Malolactic Fermentation Process Research Articles

FERMENTED FOOD PRODUCTS USING SEA BUCKTHORN FRUIT. OVERVIEW

Herbal raw materials are a unique source of biologically active substances that are of particular value in the treatment of various diseases. Sea buckthorn is one of the most famous and widespread medicinal plants, widely grown in the United States, Europe, Asia, the Russian Federation and the Republic of Belarus. Information on the phytochemical composition of fruits is quite fully covered in the scientific literature. Sea buckthorn fruits are rich in essential nutrients and biologically active substances, but remain less in demand. Thus, it is important to study and implement new ways of processing sea buckthorn fruits that can contribute to the development of new functional foods and attract the attention of consumers to these products. This review summarizes studies on the development of food products using sea buckthorn fruits and lactic acid bacterial cultures. Particular attention is paid to the process of malolactic fermentation and its influence on the content and profile of organic acids and polyphenols in the juice of sea buckthorn fruits, it is shown what changes in antioxidant properties and organoleptic characteristics occur. The role of sea buckthorn fruits and their components in the development of new probiotic dairy and non-dairy products is considered.

Transcriptional and Metabolic Response of Wine-Related Lactiplantibacillus plantarum to Different Conditions of Aeration and Nitrogen Availability

Lactic acid bacteria (LAB) perform the process of malolactic fermentation (MLF) in wine. Availability of oxygen and nitrogen nutrients could influence LAB growth, malolactic activity, and other metabolic pathways, impacting the subsequent wine quality. The impact of these two factors has received limited investigation within LAB, especially on a transcriptome level. The aim of this study was to evaluate metabolic changes in the strain Lactiplantibacillus plantarum IWBT B063, growing in synthetic grape juice medium (GJM) under different oxygen exposure conditions, and with low availability of nitrogen-based nutrients. Next-generation sequencing was used to analyze expression across the transcriptome (RNA-seq), in combination with conventional microbiological and chemical analysis. L. plantarum consumed the malic acid present in all the conditions evaluated, with a slight delay and impaired growth for nitrogen limitation and for anaerobiosis. Comparison of L. plantarum transcriptome during growth in GJM with and without O2 revealed differential expression of 148 functionally annotated genes, which were mostly involved in carbohydrate metabolism, genetic information processing, and signaling and cellular processes. In particular, genes with a protective role against oxidative stress and genes related to amino acid metabolism were differentially expressed. This study confirms the suitability of L. plantarum IWBT B063 to carry out MLF in different environmental conditions due to its potential adaption to the stress conditions tested and provides a better understanding of the genetic background of an industrially relevant strain.

Utjecaj različitih načina provođenja jabučno-mliječne fermentacije na promjene kemijskih svojstava i hlapivih komponenti crnog vina Teran (Vitis vinifera L.)

Malolactic fermentation (MLF) is a complex biochemical process playing an important role in the production of red wines. The main reasons for its implementation are the reduction of titratable acidity and the consequent increase of pH, microbiological stability of wine, and changes in aromatic and sensory properties of wine. The aim of this study was to determine the compatibility of yeast and bacteria used by different malolactic fermentation techniques and their influence on the fermentation duration, concentration of individual organic acids, aroma compounds, and on chemical and sensory properties of Teran wines. The experiment included control treatment (without MLF), spontaneous MLF, induced MLF at the beginning of alcoholic fermentation with simultaneous inoculation of yeast and bacteria (co-inoculation) and induced MLF after alcoholic fermentation (sequential MLF). In the co-inoculation treatment MLF had no negative effect on the alcoholic fermentation kinetic. Alcoholic fermentation was complete in all treatments. Co-inoculation resulted in a significantly shorter duration of the MLF process. In all MLF treatments, significant reduction of titratable acidity and the increase of pH values was noted. Wines of the spontaneous MLF treatment obtained the highest concentration of volatile acidity and ethyl acetate. In all MLF treatments a complete consumption of malic acid and a decrease in concentration of citric acid, total amount of higher alcohols, and acetaldehyde were observed. Furthermore, significantly higher concentrations of ethyl esters, diacetyl, acetoin, and 2,3-butanediol were present in wines from all MLF treatments.

Influences of acid and ethanol stresses on Oenococcus oeni SD-2a and its proteomic and transcriptional responses.

During winemaking, malolactic fermentation (MLF) is usually induced by Oenococcus oeni owing to its high resistance to wine stress factors. To ensure a controlled and efficient MLF process, starter cultures are inoculated in wine. In previous studies, O. oeni strains with sub-lethal acid or ethanol stresses showed higher freeze-drying vitality and better MLF performance. To explore the mechanisms involved, influences of acid and ethanol stresses on O. oeni SD-2a were investigated in this study to gain a better understanding of the cross-protection responses. The results showed that acid and ethanol stresses both caused damage to cell membranes and decreased cellular adenosine triphosphate concentration. At the same time, acid stress increased the uptake of glutathione, while ethanol stress led to cell depolarization. The results of comparative proteomic analysis highlighted that heat shock protein was induced with almost all acid and ethanol stresses. In addition, the expression of stress-relevant genes (hsp20, clpP, trxA, ctsR, recO, usp) increased greatly with ethanol and acid stress treatments. Finally, the viability of O. oeni was improved with acid and ethanol pretreatments after freeze-drying. This study demonstrated that acid and ethanol stresses had mixed influences on O. oeni SD-2a. Some physiological and molecular changes would contribute to a more stress-tolerant state of O. oeni, thereby improving the viability of lyophilized cells. © 2020 Society of Chemical Industry.

Compact analytical flow system for the simultaneous determination of l-lactic and l-malic in red wines

During the malolactic fermentation of red wines, l-malic acid is mainly converted to l-lactic acid. Both acids should be precisely measured during the entire process to guarantee the quality of the final wine, thus making real-time monitoring approaches of great importance in the winemaking industry. Traditional analytical methods based on laboratory procedures are currently applied and cannot be deployed on-site. In this work, we report on the design and development of a bi-parametric compact analytical flow system integrating two electrochemical biosensors that could be potentially applied in this scenario. The developed flow-system will allow for the first time the simultaneous measurement of both acids in real scenarios at the real-time and in remote way. Miniaturized thin-film platinum four-electrode chips are fabricated on silicon substrates by standard photolithographic techniques and further implemented in a polymeric fluidic structure. This includes a 15 µL flow cell together with the required fluidic channels for sample and reagent fluid management. The four-electrode chip includes counter and pseudo-reference electrodes together with two working electrodes. These are sequentially modified with electropolymerized polypyrrole membranes that entrap the specific receptors for selectively detecting both target analytes. The analytical performance of both biosensors is studied by chronoamperometry, showing a linear range from 5 × 10−6 to 1 × 10−4 M (LOD of 3.2 ± 0.3 × 10−6 M) and from 1 × 10−7 to 1 × 10−6 M (LOD of 6.7 ± 0.2 × 10−8 M) for the l-lactate and the l-malate, respectively. Both biosensors show long-term stability, retaining more than the 90% of their initial sensitivity after more than 30 days, this being a prerequisite for monitoring the whole process of the malolactic fermentation of the red wines (time between 20 and 40 days). The flow system performance is assessed with several wine samples collected during the malolactic fermentation process of three red wines, showing an excellent agreement with the results obtained with the standard method.

Identification and Characterization of the Small Heat Shock Protein Hsp20 from Oenococcus oeni SD-2a.

Oenococcus oeni can exert its function in hostile wine conditions during the malolactic fermentation process. Therefore, it is an important microbial resource for exploring resistance genes. Hsp20 is an important small heat shock protein from O. oeni. The conserved consensus motif "A-x-x-x-x-G-x-L" of Hsp20 announced its role as a member of the small heat shock protein family. The hsp20 gene from O. oeni SD-2a was cloned to create the recombinant plasmid pTriEx-Hsp20. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and the Hsp20 protein was induced by isopropyl-β-D-thiogalactoside (IPTG). The hsp20 gene from O. oeni SD-2a was successfully expressed, and a 20-kDa fusion protein was identified by SDS-PAGE. The purified Hsp20 protein was obtained using Ni-affinity chromatography. Additionally, BL21(DE3)/Hsp20 and BL21(DE3)/Ctrl were treated at high temperatures of 42 and 52°C, at pH values of 2.0-12.0, under oxidative shock with 0.1% (v/v) and 0.2% (v/v) H2O2, and under an osmotic shock of 430 and 860mM NaCl to compare the effects of heterologous expression of the Hsp20 protein from O. oeni SD-2a for stress resistance. Notably, Hsp20 overexpression showed enhanced resistance than the control strain did when confronted with different elevated stress conditions. The results demonstrated heterologous expression of the hsp20 gene from O. oeni SD-2a significantly improved the resistance of the host E. coli bacteria against stress conditions.

Robust l-malate bienzymatic biosensor to enable the on-site monitoring of malolactic fermentation of red wines

Monitoring the malolactic fermentation process is strictly required to guarantee the sensorial quality and freshness of red wines. This could be achieved by in-field and real-time continuous measurements of l-malate concentration in the fermentation tanks. The potential of a miniaturized amperometric bienzymatic biosensor as an analytical tool to be applied in such scenario is described in this paper. The biosensor comprises a thin-film gold electrode as transducer, malate dehydrogenase (MDH) and diaphorase (DP) enzymes together with nicotinamide adenine dinucleotide (NAD+) cofactor as the selective receptor and an adequate redox mediator to record the corresponding amperometric signal. Three different biosensor architectures are studied, whose main differences lie in the immobilization of the different chemical components onto the electrode surface. In all cases a fast-electrosynthethized polypyrrole (PPy) membrane is generated for this purpose. The experimental conditions are optimized and the best architecture shows a sensitivity of 1365 ± 110 mA M−1 cm−2 and a detection limit of 6.3 × 10−8 M in a concentration range of 1 × 10−7 M – 1 × 10−6 M. The biosensor presents an excellent working stability as it retains above 90% of its sensitivity after 37 days, thus enabling the monitoring of the malolactic fermentation of three red wines. The obtained results show excellent agreement with the standard colorimetric method.

New molecular evidence of wine yeast-bacteria interaction unraveled by non-targeted exometabolomic profiling

Introduction Bacterial malolactic fermentation (MLF) has a considerable impact on wine quality. The yeast strain used for primary fermentation can systematically stimulate (MLF+ phenotype) or inhibit (MLF−) bacteria and the MLF process as a function of numerous winemaking practices, but the underlying molecular evidence still remains a mystery.

Monitoring of malolactic fermentation in wine using an electrochemical bienzymatic biosensor for l-lactate with long term stability

l-lactic acid is monitored during malolactic fermentation process of wine and its evolution is strongly related with the quality of the final product. The analysis of l-lactic acid is carried out off-line in a laboratory. Therefore, there is a clear demand for analytical tools that enabled real-time monitoring of this process in field and biosensors have positioned as a feasible alternative in this regard. The development of an amperometric biosensor for l-lactate determination showing long-term stability is reported in this work. The biosensor architecture includes a thin-film gold electrochemical transducer selectively modified with an enzymatic membrane, based on a three-dimensional matrix of polypyrrole (PPy) entrapping lactate oxidase (LOX) and horseradish peroxidase (HRP) enzymes. The experimental conditions of the biosensor fabrication regarding the pyrrole polymerization and the enzymes entrapment are optimized. The biosensor response to l-lactate is linear in a concentration range of 1 × 10−6–1 × 10−4 M, with a detection limit of 5.2 × 10−7 M and a sensitivity of – (13500 ± 600) μA M−1 cm−2. The biosensor shows an excellent working stability, retaining more than 90% of its original sensitivity after 40 days. This is the determining factor that allowed for the application of this biosensor to monitor the malolactic fermentation of three red wines, showing a good agreement with the standard colorimetric method.

Temperature compensation of ultrasonic velocity during the malolactic fermentation process

Ultrasonic properties of materials present a strong dependence on temperature and in turn the ultrasonic velocity of propagation in the material under test. It is precisely for this reason that most ultrasonic measurements are often carried out with thermostated samples by using either water tanks or climate chambers. This approach is viable in a laboratory and when the measured or characterized samples are relatively small. However, this procedure is highly improbable to be applied when in situ measurements in industrial environments must be performed. This goes for the case of, for example, ultrasonic velocity measurements in wine while it is performing malolactic fermentation inside a tank of hundreds of thousands of litres. In this paper two different practical approaches to temperature compensation are studied. Then, the two temperature compensation methods are applied to the measured ultrasonic velocity values along a whole malolactic fermentation process. The results of each method are discussed.

Combine Use of Selected Schizosaccharomyces pombe and Lachancea thermotolerans Yeast Strains as an Alternative to the Traditional Malolactic Fermentation in Red Wine Production.

Most red wines commercialized in the market use the malolactic fermentation process in order to ensure stability from a microbiological point of view. In this second fermentation, malic acid is converted into l-lactic acid under controlled setups. However this process is not free from possible collateral effects that on some occasions produce off-flavors, wine quality loss and human health problems. In warm viticulture regions such as the south of Spain, the risk of suffering a deviation during the malolactic fermentation process increases due to the high must pH. This contributes to produce wines with high volatile acidity and biogenic amine values. This manuscript develops a new red winemaking methodology that consists of combining the use of two non-Saccharomyces yeast strains as an alternative to the traditional malolactic fermentation. In this method, malic acid is totally consumed by Schizosaccharomyces pombe, thus achieving the microbiological stabilization objective, while Lachancea thermotolerans produces lactic acid in order not to reduce and even increase the acidity of wines produced from low acidity musts. This technique reduces the risks inherent to the malolactic fermentation process when performed in warm regions. The result is more fruity wines that contain less acetic acid and biogenic amines than the traditional controls that have undergone the classical malolactic fermentation.

Ultrasonic velocity of water–ethanol–malic acid–lactic acid mixtures during the malolactic fermentation process

During malolactic fermentation in wines, malic acid is transformed into lactic acid by the action of lactic acid bacteria. This process can be monitored on-line by measuring the velocity of a low intensity ultrasonic wave propagating through the medium. In this work, an experimental study of ultrasonic propagation velocity in laboratory mixtures of water–ethanol–malic acid and lactic acid is presented. A good correlation was found between the ultrasonic velocity and malic and lactic acid concentrations. These results could be used to predict the end-point of the malolactic fermentation process and show the great potential of this ultrasonic technique to determine malic and lactic acid concentrations during the malolactic fermentation process.

Mutation and selection of Oenococcus oeni for controlling wine malolactic fermentation

Malolactic fermentation (MLF), mainly carried out by lactic acid bacterium, is of immense importance in winemaking allowing for improvement of microbiological stability and organoleptic characteristics of wine. However, it is difficult to address the problems of decoloration and loss of taste along with MLF induced by traditional starter culture. To further the aim at realizing a controlled and efficient MLF process, mutant of Oenococcus oeni strains after ultraviolet irradiation was selected to enhance quality of MLF. The treated strains with a lethality rate of 80–90 % were preliminarily screened, and then were secondly screened by evaluating their sensory and fermentation properties, by which O. oeni C10-1 were successfully screened. The malolactic conversion rate of O. oeni C10-1 is up to 38.81 %, which is remarkably increased when compared to original strains. Pivotally, wine fermented by O. oeni C10-1 demonstrated the highest color density of 9.240 and got the score of 5.95 in softness index, suggesting a remission of decoloration phenomenon and improved mouth feeling of wine. In all, these make it highly valuable for industrial application.

Ultrasonic monitoring of malolactic fermentation in red wines

The progress of malolactic fermentation in red wines has been monitored by using ultrasonic techniques. The evolution of ultrasonic velocity of a tone burst 1MHz longitudinal wave was measured, analyzed and compared to those parameters of oenological interest obtained simultaneously by analytical methods. Semi-industrial tanks were used during measurements pretending to be in real industrial conditions. Results showed that the ultrasonic velocity mainly changes as a result of the conversion by lactic acid bacteria of malic acid into lactic acid and CO2. Overall, the present study has demonstrated the potential of the ultrasonic technique in monitoring the malolactic fermentation process.

The application of malolactic fermentation process to create good-quality grape wine produced in cool-climate countries: a review

The work presents the essence of the secondary fermentation occurring in the course of grape wines production—malolactic fermentation (MLF). The selection of wine yeast and bacteria preparations, scenario of malolactic bacteria inoculation as well as control system of the process conditions are proposed to create the specific character and style of grape wines produced in cool-climate countries, in which the acidity of grape must is often significantly enhanced and the concentration of aroma compounds is considerably low. The role played by appropriately run MLF is presented, along with the advantages and disadvantages of the process, benefits, and technological drawbacks resulting from the introduction of this procedure to the vinification process. Moreover, methods to initiate and run MLF as well as interactions occurring between microorganisms used in wine production, i.e., wine yeasts from the genus Saccharomyces and wine bacteria from the genus Oenococcus, are also presented and discussed.

Role of Malolactic Fermentation on the Quality of Mango (Mangifera indica L.) Wine

The interactions between three Saccharomyces cerevisiae strains individually and with Oenococcus oeni during the process of malolactic acid fermentation (MLF) was investigated in mango wine. In all the treatments, alcoholic fermentation (AF) was completed between 10 and 12 d, and the yeast populations were not altered by the bacterial inoculation. Compared with sequential inoculation, the wine with simultaneous inoculation showed higher ethyl acetate content (26.6–41.1 mg/L). The total concentrations of higher alcohols were in the range of 350.9–372.4 mg/L. There were no significant (P < 0.05) differences in the concentrations of 1-propanol (12.6–13.4 mg/L); however, differences in the concentrations of isobutanol and amyl alcohol (40.4–50.1 and 241.4–269.0 (mg/L), respectively) were significant (P < 0.05). A significant decrease in acetaldehyde was observed in simultaneously inoculated wine in the range of 14.1–42.5 (mg/L), but no significant (P < 0.05) difference in methanol content was observed. Wine inoculated with O. oeni showed better sensory scores to that of control wine. Irrespective of the yeast strain used, simultaneous treatment showed better sensorial attributes in flavor, fruity aroma, and overall acceptability. Inoculation of O. oeni to mango juice or wine had no significant influence on the volatile aroma composition of the final wine.

Biogenic amines and ethyl carbamate in primitivo wine: survey of their concentrations in commercial products and relationship with the use of malolactic starter.

This research was conducted to determine the biogenic amine (BA) and ethyl carbamate (EC) concentrations in commercial Primitivo wine samples and the influence of the use of malolactic starter culture on concentrations of these potentially hazardous compounds in this wine. One hundred sixty bottles of wine from eight producers in the Apulia region of southern Italy were purchased at retail and analyzed. The most common BAs were histamine, 2-phenylethylamine, tyramine, cadaverine, putrescine, spermine, and spermidine. Putrescine (derived from ornithine) was the most abundant BA in all commercial Primitivo wines (5.41 to 9.51 mg/liter), 2-phenylethylamine was detected in only two commercial wines (at less than 2.12 mg/liter), and histamine was found at concentrations of 1.49 to 16.34 mg/liter. The concentration of EC in commercial Primitivo wine was 6.81 to 15.62 ppb, which is not considered dangerous for human health. Malolactic fermentation (MLF) affected the concentrations of BAs and EC differently. For EC, no significant differences were detected between samples of wine produced by spontaneous fermentation and wine that was inoculated malolactic starter. Mean EC concentrations were 12 and 14 ppb in two batches tested (always 18 ppb or less), regardless of whether the malolactic starter was added. Although present at trace levels in wine before the MLF, histamine accumulated during the MLF process, regardless of whether the malolactic starter was added. However, the increase in histamine was higher in wines without the malolactic starter. The concentrations of putrescine and cadaverine increased after MLF, especially in the wine that spontaneously fermented. The use of a selected malolactic starter resulted in reductions in BA concentrations in wine produced by this guided MLF compared with wine produced by spontaneous MLF.

Effects of a bacteriocin‐like substance produced by Leuconostoc mesenteroides subsp. cremoris on spoilage strain Lactobacillus fructivorans and various pathogens

SummaryCertain lactic acid bacteria present in the natural flora of the wine fermentation medium perform the malolactic fermentation process and other biochemical activities which lead to the formation of the outstanding aroma and bouquet of wine. However, because of the spoilage phenomena caused also by certain lactic cultures, off‐odours formed via metabolic side reactions cause severe economic losses in the wine industry. Several control mechanisms were introduced in order to eliminate these spoilage cultures, but none of these has been implemented as an efficient and healthy process. A partially purified bacteriocin‐like inhibitory substance (BLIS) with a molecular weight of 32 000 Da, obtained from a Leuconostoc mesenteroides subsp. cremoris isolate belonging to the native flora of wines of the Cappadocia region, was studied for its inhibitory activity on the spoilage strain, Lactobacillus fructivorans, isolated from the same wine flora and several other pathogens, and was found to be efficient in eliminating both the spoilage strain and the pathogens.

Functional Analyses of the Malolactic Wine Yeast ML01

Deacidification of grape musts and wines is important for the production of well-balanced wines. The bacterial malolactic fermentation (MLF) process is unreliable and stuck MLF often leads to spoilage of wines and the production of biogenic amines. The genetically engineered wine yeast, ML01, is a Prise de Mousse strain that contains the malate transport gene (<i>mae1</i>) from <i>Schizosaccharomyces pombe</i> and the malolactic gene (<i>mleA</i>) from <i>Oenococcus oeni</i>, stably integrated into the genome at the <i>URA3</i> locus. Both genes were isolated from wine-related microorganisms and are expressed under control of the <i>Saccharomyces cerevisiae PGK1</i> promoter and terminator sequences. ML01 is capable of decarboxylating up to 9.2 g/L of malate to equimolar amounts of lactate in Chardonnay grape must during the alcoholic fermentation. ML01 contains no antibiotic resistance marker genes or vector DNA sequences. The presence of the malolactic cassette in the genome does not affect growth, ethanol production, fermentation kinetics, or metabolism of ML01. Wines produced by the ML01 yeast have lower volatile acidity and improved color properties than wines produced with the parental yeast and a bacterial MLF. Analysis of the volatile compounds, sensory analyses, and industrial production of wine indicate that ML01 is suitable for the commercial production of quality wine.

Strategies for enhanced malolactic fermentation in wine and cider maturation

AbstractMalolactic fermentation (the conversion of malate to lactate) has been recognised as a desired process in wine and cider making and is being progressively developed. It not only concerns the conversion of malate but also involves flavour‐related biotransformations catalysed by malolactic fermentation bacteria. This review considers strategies to improve the reliability and performance of malolactic fermentation and documents the development of cell propagation systems and maturation processes, especially in relation to the physiology and biochemistry of malolactic fermentation bacteria. This includes the use of starter cultures and high cell concentrations for propagation and biotransformation, the types of bioreactors and their operational modes, especially those associated with the process of malolactic fermentation. Oenococcus oeni, the predominant organism associated with malolactic fermentation, is an acidophilic bacterium and is able to grow in wine at pH 3.5 or lower in the presence of ethanol and sulphite. As malolactic fermentation takes place in a highly alcoholic (up to 13% v/v) and highly acidic (pH 3.5 or lower) environment, slow growth and poor yields are frequently encountered when starter cultures are used. As a result, it requires several weeks or even months in such conditions to achieve full maturation. The malolactic fermentation process is also affected by temperature, malate concentration, nutrient composition and cell concentration. With an improved understanding of malolactic fermentation and the use of high cell concentrations, appropriate bioreactor designs and various operational modes, process innovation involving the separation of cell propagation from the maturation process now looks feasible. The review assesses the performance of malolactic fermentation systems and the relative benefits of high‐cell‐concentration biotransformation systems (free cells, immobilised cells or membrane bioreactor) to achieve malolactic fermentation with high productivity. Copyright © 2006 Society of Chemical Industry