Food Fermentation Industry Research Articles

Characterization of newly isolated Bacillus cereus D3 Co-producing α-amylase and protease and its application in food raw materials

Bacillus cereus, which effectively secretes various extracellular enzymes, has been widely used in the food, feed, and fermentation industries. One strain co-producing α-amylase and protease was newly isolated from Daqu and identified as B. cereus D3. It was shown that the production of α-amylase and protease co-produced by B. cereus D3 reached 13.89 ± 0.07 and 2468.45 ± 38.96 U/mL through optimization of fermentation conditions (nutrients (g/L): 20 of glucose, 20 of urea, 0.7 of MnCl2; incubation conditions: 24 h of incubation, initial medium pH of 7.0, temperature of 37 °C, 8% inoculum size, and 30% working volume). Moreover, the enzymatic genetic co-expression exhibited a similar pattern to the changes in enzymatic activities (growth-phase-dependent pattern). The two enzymes performed excellent thermal stability and were stable over wide pH ranges of 6.0–11.0 and 2.0–8.0, respectively. Finally, the hydrolysis degrees of raw materials including rice, sorghum, corn, and soybeans by α-amylase from B. cereus D3 reached 66.59 ± 4.21, 57.99 ± 4.39, 57.44 ± 4.67, and 38.01% ± 1.73%, respectively, producing glucose and maltose as the main products. The hydrolysis degrees of proteinous materials including mackerel heads and soybeans by protease from B. cereus D3 were 39.93 ± 1.99 and 30.44% ± 1.39%. SDS-PAGE analysis displayed extensive breakdown of macromolecular proteins, forming peptides of varied sizes. This is a systematic study on the co-production of α-amylase and protease by B. cereus, which is crucial to widen the understanding of this species co-producing multi-enzymes and explore its potential application in the food fermentation industry.

Transcriptome Analysis Reveals the Variations in Enzyme Production of Saccharopolyspora rosea A22 under Different Temperatures.

Saccharopolyspora is a key microorganism in the fermentation of traditional fermented foods, capable of producing saccharifying and liquefying enzymes at elevated temperatures. However, the specific mechanisms and regulatory pathways governing Saccharopolyspora's response to ambient temperatures are not yet fully understood. In this study, the morphological differences in Saccharopolyspora rosea screened from traditional handmade wheat Qu at different temperatures were initially explored. At 37 °C, the mycelium exhibited abundant growth and radiated in a network-like pattern. As the temperature increased, the mycelium aggregated into clusters. At 50 °C, it formed highly aggregated ellipsoidal structures, with the mycelium distributed on the spherical surface. Subsequently, we assessed the biomass, saccharifying enzyme activity and liquefying enzyme activity of Saccharopolyspora rosea cultured at 37 °C, 42 °C and 50 °C. Furthermore, transcriptome analysis demonstrated that Saccharopolyspora rosea employs mechanisms related to the carbon metabolism, the TCA cycle, glycine, serine and threonine metabolisms, and microbial metabolism in diverse environments to coordinate its responses to changes in environmental temperature, as verified by the expression of typical genes. This study enhances our understanding of the differences in high-temperature enzyme production by Saccharopolyspora, and offers valuable guidance for the traditional fermented food industry to drive innovation.

The MarR family regulator RmaH mediates acid tolerance of Lactococcus lactis through regulating peptidoglycan modification genes

Lactococcus lactis, widely used in the food fermentation industry, has developed various ways to regulate acid adaptation in the process of evolution. The investigation into how peptidoglycan (PG) senses and responds to acid stress is an expanding field. Here, we addressed the regulation of murT-gatD genes which are responsible for the amidation of PG D-Glu. We found that lactic acid stress reduced murT-gatD expression, and overexpressing these genes notably decreased acid tolerance of L. lactis NZ9000, possibly due to a reduction in PG's negative charge, facilitating the influx of extracellular protons into the cell. Subsequently, using a combination of DNA pull-down assay and electrophoretic mobility shift assay (EMSA), we identified a novel MarR family regulator, RmaH, as an activator of murT-gatD transcription. Further MEME motif prediction, EMSA verification and fluorescent protein reporter assay showed that RmaH directly bound to the DNA motif 5'-KGVAWWTTTTGCT-3' located in the upstream region of murT-gatD. Beyond the mechanistic investigation of RmaH activation of murT-gatD, this study provides new insight into how peptidoglycan modification is regulated and responds to lactic acid stress.

Novel cell wall polysaccharide genotypes and structures of lactococcal strains isolated from milk and fermented foods

The biosynthetic machinery for cell wall polysaccharide (CWPS) formation in Lactococcus lactis and Lactococcus cremoris is encoded by the cwps locus. The CWPS of lactococci typically consists of a neutral rhamnan component, which is embedded in the peptidoglycan, and to which a surface-exposed side chain oligosaccharide or polysaccharide pellicle (PSP) component is attached. The rhamnan component has been shown for several lactococcal strains to consist of a repeating rhamnose trisaccharide subunit, while the side chain is diverse in glycan content, polymeric status and glycosidic linkage architecture. The observed structural diversity of the CWPS side chain among lactococcal strains is reflected in the genetic diversity within the variable 3′ region of the corresponding cwps loci. To date, four distinct cwps genotypes (A, B, C, D) have been identified, while eight subtypes (C1 through to C8) have been recognized among C-genotype strains. In the present study, we report the identification of three novel subtypes of the lactococcal cwps C genotypes, named C9, C10 and C11. The CWPS of four isolates representing C7, C9, C10 and C11 genotypes were analysed using 2D NMR to reveal their unique CWPS structures. Through this analysis, the structure of one novel rhamnan, three distinct PSPs and three exopolysaccharides were elucidated. Results obtained in this study provide further insights into the complex nature and fascinating diversity of lactococcal CWPSs. This highlights the need for a holistic view of cell wall-associated glycan structures which may contribute to robustness of certain strains against infecting bacteriophages. This has clear implications for the fermented food industry that relies on the consistent application of lactococcal strains in mesophilic production systems.

Advancements in Fermented Beverage Safety: Isolation and Application of Clavispora lusitaniae Cl-p for Ethyl Carbamate Degradation and Enhanced Flavor Profile.

Ethyl carbamate (EC) is a natural by-product in the production of fermented food and alcoholic beverages and is carcinogenic and genotoxic, posing a significant food safety concern. In this study, Clavispora lusitaniae Cl-p with a strong EC degradation ability was isolated from Daqu rich in microorganisms by using EC as the sole nitrogen source. When 2.5 g/L of EC was added to the fermentation medium, the strain decomposed 47.69% of ethyl carbamate after five days of fermentation. It was unexpectedly found that the strain had the ability to produce aroma and ester, and the esterification power reached 30.78 mg/(g·100 h). When the strain was added to rice wine fermentation, compared with the control group, the EC content decreased by 41.82%, and flavor substances such as ethyl acetate and β-phenylethanol were added. The EC degradation rate of the immobilized crude enzyme in the finished yellow rice wine reached 31.01%, and the flavor substances of yellow rice wine were not affected. The strain is expected to be used in the fermented food industry to reduce EC residue and improve the safety of fermented food.

An effective strategy for improving the freeze-drying survival rate of Lactobacillus curvatus and its potential protective mechanism

Freeze-drying is a common method to prepare dried starter cultures with high stability and a long storage time. However, the bacterial cells suffer varying degrees of damage during the freeze-drying process. This study aimed to provide an effective strategy to improve the survival rate of Lactobacillus curvatus and to explore its potential protective mechanisms. The survival rates of bacterial cells in different cryoprotectants before and after freeze-drying were measured using the plate count method. The protective effects of composite cryoprotectants on bacterial cells were investigated from multiple perspectives, including key enzyme activities, the internal morphology of bacterial cells, and cell membrane integrity. Results showed that the freeze-drying survival rate of bacterial cells in the composite cryoprotectants consisting of trehalose, oleic acid and skimmed milk (85.38%) was significantly improved compared to that in PBS solution (2.04%). The addition of composite cryoprotectants could reduce damage to the cell wall and cell membrane, maintain the normal morphology of bacterial cells and cell membrane integrity, and improve key enzyme activities (lactate dehydrogenase, pyruvate kinase and ATPases) during the freeze-drying process. The viability of freeze-dried cultures was also monitored at different temperatures during storage, and the cell survival of freeze-dried cultures at −20 °C was the highest, reaching 61.13%. The findings obtained in this work offer a new method to improve the viability of Lactobacillus curvatus after freeze-drying, and may promote the wide use of freeze-dried cultures in the food fermentation industry.

Halophilic lactic acid bacteria - Play a vital role in the fermented food industry.

Halophilic lactic acid bacteria have been widely found in various high-salt fermented foods. The distribution of these species in salt-fermented foods contributes significantly to the development of the product's flavor. Besides, these bacteria also have the ability to biosynthesize bioactive components which potentially apply to different areas. In this review, insights into the metabolic properties, salt stress responses, and potential applications of these bacteria have been have been elucidated. The purpose of this review highlights the important role of halophilic lactic acid bacteria in improving the quality and safety of salt-fermented products and explores the potential application of these bacteria.

Optimization of Tempe Fermentation With the Addition of a Combination of Yeast and Starch Flour: Effect on Process Speed and Product Freshness Period

This research explores the potential for adding a combination of tempeh yeast and starch to the tempeh fermentation process to speed up fermentation and extend the product's freshness period. Through an experimental approach, we investigated the effect of the proportion of yeast and starch on the fermentation speed and shelf life of tempeh. We found that a yeast to starch ratio of 1:2 significantly accelerated the fermentation process, while a ratio of 1:1 was effective in extending the shelf life of tempeh up to 80 hours. Organoleptic tests show positive consumer acceptance of the tempeh variations produced. This research makes a significant contribution to tempeh processing technology, offering practical solutions for the fermented food industry to improve the quality and attractiveness of tempeh products on the market.

Transcriptome analysis of the response of Lactiplantibacillus plantarumIMAU10120 to infection by phage P2

Bacteriophage infection of lactic acid bacteria remains a significant problem in the food fermentation industry. To improve understanding about the underlying mechanism of phage–host infection, we used RNA sequencing technology to investigate the effect of phage infection on the expression of host genes based on the one‐step growth curve. Results identified a total of 3207 genes, of which 178 showed a significant differential expression in phage‐infected host. Moreover, bioinformatic analyses unravelled that these genes regulated many processes in host bacteria, including ribosome, RNA polymerase, DNA replication and metabolic pathways. This study further elucidates the response of host bacteria to phage infection. These results provide valuable data for developing effective phage control measures and improving the success rate of fermentation in the dairy industry.

Genomic and biological characteristics of a novel phage induced from Limosilactobacillus fermentum

Bacteriophage infection of starter cultures remains a major threat to the food fermentation industry. To date, adequate information regarding the genome and characteristics of the Limosilactobacillus fermentum (L. fermentum) phage is lacking. The phage LFP01 was obtained by inducing L. fermentum IMAU32646 with mitomycin-C. The results showed that this phage contained a linear double-stranded DNA genome spanning 40,562 bp with a GC content of 46.25%. It comprises 54 coding sequences, which are grouped into three potential functional modules: DNA replication, cell lysogeny/lysis, and virion morphogenesis. Taxonomic analysis further confirmed that this phage belonged to a novel virus genus within the Caudoviricetes class. The optimal multiplicity of infection was 0.1, the latent period was 45 min, and the burst size was 158 ± 2.65 PFU/cell. This phage exhibited good environmental tolerance at 0–42 °C and a pH of 4–11. High temperature (50 °C) and strongly acidic (pH 2–3) conditions significantly influenced the adsorption capacity of the phage, but divalent cations and chloramphenicol had no similar effect. The findings of this study could enrich the database of phages and broaden the understanding of phage diversity.

Lactococcus lactis in Dairy Fermentation—Health-Promoting and Probiotic Properties

The use of lactic acid bacteria (LAB) in the fermentation process to produce fermented foods has a long history. Furthermore, LAB are beneficial microorganisms known for their health-promoting characteristics. During fermentation, LAB have the capacity to produce significant amounts of bioactive substances, such as peptides, bacteriocins, lactic acid, exopolysaccharides (EPSs), enzymes, and others. Lactococcus lactis as one of the best-known and well-characterized species of LAB serves as a model organism for studying LAB. For a very long time, L. lactis has been used in milk fermentation, both in well-monitored industrial settings and on a small scale in traditional operations. Furthermore, L. lactis is a vital microorganism in the dairy food fermentation industry due to its role in acidification, flavor development, and the creation of various dairy products, including cheese, fermented butter, and others. The novelty of this review is the comprehensive and organized presentation of the main benefits of the use of L. lactis in milk fermentation processes including technological and safety features relevant for the dairy industry, probiotic potential, the ability to produce bioactive compounds (e.g., bacteriocins, GABA), and the recent development of such bacteria research methods like whole genome sequencing (WGS).

Effect of caramel colors addition on the color, physicochemical properties, and flavor of Huangjiu

Caramel color is a food colorant widely used in the fermented beverage industries. In this context, this study investigated the effects of caramel color on the quality of Huangjiu in terms of color, physicochemical properties, sensory characteristics, and the release of volatile organic compounds (VOCs). The results showed that Class IV caramel colors with the highest color intensity had the lowest coloring ability in Huangjiu due to precipitation. Caramel colors significantly affected the total sugar, titratable acidity, and amino nitrogen of Huangjiu. Sensory analyses indicated that the three classes of caramel colors significantly affected 10 sensory attributes and the aroma release of Huangjiu. The headspace concentrations of the 40 VOCs in Huangjiu changed significantly. Principal component analysis (PCA) and cluster analysis showed that Classes I and III caramel colors had similar effects on the VOCs in Huangjiu. Partial least-squares discriminant analysis (PLS-DA) showed that 9 compounds made important contributions to distinguishing different classes of caramel-colored Huangjiu samples. This study provides a theoretical foundation for the scientific application of caramel color in Huangjiu and other fermented food industries.

Nutritional Values and Bio-Functional Properties of Fungal Proteins: Applications in Foods as a Sustainable Source.

From the preparation of bread, cheese, beer, and condiments to vegetarian meat products, fungi play a leading role in the food fermentation industry. With the shortage of global protein resources and the decrease in cultivated land, fungal protein has received much attention for its sustainability. Fungi are high in protein, rich in amino acids, low in fat, and almost cholesterol-free. These properties mean they could be used as a promising supplement for animal and plant proteins. The selection of strains and the fermentation process dominate the flavor and quality of fungal-protein-based products. In terms of function, fungal proteins exhibit better digestive properties, can regulate blood lipid and cholesterol levels, improve immunity, and promote gut health. However, consumer acceptance of fungal proteins is low due to their flavor and safety. Thus, this review puts forward prospects in terms of these issues.

ÉPOP (traditional starter culture): a complex composition of plant resources prepared by the Misings of Assam, Northeast India

Ethnic practices with relation to starter culture reflect a region-specific traditions; therefore, documentation of such knowledge is key to improvise basic understanding as how traditions, cultures, and processes are linked to local dietary systems, food and nutrition security, and social connection. Considering this, the present study aims to investigate the Mising ethnic tribal group, Assam state in Northeast India, with a focus to investigate mode of starter culture making for preparing local beverage along with plant species being used, time of collection, procedure of making starter culture, and how it is preserved. The community maintains an agrarian culture and is closely associated with forest-based natural resources. This group of people consumes various foods prepared from wild plants and crops along with meat and fish. Apong is one of the most important components of their culture, used as a beverage made up of rice by using starter culture locally known as ÉPOP. Detailed process of making of starter culture was documented. A total of 31 plant species varying from 22 families belonging to both flowering and non-flowering plants were recorded used by the community. Eight species also comprised market potential for diverse uses besides being used in starter culture preparation. The source of the collected resources varied from fallow land to forest, with the highest collection concentration in the forest (35.5%), mostly collected in the afternoon (51.61%) to ensure a quality of the plant material. The collected plant materials are mixed with soaked glutinous rice and made into starter cultures of different sizes that have a shelf life of 6 months. The study highlights that use of starter culture is consistent and harmless with distinct flavor and taste that apt to local appetite. Conserving community knowledge on starter culture making, however, requires appropriate policy planning and action line. The investigation not only increases our understanding on local food tradition but also has potential for applications in fermented food industry and thus opens up a new line of research in future.

Alterations in the multilevel structure and depolymerization behavior of gluten induced by selenium in fermented dough

This study was conducted to evaluate the alterations of multilevel structure of gluten induced by selenium (Se) and their relationship with the properties of the fermented dough. The results of molecular weight distributions indicated that the degree of gluten aggregation decreased as the Se content in the fermented dough increased. Moreover, the elevated Se content led to a higher percentage of β-sheet in the dough proteins, indicating a more stable structure after depolymerization. Intermolecular interactions indicated that Se disrupted covalent cross-linking between gluten by affecting disulfide (SS) bonds. The levels of glutathione (GSH), free sulfhydryl and SS bond in the dough increased with increasing Se content. This suggested that more GSH was produced by Se induced yeast, which reacted with intermolecular SS in gluten, resulting in reduced gluten cross-linking. Meanwhile, Se affected the viscoelasticity and enhanced the height of the fermented dough. Improved quality of the steamed bread was found with even pore distributions and reduced hardness. This study has the potential to expand the application of Se in the fermented food industry and offer practical ways to increase Se consumption in Se-deficient areas.

Novel Strain of Paenibacillus phyllosphaerae CS-148 for the Direct Hydrolysis of Raw Starch into Glucose: Isolation and Fermentation Optimization.

The conventional process for converting starch to glucose is energy-intensive. To lower the cost of this process, a novel strain of Paenibacillus phyllosphaerae CS-148 was isolated and identified, which could directly hydrolyze raw starch into glucose and accumulate glucose in the fermentation broth. The effects of different organic and inorganic nitrogen sources, the culture temperature, the initial pH, and the agitation speed on the yield of glucose were optimized through the one-factor-at-a-time method. Nine factors were screened by Plackett-Burman design, and three factors (raw corncob starch, yeast extract and (NH4)2SO4) had significant effects on glucose yield. Three significant factors were further optimized using Box-Behnken design. Under the optimized fermentation conditions (raw corncob starch 40.4 g/L, yeast extract 4.27 g/L, (NH4)2SO4 4.39 g/L, KH2PO4 2 g/L, MgSO4`7H2O 2 g/L, FeSO4`7H2O 0.02 g/L, NaCl 2 g/L, KCl 0.5 g/L, inoculums volume 4%, temperature 35 °C, agitation rate 150 rpm, and initial pH 7.0), the maximum glucose yield reached 17.32 ± 0.46 g/L, which is 1.33-fold compared to that by initial fermentation conditions. The maximum conversion rate and glucose productivity were 0.43 ± 0.01 g glucose/g raw corn starch and 0.22 ± 0.01 g/(L·h), respectively. These results implied that P. phyllosphaerae CS-148 could be used in the food industry or fermentation industry at a low cost.

Reviewing the Source, Physiological Characteristics, and Aroma Production Mechanisms of Aroma-Producing Yeasts.

Flavor is an essential element of food quality. Flavor can be improved by adding flavoring substances or via microbial fermentation to impart aroma. Aroma-producing yeasts are a group of microorganisms that can produce aroma compounds, providing a strong aroma to foods and thus playing a great role in the modern fermentation industry. The physiological characteristics of aroma-producing yeast, including alcohol tolerance, acid tolerance, and salt tolerance, are introduced in this article, beginning with their origins and biological properties. The main mechanism of aroma-producing yeast is then analyzed based on its physiological roles in the fermentation process. Functional enzymes such as proteases, lipases, and glycosidase are released by yeast during the fermentation process. Sugars, fats, and proteins in the environment can be degraded by these enzymes via pathways such as glycolysis, methoxylation, the Ehrlich pathway, and esterification, resulting in the production of various aromatic esters (such as ethyl acetate and ethyl caproate), alcohols (such as phenethyl alcohol), and terpenes (such as monoterpenes, sesquiterpenes, and squalene). Furthermore, yeast cells can serve as cell synthesis factories, wherein specific synthesis pathways can be introduced into cells using synthetic biology techniques to achieve high-throughput production. In addition, the applications of aroma yeast in the food, pharmaceutical, and cosmetic industries are summarized, and the future development trends of aroma yeasts are discussed to provide a theoretical basis for their application in the food fermentation industry.

Expanding the genetic programmability of Lactiplantibacillus plantarum.

Lactobacilli are ubiquitous in nature and symbiotically provide health benefits for countless organisms including humans, animals and plants. They are vital for the fermented food industry and are being extensively explored for healthcare applications. For all these reasons, there is considerable interest in enhancing and controlling their capabilities through the engineering of genetic modules and circuits. One of the most robust and reliable microbial chassis for these synthetic biology applications is the widely used Lactiplantibacillus plantarum species. However, the genetic toolkit needed to advance its applicability remains poorly equipped. This mini-review highlights the genetic parts that have been discovered to achieve food-grade recombinant protein production and speculates on lessons learned from these studies for L. plantarum engineering. Furthermore, strategies to identify, create and optimize genetic parts for real-time regulation of gene expression and enhancement of biosafety are also suggested.

From genotype to phenotype: computational approaches for inferring microbial traits relevant to the food industry.

When selecting microbial strains for the production of fermented foods, various microbial phenotypes need to be taken into account to achieve target product characteristics, such as biosafety, flavor, texture, and health-promoting effects. Through continuous advances in sequencing technologies, microbial whole-genome sequences of increasing quality can now be obtained both cheaper and faster, which increases the relevance of genome-based characterization of microbial phenotypes. Prediction of microbial phenotypes from genome sequences makes it possible to quickly screen large strain collections in silico to identify candidates with desirable traits. Several microbial phenotypes relevant to the production of fermented foods can be predicted using knowledge-based approaches, leveraging our existing understanding of the genetic and molecular mechanisms underlying those phenotypes. In the absence of this knowledge, data-driven approaches can be applied to estimate genotype-phenotype relationships based on large experimental datasets. Here, we review computational methods that implement knowledge- and data-driven approaches for phenotype prediction, as well as methods that combine elements from both approaches. Furthermore, we provide examples of how these methods have been applied in industrial biotechnology, with special focus on the fermented food industry.

청국장 발효 중 surfactin 생산량 품질지표 설정

Surfactin, one of the most powerful biosurfactants, can be widely applied in agriculture, food, and pharmaceutics. The purpose of the present study was to establish suitable indicators for a rapid detection method that can confirm the surfactin productivity of cheonggukjang. In the present study, changes in the total number of bacteria were quantified according to the fermentation time of chenggukjang. Furthermore, physicochemical factors, such as pH, color value, surface tension, refractive index, absorbance, and protein contents, were assessed in chenggukjang viscous substances, and a correlation analysis between the physicochemical factors and surfactin contents was performed. We found that the protein contents gradually increased up to 48 h (4.21±0.11 mg/mL) without a change in the surface tension. Furthermore, the refractive index, absorbance at 280 nm, and color value were significantly increased as the fermentation time increased; however, there were no statistically significant differences after 36 h of fermentation. Interestingly, this result exhibited a tendency similar to the surfactin content according to fermentation time. These findings suggest that surfactin can be used as a suitable quality indicator and may provide an experimental basis for the fermented food industry.