方面的应用进展。最后,对全球和我国食品发酵产业面临的挑战和未来发展趋势进行了总结和展望,以期为食品发酵的技术革新和工业化应用提供参考。     

Natural genetic transformation: A novel tool for efficient genetic engineering of the dairy bacterium Streptococcus thermophilus

Streptococcus thermophilus is widely used for the manufacture of yoghurt and Swiss or Italian-type cheeses. These products have a market value of approximately 40 billion dollars per year, making S. thermophilus a species that has major economic importance. Even though the fermentation properties of this bacterium have been gradually improved by classical methods, there is great potential for further improvement through genetic engineering. Due to the recent publication of three complete genome sequences, it is now possible to use a rational approach for designing S. thermophilus starter strains with improved properties. Progress in this field, however, is hampered by a lack of genetic tools. Therefore, we developed a system, based on natural transformation, which makes genetic manipulations in S. thermophilus easy, rapid, and highly efficient. The efficiency of this novel tool should make it possible to construct food-grade mutants of S. thermophilus, opening up exciting new possibilities that should benefit consumers as well as the dairy industry.     

工业微生物的噬菌体感染与防治策略

以细菌为基础的生物技术在蓬勃发展的同时也不断受到噬菌体感染的威胁,噬菌体感染已成为微生物发酵过程中的一个顽疾,其实质是噬菌体与细菌之间复杂的共进化关系。在漫长的进化过程中,噬菌体已经形成了多种针对细菌抗性系统的逃逸机制。合理的工厂设计、菌株的轮换策略和传统的基因工程方法能在一定程度上降低噬菌体感染的风险,但仍然无法避免。基于CRISPR-Cas系统的防治策略仅需噬菌体的序列信息就可以理性设计噬菌体抗性菌株,且可以通过叠加效应不断增强菌种抗性,从而避免噬菌体的逃逸;群体感应信号分子则可以从整体水平上调节细菌的噬菌体抗性。这些新发现为噬菌体感染问题的解决带了新的希望,而噬菌体基因组编辑技术和合成生物学的快速发展则将进一步加深人们对噬菌体感染防治领域的认识。     

Typification of Oenococcus oeni strains by multiplex RAPD-PCR and study of population dynamics during malolactic fermentation

AIMS: The goal of this study was to develop a reproducible method for molecular typing strains of Oenococcus oeni, and also to apply it in the study of population dynamics of these strains during malolactic fermentation of wine. METHODS AND RESULTS: A new method of multiplex randomly amplified polymorphic DNA (RAPD)-PCR has been developed, based on the combination of one random 10-mer and one specific 23-mer oligonucleotide in a single PCR. This method generates unique and discriminant DNA profiles for strains of O. oeni. The strains of this species were also clearly distinguished from other species of lactic acid bacteria. The method was applied to study the dynamics of O. oeni strains during malolactic fermentation, in three vintages in the same cellar. CONCLUSIONS: A fast and reliable method for typing strains of O. oeni has been designed and optimized. It improves the reproducibility and rapidity of conventional RAPD-PCR, and it has been validated monitoring the population dynamics during malolactic fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This method will be a good tool to study the population dynamics of bacteria during malolactic fermentation and to evaluate the performance of new malolactic starter cultures and their dominance over the native microbiota.     

Autochthonous fermentation starters for the industrial production of Negroamaro wines

The aim of the present study was to establish a new procedure for the oenological selection of Saccharomyces cerevisiae strains isolated from natural must fermentations of an important Italian grape cultivar, denoted as “Negroamaro”. For this purpose, 108 S. cerevisiae strains were selected as they did not produce H₂S and then assayed by microfermentation tests. The adopted procedure made it possible to identify 10 strains that were low producers of acetic acid and hydrogen sulphide and showed that they completed sugar consumption during fermentation. These strains were characterized for their specific oenological and technological properties and, two of them, strains 6993 and 6920, are good candidates as industrial starter cultures. A novel protocol was set up for their biomass production and they were employed for industrial-scale fermentation in two industrial cellars. The two strains successfully dominated the fermentation process and contributed to increasing the wines’ organoleptic quality. The proposed procedure could be very effective for selecting “company-specific” yeast strains, ideal for the production of typical regional wines. “Winery” starter cultures could be produced on request in a small plant just before or during the vintage season and distributed as a fresh liquid concentrate culture.