FT-ICR-MS reveals the molecular imprints of the brewing process.

The study of fermentation and brewing has a long history of pioneering discoveries that continue to influence modern industrial food production. Since then, numerous research endeavors have yielded conventional criteria that guide contemporary brewing practices. However, the intricate open challenges faced today necessitate a more exhaustive understanding of the process at the molecular scale. We have developed an ultra-high-resolution mass spectrometric analysis (FT-ICR-MS) of the brewing process that can rapidly and comprehensively resolve thousands of molecules. This approach allows us to track molecular fluctuation during brewing at the level of chemical compositions. Employing biological triplicates, our investigation of two brewing lines that are otherwise identical except for the malt used revealed over 8,000 molecular descriptors of the brewing process. Metabolite imprints of both the similarities and differences arising from deviating malting temperatures were visualized. Additionally, we translated traditional brewing attributes such as the EBC-value, free amino nitrogen, pH-value, and concentration curves of specific molecules, into highly correlative molecular patterns consisting of hundreds of metabolites. These in-depth molecular imprints provide a better understanding of the molecular circumstances leading to various changes throughout the brewing process. Such chemical maps go beyond the observation of traditional brewing attributes and are of great significance in the investigation strategies of current open challenges in brewing research. The molecular base of knowledge, along with advancements in technological and data integration schemes, can facilitate the efficient monitoring of brewing and other productions processes.

A new hypothesis for the origin of the lager yeast Saccharomyces pastorianus.

Saccharomyces pastorianus, which is responsible for the production of bottom-fermented lager beer, is a hybrid species that arose from the mating of the top-fermenting ale yeast Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus around the start of the 17th century. Based on detailed analysis of Central European brewing records, we propose that the critical event for the hybridization was the introduction of top-fermenting S. cerevisiae into an environment where S. eubayanus was present, rather than the other way around. Bottom fermentation in parts of Bavaria preceded the proposed hybridization date by a couple of hundred years and we suggest that this was carried out by mixtures of yeasts, which may have included S. eubayanus. A plausible case can be made that the S. cerevisiae parent came either from the Schwarzach wheat brewery or the city of Einbeck, and the formation of S. pastorianus happened in the Munich Hofbräuhaus between 1602 and 1615 when both wheat beer and lager were brewed contemporaneously. We also describe how the distribution of strains from the Munich Spaten brewery, and the development by Hansen and Linder of methods for producing pure starter cultures, facilitated the global spread of the Bavarian S. pastorianus lineages.

Principles of engagement on research and other collaborations between the brewing sector and research entities: the FACT Principles.

Public-private partnerships are subject to intense scrutiny. This is specifically the case for sensitive health-related topics such as alcohol consumption. The brewing sector and representatives of the scientific community therefore stressed the need for specific principles for the proper and transparent governance of research and other collaborations between the brewing sector and research entities. At a 1-day seminar, a group of scientists and representatives from the brewing and food sector reached a consensus for such principles. They adhere to the following four fundamental conditions: Freedom of research, Accessibility, Contextualisation and Transparency. The points of focus in the FACT principles are open science, meaning that the methods and results are made accessible and reusable, and relationships are clearly disclosed. Actions to be taken for dissemination and implementation of the FACT Principles are, for instance, publishing them on public websites, including them in formal research agreements, and citing them in scientific publications. Scientific journals and (research) societies are encouraged to support the FACT Principles. In conclusion, the FACT Principles provide a framework for increased transparency and control of funding-related bias in research and other collaborations between the brewing sector and research entities. Monitoring their use and evaluating their impact will help to further refine and enforce the FACT Principles in the future.

Assessment of Tannin Tolerant Non-Saccharomyces Yeasts Isolated from Miang for Production of Health-Targeted Beverage Using Miang Processing Byproducts.

This research demonstrated an excellent potential approach for utilizing Miang fermentation broth (MF-broth), a liquid residual byproduct from the Miang fermentation process as a health-targeted beverage. One hundred and twenty yeast strains isolated from Miang samples were screened for their potential to ferment MF-broth and four isolates, P2, P3, P7 and P9 were selected, based on the characteristics of low alcoholic production, probiotic properties, and tannin tolerance. Based on a D1/D2 rDNA sequence analysis, P2 and P7 were identified to be Wikerhamomyces anomalus, while P3 and P9 were Cyberlindnera rhodanensis. Based on the production of unique volatile organic compounds (VOCs), W. anomalus P2 and C. rhodanensis P3 were selected for evaluation of MF-broth fermentation via the single culture fermentation (SF) and co-fermentation (CF) in combination with Saccharomyces cerevisiae TISTR 5088. All selected yeasts showed a capability for growth with 6 to 7 log CFU/mL and the average pH value range of 3.91-4.09. The ethanol content of the fermented MF-broth ranged between 11.56 ± 0.00 and 24.91 ± 0.01 g/L after 120 h fermentation, which is categorized as a low alcoholic beverage. Acetic, citric, glucuronic, lactic, succinic, oxalic and gallic acids slightly increased from initial levels in MF-broth, whereas the bioactive compounds and antioxidant activity were retained. The fermented MF-broth showed distinct VOCs profiles between the yeast groups. High titer of isoamyl alcohol was found in all treatments fermented with S. cerevisiae TISTR 5088 and W. anomalus P2. Meanwhile, C. rhodanensis P3 fermented products showed a higher quantity of ester groups, ethyl acetate and isoamyl acetate in both SF and CF. The results of this study confirmed the high possibilities of utilizing MF-broth residual byproduct in for development of health-targeted beverages using the selected non-Saccharomyces yeast.

Multi-Response Optimization of the Malting Process of an Italian Landrace of Rye (Secale cereale L.) Using Response Surface Methodology and Desirability Function Coupled with Genetic Algorithm.

Rye is used in some applications in the food and beverage industry and for the preparation of functional foods. It is an interesting raw material in malting and brewing due to its characteristic contribution to the beer's color, turbidity, foam and aroma. The aim of this work was to optimize the micro-malting process of a rye landrace. The response surface methodology (RSM) was applied to study the influence of three malting parameters (germination time, germination temperature and degree of steeping) on the quality traits of malted rye. Long germination times at high temperatures resulted in an increase in the extract and Kolbach index. The model for the apparent attenuation limit showed a particular pattern, whereby time and temperature inversely influenced the response. The lowest viscosities were determined in the worts produced from highly modified malts. Optimization of the variables under study was achieved by means of a desirability function and a genetic algorithm. The two methodologies provided similar results. The best combination of parameters to optimize the malting process on the rye landrace under study was achieved at 6 days, 12 °C and 44 g/100 g.

Beer fermentation performance and sugar uptake of Saccharomycopsis fibuligera-A novel option for low-alcohol beer.

There is a growing trend for beers with novel flavor profiles, as consumers demand a more diversified product range. Such beers can be produced by using non-Saccharomyces yeasts. The yeast species Saccharomycopsis fibuligera is known to produce exceptionally pleasant plum and berry flavors during brewer's wort fermentation while its mycelia growth is most likely a technological challenge in industrial-scale brewing. To better understand and optimize the physiological properties of this yeast species during the brewing process, maltose and maltotriose uptake activity trials were performed. These revealed the existence of active transmembrane transporters for maltose in addition to the known extracellular amylase system. Furthermore, a single cell isolate of S. fibuligera was cultured, which showed significantly less mycelial growth during propagation and fermentation compared to the mother culture and would therefore be much more suitable for application on an industrial scale due to its better flocculation and clarification properties. Genetic differences between the two cultures could not be detected in a (GTG)5 rep-PCR fingerprint and there was hardly any difference in the fermentation process, sugar utilization and flavor profiles of the beers. Accordingly, the characteristic plum and berry flavor could also be perceived by using the culture from the single cell isolate, which was complemented by a dried fruit flavor. A fermentation temperature of 20°C at an original gravity of 10 °P proved to be optimal for producing a low-alcohol beer at around 0.8% (v/v) by applying the S. fibuligera yeast culture from the single cell isolate.

Investigations into metabolic properties and selected nutritional metabolic byproducts of different non-Saccharomyces yeast strains when producing nonalcoholic beer.

Nonalcoholic beers are becoming increasingly popular, in part due to consumers' awareness of a healthier lifestyle. Additionally, consumers are demanding diversification in the product range, which can be offered by producing nonalcoholic beers using non-Saccharomyces yeasts for fermentation to create a wide variety of flavors. So far, little is known about the nutritionally relevant byproducts that these yeasts release during wort fermentation and whether these yeasts can be considered safe for food fermentations. To gain insights into this, the B vitamins of four different nonalcoholic beers fermented with the yeast species Saccharomycodes ludwigii, Cyberlindnera saturnus (two strains), and Kluyveromyces marxianus were analyzed. Furthermore, a total of 16 beers fermented with different non-Saccharomyces yeast strains were analyzed for biogenic amines. Additionally, stress tolerance tests were performed at 37°C and in synthetic human gastric juice in vitro. B vitamins were found in the four nonalcoholic beers in nutritionally relevant amounts so they could serve as a supplement for a balanced diet. Biogenic amines remained below the limit of determination in all 16 beers, and thus likely had no influence, while the stress tolerance tests gave a first indication that seven yeast strains could possibly tolerate the human gastric juice milieu.

Archeochemistry reveals the first steps into modern industrial brewing.

A historical beer, dated to the German Empire era, was recently found in northern Germany. Its chemical composition represents a unique source of insights into brewing culture of the late nineteenth century when pioneer innovations laid the foundations for industrial brewing. Complementary analytics including metabolomics, microbiological, sensory, and beer attribute analysis revealed its molecular profile and certify the unprecedented good storage condition even after 130 years in the bottle. Comparing its chemical signature to that of four hundred modern brews allowed to describe molecular fingerprints teaching us about technological aspects of historical beer brewing. Several critical production steps such as malting and germ treatment, wort preparation and fermentation, filtration and storage, and compliance with the Bavarian Purity Law left detectable molecular imprints. In addition, the aging process of the drinkable brew could be analyzed on a chemical level and resulted in an unseen diversity of hops- and Maillard-derived compounds. Using this archeochemical forensic approach, the historical production process of a culturally significant beverage could be traced and the ravages of time made visible.

Influence of Varying Fermentation Parameters of the Yeast Strain Cyberlindnera saturnus on the Concentrations of Selected Flavor Components in Non-Alcoholic Beer Focusing on (E)-ß-Damascenone.

The diversification of beer flavor is becoming increasingly popular, especially in the field of non-alcoholic beers, where sales are growing steadily. While flavor substances of traditional beers can largely be traced back to defined secondary metabolites, the production of non-alcoholic beers with non-Saccharomyces yeasts generates novel fruity flavors, some of which cannot yet be assigned to specific flavor substances. In a recently published study, besides pear, cool mint sweets, and banana-like flavor, distinctive red berry and apple flavors were perceived in a non-alcoholic beer fermented with the yeast strain Cyberlindnera saturnus TUM 247, whose secondary metabolites were to be elucidated in this study. The trials were carried out using response surface methodology to examine the fermentation properties of the yeast strain and to optimize the beer with maximum fruitiness but minimal off-flavors and ethanol content. It turned out that a low pitching rate, a moderate fermentation temperature, and an original gravity of 10.5 °P gave the optimal parameters. Qualitative analysis of the secondary metabolites, in addition to standard analysis for traditional beers, was first performed using headspace-gas chromatography with olfactometry. (E)-ß-damascenone emerged as the decisive substance for the red berry and apple flavor and so this substance was then quantitated. Although (E)-ß-damascenone is a well-known secondary metabolite in beer and this substance is associated with apple or cooked apple- and berry-like flavors, it has not yet been reported as a main flavor component in non-alcoholic beers.

Hidden in its color: A molecular-level analysis of the beer's Maillard reaction network.

We here report a comprehensive non-targeted analytical approach to describe the Maillard reaction in beer. By Fourier-transform ion cyclotron mass spectrometry (FT-ICR-MS), we were able to assign thousands of unambiguous molecular formulae to the mass signals and thus directly proceed to the compositional space of 250 analyzed beer samples. Statistical data analyses of the annotated compositions showed that the Maillard reaction is one of the driving forces of beer's molecular diversity leading to key compositional changes in the beer metabolome. Different visualization methods allowed us to map the systematic nature of Maillard reaction derived compounds. The typical molecular pattern, validated by an experimental Maillard reaction model system, pervades over 2,800 (40%) of the resolved small molecules. The major compositional changes were investigated by mass difference network analysis. We were able to reveal general reaction sequences that could be assigned to successive Maillard intermediate phase reactions by shortest path analysis.

Mashes to Mashes, Crust to Crust. Presenting a novel microstructural marker for malting in the archaeological record.

The detection of direct archaeological remains of alcoholic beverages and their production is still a challenge to archaeological science, as most of the markers known up to now are either not durable or diagnostic enough to be used as secure proof. The current study addresses this question by experimental work reproducing the malting processes and subsequent charring of the resulting products under laboratory conditions in order to simulate their preservation (by charring) in archaeological contexts and to explore the preservation of microstructural alterations of the cereal grains. The experimentally germinated and charred grains showed clearly degraded (thinned) aleurone cell walls. The histological alterations of the cereal grains were observed and quantified using reflected light and scanning electron microscopy and supported using morphometric and statistical analyses. In order to verify the experimental observations of histological alterations, amorphous charred objects (ACO) containing cereal remains originating from five archaeological sites dating to the 4th millennium BCE were considered: two sites were archaeologically recognisable brewing installations from Predynastic Egypt, while the three broadly contemporary central European lakeshore settlements lack specific contexts for their cereal-based food remains. The aleurone cell wall thinning known from food technological research and observed in our own experimental material was indeed also recorded in the archaeological finds. The Egyptian materials derive from beer production with certainty, supported by ample contextual and artefactual data. The Neolithic lakeshore settlement finds currently represent the oldest traces of malting in central Europe, while a bowl-shaped bread-like object from Hornstaad-Hörnle possibly even points towards early beer production in central Europe. One major further implication of our study is that the cell wall breakdown in the grain's aleurone layer can be used as a general marker for malting processes with relevance to a wide range of charred archaeological finds of cereal products.

Screening for new brewing yeasts in the non-Saccharomyces sector with Torulaspora delbrueckii as model.

This study describes a screening system for future brewing yeasts focusing on non-Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off-flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by-products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre-fermentation as a bio-flavouring agent for beers that have been post-fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour-forming properties.

The influence of serial repitching of Saccharomyces pastorianus on its karyotype and protein profile during the fermentation of gluten-free buckwheat and quinoa wort.

Gluten-free beer-like beverages from malted buckwheat and quinoa are somehow close to their commercial production, but rather high expenses are expected due to the relatively high price of grain, some technological adaptations of process and the need for external enzyme supplementation during mashing. One of the common and efficient cost reduction measures in the industrial scale is serial repitching of the yeast biomass, which has not been studied for the buckwheat and quinoa wort fermentation before. In that manner we have monitored possible changes in yeast's proteins and chromosomal DNA during eleven serial repitchings of the yeast Saccharomyces pastorianus strain TUM 34/70 for fermentation of the barley, buckwheat and quinoa wort. Karyotypes showed changes in regard to the raw materials used and many responsible candidate proteins are suggested which could cause these differences. Different relative expressions of some protein bands were also linked to the proteins involved in yeast stress response and proteins involved in fermentation performance. Results suggest that serial repitching of the strain TUM 34/70 seems suitable for the production of gluten-free beer-like beverages from buckwheat and quinoa.

Teff (Eragrostis tef) as a raw material for malting, brewing and manufacturing of gluten-free foods and beverages: a review.

The demand for gluten-free foods is certainly increasing. Interest in teff has increased noticeably due to its very attractive nutritional profile and gluten-free nature of the grain, making it a suitable substitute for wheat and other cereals in their food applications as well as foods for people with celiac disease. The main objective of this article is to review researches on teff, evaluate its suitability for different food applications, and give direction for further research on its applications for health food market. Teff is a tropical low risk cereal that grows in a wider ecology and can tolerate harsh environmental conditions where most other cereals are less viable. It has an excellent balance of amino acid composition (including all 8 essential amino acids for humans) making it an excellent material for malting and brewing. Because of its small size, teff is made into whole-grain flour (bran and germ included), resulting in a very high fiber content and high nutrient content in general. Teff is useful to improve the haemoglobin level in human body and helps to prevent malaria, incidence of anaemia and diabetes. The nutrient composition of teff grain indicates that it has a good potential to be used in foods and beverages worldwide. The high levels of simple sugars and α-amino acids as a result of breakdown of starch and protein, respectively, are essential for fermentation and beer making.

Influence of malting conditions on sorghum (Sorghum bicolor (L.) Moench) as a raw material for fermented beverages.

There has been recently increased interest in sorghum to substitute the gluten containing cereals in the diet of people suffering from celiac disease. The response surface methodology was used to determine the influence of malting parameters (degree of steeping, germination temperature and time) on sorghum (Sorghum bicolor (L.) Moench). Each parameter was varied at three levels. Malting attributes, considered important to produce high quality malt for the production of lactic acid fermented beverages, were analyzed. The optimized conditions were: degree of steeping 41%, germination temperature 27℃ after 7 days of germination. Under these conditions, the following values of the studied attributes can be obtained: α-amylase 139 U/g, ß-amylase 60 U/g, extract 83.8%, free amino nitrogen 117.8 mg/100 g, Kolbach index 26.6%, water-extractable arabinoxylan 0.3 g/L and vitamin B2 114.9 µg/L. Among the tested parameters, the germination time had the highest effect on malting attributes. Although the activity of amylolytic enzymes α- and ß-amylase were low, the value of extract was high and comparable to that of barley malt. The obtained results showed that sorghum malt is a promising raw material for the production of lactic acid fermented beverages.