Fermentation of plant-based dairy alternatives by lactic acid bacteria

Ethical, environmental and health concerns around dairy products are driving a fast-growing industry for plant-based dairy alternatives, but undesirable flavours and textures in available products are limiting their uptake into the mainstream. The molecular processes initiated during fermentation by lactic acid bacteria in dairy products is well understood, such as proteolysis of caseins into peptides and amino acids, and the utilisation of carbohydrates to form lactic acid and exopolysaccharides. These processes are fundamental to developing the flavour and texture of fermented dairy products like cheese and yoghurt, yet how these processes work in plant-based alternatives is poorly understood. With this knowledge, bespoke fermentative processes could be engineered for specific food qualities in plant-based foods. This review will provide an overview of recent research that reveals how fermentation occurs in plant-based milk, with a focus on how differences in plant proteins and carbohydrate structure affect how they undergo the fermentation process. The practical aspects of how this knowledge has been used to develop plant-based cheeses and yoghurts is also discussed.     

Identification of industrial strains of lactic acid bacteria by methods of molecular genetic typing

Various methods currently used in microbiology for determining taxonomic state of bacteria are discussed. The main focus is aimed at identifying and gene typing of lactic acid bacteria, used as starter cultures for industrial process of production of sour milk products, meat products, and probiotics.     

Identification of industrial strains of lactic acid bacteria by methods of molecular genetic typing

Various methods currently used in microbiology for determining taxonomic state of bacteria are discussed. The main focus is aimed at identifying and gene typing of lactic acid bacteria, used as starter cultures for industrial process of production of sour milk products, meat products, and probiotics.     

Occurrence and function of yeasts in Asian indigenous fermented foods

In the Asian region, indigenous fermented foods are important in daily life. In many of these foods, yeasts are predominant and functional during the fermentation. The diversity of foods in which yeasts predominate ranges from leavened bread-like products such as nan and idli, to alcoholic beverages such as rice and palm wines, and condiments such as papads and soy sauce. Although several products are obtained by natural fermentation, the use of traditional starter cultures is widespread. This minireview focuses on the diversity and functionality of yeasts in these products, and on opportunities for research and development.     

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.     

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.     

Microbial ecology and quality assurance in food fermentation systems. The case of kefir grains application

Fermentation technology has become a modern method for food production the last decades as a process for enhancing product stability, safety and sensory standards. The main reason for this development is the increasing consumers’ demand for safe and high quality food products. The above has led the scientific community to the thorough study for the appropriate selection of specific microorganisms with desirable properties such as bacteriocin production, and probiotic properties. The main food products produced through fermentation activity are bread, wine, beer cheese and other dairy products. The microorganisms conducting the above processes are mainly yeasts and lactic acid bacteria. The end products of carbohydrate catabolism by these microorganisms contribute not only to preservation as it was believed years ago, but also to the flavour, aroma and texture and to the increase of the nutritional quality by thereby helping determine unique product characteristics. Thus, controlling the function of specific microorganisms or the succession of microorganisms that dominate the microflora is therefore advantageous, because it can increase product quality, functionality and value. Throughout the process of the discovery of microbiological diversity in various fermented food systems, the development of starter culture technology has gained more scientific attention, and it could be used for the control of the manufacturing operation, and management of product quality. In the frame of this review the presentation of the quality enhancement of most consumed fermented food products around the world is attempted and the new trends in production of fermented food products, such as bread is discussed. The review is focused in kefir grains application in bread production.     

Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products

Flavour development in dairy fermentations, most notably cheeses, results from a series of (bio)chemical processes in which the starter cultures provide the enzymes. Particularly the enzymatic degradation of proteins (caseins) leads to the formation of key-flavour components, which contribute to the sensory perception of dairy products. More specifically, caseins are degraded into peptides and amino acids and the latter are major precursors for volatile aroma compounds. In particular, the conversion of methionine, the aromatic and the branched-chain amino acids are crucial. A lot of research has focused on the degradation of caseins into peptides and free amino acids, and more recently, enzymes involved in the conversion of amino acids were identified. Most data are generated on Lactococcus lactis, which is the predominant organism in starter cultures used for cheese-making, but also Lactobacillus, Streptococcus, Propionibacterium and species used for surface ripening of cheeses are characterised in their flavour-forming capacity. In this paper, various enzymes and pathways involved in flavour formation will be highlighted and the impact of these findings for the development of industrial starter cultures will be discussed.     

Use of isolated kefir starter cultures in kefir production

Kefir is a beverage produced by lactic-alcoholic fermentation of milk using kefir grain. For the first time in Iran, the microbial flora of kefir grain was isolated and identified (Motaghi et al. 1997). In this paper various ratios of starter cultures of kefir grains were investigated. Various ratios of lactic acid bacteria, yeasts and acetic acid bacteria were tested and the quality (colour, smell, flavour, acidity, effervescence and viscosity) of the product was assessed. At constant incubation time and temperature (24 h, 25 °C using homogenised milk with 2.5% fat), samples with various ratios of starter culture (3–5% w/v) were examined and analysed for protein, fat, sugar, alcohol, carbon dioxide, acidity, density, and riboflavin content. Samples produced with 3% (v/v) bacterial mixed culture and 2% (v/v) yeast (K3 procedure) culture were considered as best with respect to quality and organoleptic quality. The comparison of the results with the organoleptic tests of previous studies showed that the kefir produced with kefir grain is more desirable as compared with kefir produced with starter cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Thermally-dried free and immobilized kefir cells as starter culture in hard-type cheese production

In an attempt to seek for suitable dried cultures, thermally-dried kefir was employed as starter in hard-type cheese production and tested in cheeses ripened at 5, 18 and 22 °C. Both free and immobilised on casein kefir cells were used and compared to cheese made without starter culture. Cheese products made with free cells of kefir culture were characterized by longer preservation time, improved aroma, taste, texture characteristics and increased degree of openness. Volatile profiles obtained by GC/MS analysis revealed a 216% increase in total concentration of esters, organic acids, alcohols and carbonyl compounds between cheeses prepared with and without kefir culture.     

The cocoa bean fermentation process: from ecosystem analysis to starter culture development

Cocoa bean fermentation is still a spontaneous curing process to facilitate drying of nongerminating cocoa beans by pulp removal as well as to stimulate colour and flavour development of fermented dry cocoa beans. As it is carried out on farm, cocoa bean fermentation is subjected to various agricultural and operational practices and hence fermented dry cocoa beans of variable quality are obtained. Spontaneous cocoa bean fermentations carried out with care for approximate four days are characterized by a succession of particular microbial activities of three groups of micro‐organisms, namely yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB), which results in well‐fermented fully brown cocoa beans. This has been shown through a plethora of studies, often using a multiphasic experimental approach. Selected strains of several of the prevailing microbial species have been tested in appropriate cocoa pulp simulation media to unravel their functional roles and interactions as well as in small plastic vessels containing fresh cocoa pulp‐bean mass to evaluate their capacity to dominate the cocoa bean fermentation process. Various starter cultures have been proposed for successful fermentation, encompassing both cocoa‐derived and cocoa nonspecific strains of (hybrid) yeasts, LAB and AAB, some of which have been implemented on farms successfully.     

Engineering metabolic highways in Lactococci and other lactic acid bacteria

Lactic acid bacteria (LAB) are widely used in industrial food fermentations and are receiving increased attention for use as cell factories for the production of food and pharmaceutical products. Glycolytic conversion of sugars into lactic acid is the main metabolic highway in these Gram-positive bacteria and Lactococcus lactis has become the model organism because of its small genome, genetic accessibility and simple metabolism. Here we discuss the metabolic engineering of L. lactis and the value of metabolic models compared with other LAB, with a particular focus on the food-grade production of metabolites involved in flavour, texture and health.     

Autochthonous fungi are central components in microbial community structure in raw fermented sausages

Raw meat sausage represents a unique ecological niche rich in nutrients for microbial consumption, making it particularly vulnerable to microbial spoilage. Starter cultures are applied to improve product stability and safety as well as flavour characteristics. However, the influence of starter cultures on microbial community assembly and succession throughout the fermentation process is largely unknown. In particular the effect on the fungal community has not yet been explored. We evaluate the microbiological status of four different raw meat sausages using high-throughput 16S rRNA gene and ITS2 gene sequencing. The objective was to study temporal changes of microbial composition during the fermentation process and to identify potential keystone species that play an important role within the microbial community. Our results suggest that fungi assigned to the species Debaryomyces hansenii and Alternaria alternata play a key role in microbial community dynamics during fermentation. In addition, bacteria related to the starter culture Lactobacillus sakei and the spoilage-associated genera Acinetobacter, Pseudomonas and Psychrobacter are central components of the microbial ecosystem in raw fermented sausages. Elucidating the exact role and interactions of these microorganisms has the potential to have direct impacts on the quality and safety of fermented foods.     

Kimchi microflora: history,current status,and perspectives for industrial kimchi production

Kimchi, a traditional Korean food made by the fermentation of vegetables, has become popular globally because of its organoleptic, beneficial, and nutritional properties. Spontaneous kimchi fermentation in unsterilized raw materials leads to the growth of various lactic acid bacteria (LAB), which results in variations in the taste and sensory qualities of kimchi products and difficulties in the standardized industrial production of kimchi. Raw materials, kimchi varieties, ingredients, and fermentation conditions have significant effects on the microbial communities and fermentative characteristics of kimchi during fermentation. Heterofermentative LAB belonging to the genera Leuconostoc, Lactobacillus, and Weissella are likely to be key players in kimchi fermentation and have been subjected to genomic and functional studies to gain a better understanding of the fermentation process and beneficial effects of kimchi. The use of starter cultures has been considered for the industrial production of high quality, standardized kimchi. Here, we review the composition and biochemistry of kimchi microflora communities, functional and genomic studies of kimchi LAB, and perspectives for industrial kimchi production.     

Lactic acid bacteria producing B-group vitamins: a great potential for functional cereals products

Wheat contains various essential nutrients including the B group of vitamins. However, B group vitamins, normally present in cereals-derived products, are easily removed or destroyed during milling, food processing or cooking. Lactic acid bacteria (LAB) are widely used as starter cultures for the fermentation of a large variety of foods and can improve the safety, shelf life, nutritional value, flavor and overall quality of the fermented products. In this regard, the identification and application of strains delivering health-promoting compounds is a fascinating field. Besides their key role in food fermentations, several LAB found in the gastrointestinal tract of humans and animals are commercially used as probiotics and possess generally recognized as safe status. LAB are usually auxotrophic for several vitamins although certain strains of LAB have the capability to synthesize water-soluble vitamins such as those included in the B group. In recent years, a number of biotechnological processes have been explored to perform a more economical and sustainable vitamin production than that obtained via chemical synthesis. This review article will briefly report the current knowledge on lactic acid bacteria synthesis of vitamins B2, B11 and B12 and the potential strategies to increase B-group vitamin content in cereals-based products, where vitamins-producing LAB have been leading to the elaboration of novel fermented functional foods. In addition, the use of genetic strategies to increase vitamin production or to create novel vitamin-producing strains will be also discussed.     

The effect of Debina grapevine indigenous yeast strains of <Emphasis Type="Italic">Metschnikowia</Emphasis> and <Emphasis Type="Italic">Saccharomyces</Emphasis> on wine flavour

The spontaneous alcoholic fermentation of grape must is a complex microbiological process involving a large number of various yeast species, to which the flavour of every traditional wine is largely attributed. Whilst Saccharomyces cerevisiae is primarily responsible for the conversion of sugar to alcohol, the activities of various non-Saccharomyces species enhance wine flavour. In this study, indigenous yeast strains belonging to Metschnikowia pulcherrima var. zitsae as well as Saccharomyces cerevisiae were isolated and characterized from Debina must (Zitsa, Epirus, Greece). In addition, these strains were examined for their effect on the outcome of the wine fermentation process when used sequentially as starter cultures. The resulting wine, as analyzed over three consecutive years, was observed to possess a richer, more aromatic bouquet than wine from a commercial starter culture. These results emphasize the potential of employing indigenous yeast strains for the production of traditional wines with improved flavour.     

The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology

The food industry is constantly striving to develop new products to fulfil the ever changing demands of consumers and the strict requirements of regulatory agencies. For foods based on microbial fermentation, this pushes the boundaries of microbial performance and requires the constant development of new starter cultures with novel properties. Since the use of ingredients in the food industry is tightly regulated and under close scrutiny by consumers, the use of recombinant DNA technology to improve microbial performance is currently not an option. As a result, the focus for improving strains for microbial fermentation is on classical strain improvement methods. Here we review the use of these techniques to improve the functionality of lactic acid bacteria starter cultures for application in industrial-scale food production. Methods will be described for improving the bacteriophage resistance of specific strains, improving their texture forming ability, increasing their tolerance to stress and modulating both the amount and identity of acids produced during fermentation. In addition, approaches to eliminating undesirable properties will be described. Techniques include random mutagenesis, directed evolution and dominant selection schemes.     

Determination of aspartase activity in dairy Propionibacterium strains

Propionic acid bacteria (PAB) are important as starter cultures for the dairy industry in the manufacture of Swiss-type cheeses, in which they are involved in the formation of eyes and are responsible for the typical flavour and aroma. These characteristics are mainly due to the classical propionic acid fermentation, but also the conversion of aspartate to fumarate and ammonia by the enzyme aspartase and the subsequent reduction of fumarate to succinate, which occur in dairy Propionibacterium freudenreichii ssp. shermanii and ssp. freudenreichii starter strains. Additionally, the metabolism of free amino acids may be partly responsible for secondary fermentation and the subsequent split defects in cheese matrix. Here a method for aspartase activity was established and a number of dairy propionibacteria belonging to P. freudenreichii ssp. shermanii and freudenreichii were screened for this enzyme activity. A wide range of aspartase activity could be found in PAB isolates originating from cheese. The majority, i.e. 70% of the 100 isolates tested, showed very low levels of aspartate activity.     

Effect of aflatoxin B-1 on the proteolytic activity of some lactic-acid bacteria

The proteolytic activity of five (ATCC) strains of lactic acid bacteria were investigated in the presence of different concentrations of aflatoxin B-1. The presented data revealed that aflatoxin in milk can affect the lactic acid bacteria which are used in the manufacture of dairy products. Such effect depends on toxin concentration and the species of lactic acid bacteria.This investigation is of practical value because it may explain the effect which occurs during cheese manufacture. This defect can be characterized by off flavour which can be very undesirable for a ripened cheese.     

Engineering lactic acid bacteria for increased industrial functionality

Based on their spoilage-preventing and flavor-contributing characteristics, lactic acid bacteria (LAB) are employed as starter cultures for the fermentation of foods and feeds. In addition, several specific LAB strains are marketed on basis of their beneficial effects on the consumer's health, representing an explosively growing market for the products containing these so-called probiotics. Due to this extensive industrial use there is a strong interest in unraveling the molecular mechanisms involved in industrial robustness, cognate stress resistance, and health-promoting phenotypes of these LAB that may vary drastically between different starter and probiotic strains currently marketed. This review describes some of the post-genomic tools developed, as well as their employment for the identification of bacterial effector molecules involved in the aforementioned industrially relevant phenotypes. Furthermore, it addresses possible strategies to exploit such knowledge into the rational design of LAB strains with increased industrial functionality.