Low-salt Fermentation Research Articles

High efficiency production of 5-hydroxyectoine using metabolically engineered Escherichia coli

The 5-hydroxyectoine is a natural protective agent with long-lasting moisturising and radiation resistance properties. It can be naturally synthesized by some extremophiles using the “bacterial milking” process, but this can corrode bioreactors and downstream purification may cause environmental pollution. In this study, an engineered Escherichia coli (E. coli) strain was constructed for the 5-hydroxyectoine production. First, three ectoine hydroxylases were characterised and the enzyme from Halomonas elongata was the most effective. The L-2,4-diaminobutyrate transaminase mutant was introduced into the engineered strain, which could accumulate 2.8 g/L 5-hydroxyectoine in shake flasks. By activating the glyoxylate cycle and balancing the α-ketoglutarate distribution, the 5-hydroxyectoine titer was further increased to 3.4 g/L. Finally, the optimized strain synthesized 58 g/L 5-hydroxyectoine via a semi-continuous feeding process in a NaCl-free medium. Overall, this study reported the highest titer of 5-hydroxyectoine synthesized by E. coli and established a low-salt fermentation process through the aforementioned efforts.

An effective means to improve the flavor quality of traditional fermented sour meat: The salt reduction strategy

The flavor formation of fermented meat products is significantly influenced by salt. In this study, the flavor analysis methods of electronic tongue and nose, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry were utilized to explore the effect of salt content on the formation of sour meat flavor. The results demonstrated a significant decrease in pH value with the reduction of salt content (P < 0.05), while there was a significant rise in thiobarbituric acid reactive substances (TBARS) value associated with the decline in salt content (P < 0.05). Alcohols, aldehydes, and esters such as 1-octen-3-ol, 3-methyl-1-butanol, hexanal, heptanal, ethyl octanoate, ethyl heptanoate, etc. were considered to have significant contributions to the overall flavor of sour meat. The rapid accumulation of these aroma compounds under low-salt conditions was regarded as one of the key factors in the overall flavor enhancement of sour meat products. Additionally, the low-salt fermentation also promoted the accumulation of free amino acids and free fatty acids in sour meat products, thereby facilitating the formation of more complex aroma compounds. This study offers new technological ways to improve the conventional method of fermenting sour meat and offers theoretical justification for studies on salt reduction in conventionally fermented meat products.

Dynamic analysis of microbial community, flavor components of low-salt fermented red pepper sauce and exploring the key flavours formation

Fermented red pepper (FRP) sauce serves as a representative condiment, and the traditional fermentation of FRP requires high salt content, which is detrimental to human health, thus developing low-salt FRP is a trend. However, the effects of low-salt fermentation on quality and flavor substances in FRP sauce are poorly understood. The aim of this study is to reveal the dynamic changes of microbial community and flavor compounds in low-salt FRP sauce, analyze the interactions between microorganisms and key flavor compounds, and characterize the metabolic pathways of the main flavor components. The results showed that Levilactobacillus, Companilactobacillus and Candida were the core dominant genera in low-salt FRP sauce based on the analysis of the data of amplicon sequence variants. Further, the amplicon data were subjected to spearman correlation analysis found that Levilactobacillus was negatively correlated with other genera, while Companilactobacillus was proved to be remarkably positively correlated with other microorganisms at the strain level. Additionally, 18 volatile compounds and 40 non-volatile compounds were identified as the key flavor substances by HPLC, HS-SPME-GC-MS and UHPLC-QE-MS. Finally, combined metabolomics and metagenomics data revealed that the metabolism of various amino acids was the main pathway for flavor formation, and Companilactobacillus and Candida were significantly positively correlated with flavor formation, which were therefore identified as the core genera for flavor production. These findings deepened our understanding of the effects of low-salt fermentation on microbial communities and flavor metabolites in FRP sauce, which will accelerate the development of reduced-salt fermented foods in the future.

Amylase, protease, and lipase-producing microbes of local origin as potential starter cultures for low-salt moromi fermentation

High salt concentration (18%-22% NaCl) is essential for halophilic microbes to grow and develop aroma during soy sauce fermentation. Due to health concerns, recent efforts have focused on reducing salt during fermentation by introducing novel yeast species. However, the natural fermentation of soy sauce involves a more complex microbial community essential for achieving optimal sensory qualities. This study aimed to select potential bacterial and fungal species from traditional soy sauce factories in Indonesia for use in low-salt fermentation. Isolates that showed positive enzymatic activities at 20%, as well as 5% NaCl, were subjected to growth profile characterisation in tryptic soy broth (TSB) with different salt concentrations (0%, 5%, and 20%) and pH (4.0, 5.0, and 6.0). A total of 47 bacterial and 39 fungal isolates were obtained. Fourteen bacterial and 12 fungal isolates demonstrated positive enzymatic activities at 20% and 5% NaCl. The majority of bacterial strains were identified as Bacillus subtilis, while most of the fungal isolates belonged to the Aspergillus genus (Aspergillus oryzae and Aspergillus tamarii). The majority of Bacillus isolates performed rapid growth in 5% NaCl and relatively low pH (5.0), indicating their potential application in low-salt soy sauce production.

Novel insight into flavor and quality formation in naturally fermented low-salt fish sauce based on microbial metabolism

Low-salt fermentation is an effective way to shorten the fermentation time of fish sauce. In this study, the changes of microbial community, flavor, and quality during the natural fermentation of low-salt fish sauce were studied, followed by the elucidation of flavor and quality formation mechanisms based on microbial metabolism. The 16S rRNA gene high-throughput sequencing showed that both richness and evenness of microbial community were reduced during fermentation. The microbial genera, including Pseudomonas, Achromobacter, Stenotrophomonas, Rhodococcus, Brucella, and Tetragenococcus were more suitable for the fermentation environment, and obviously increased along with the fermentation. There were a total of 125 volatile substances identified by HS-SPME-GC–MS, of which 30 substances were selected as the characteristic volatile flavor substances, mainly including aldehydes, esters, and alcohols. Large amounts of free amino acids were produced in the low-salt fish sauce, especially umami and sweet amino acids, as well as high concentrations of biogenic amines. Correlation network constructed by the Pearson’s correlation coefficient showed that most characteristic volatile flavor substances were significantly positively correlated with Stenotrophomonas, Achromobacter, Rhodococcus, Tetragenococcus, and Brucella. Stenotrophomonas and Tetragenococcus were significantly positively correlated with most free amino acids, especially umami and sweet amino acids. Pseudomonas and Stenotrophomonas were positively correlated with most biogenic amines, especially histamine, tyramine, putrescine, and cadaverine. Metabolism pathways suggested that the high concentrations of precursor amino acids contributed to the production of biogenic amines. This study indicates that the spoilage microorganisms and biogenic amines in the low-salt fish sauce need to be further controlled, and the strains belonging to Tetragenococcus can be isolated as potential microbial starters for the production of low-salt fish sauce.

Effects of Low-Temperature and Low-Salt Fermentation on the Physicochemical Properties and Volatile Flavor Substances of Chinese Kohlrabi Using Gas Chromatography–Ion Mobility Spectrometry

To explore the effect of low-temperature and low-salt fermentation on the volatile flavor substances of Chinese kohlrabi, low-temperature and low-salt fermented Chinese kohlrabi (LSCK) and traditional high-salt fermented Chinese kohlrabi (HSCK) were produced. The physicochemical and texture properties of the two kinds of Chinese kohlrabies were evaluated. Headspace gas chromatography-ion mobility spectrometry (GC-IMS) and electronic nose (E-nose) were used to analyze the volatile flavor substances of the kohlrabi. The results showed that the total acid content significantly decreased (p &lt; 0.05), while protein and reducing sugar contents significantly increased (p &lt; 0.05) by low-temperature and low-salt fermentation. A total of 114 volatile flavor substances were identified. The alcohol, ketone, pyrazine, ether, and nitrile contents in LSCK were significantly higher than those in HSCK (p &lt; 0.05). Moreover, the unpleasant flavor from the 3-methylbutyric acid formation was effectively depressed in LSCK. The principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA) models established by multivariate statistical analysis significantly distinguished the two types of kohlrabies. Multivariate statistical analysis suggested that 16 volatile flavor substances with VIP &gt;1, including tetrahydrothiophene, ethyl 3-(methylthio)propanoate, 3-methylbutyric acid, hexanenitrile, and 3-methyl-3-buten-1-ol, could be used as potential biomarkers for identifying LSCK and HSCK. The E-nose analysis further demonstrated that there was a significant difference in overall flavor between the LSCK and HSCK. The present study provides support for the development of green processing technology and new low-salt Chinese kohlrabi products.

Moromi mash dysbiosis trigged by salt reduction is relevant to quality and aroma changes of soy sauce

Health concerns related to excessive salt consumption have increased the demand for foods with reduced salt content. However, it is a challenge to perform low salt fermentation of high salt liquid-state (HSL) soy sauce due to the interplay between salt and microorganisms. In this study, ≤12 % (w/v) NaCl led to failed fermentation of HSL soy sauce. At 9 % (w/v) NaCl, amino acid nitrogen decreased to 0.31 g/100 mL, total acid increased to 10.1 g/L, and biogenic amines increased to 904.49 mg/L. With reduced salt, the total number of bacteria (1–2 orders of magnitude) and spoilage bacteria (Bacillus, Kurthia, Staphylococcus saprophyticus, and Lactobacillus pobuzihii) increased, and the total number of functional microorganisms (Weissella, Zygosaccharomyces, and Candida) decreased. Unacceptable volatiles contents were higher in reduced-salt soy sauce than in normal salt soy sauce. Most of the unacceptable volatiles were positively correlated with spoilage bacteria.

Low-salt fermentation improves flavor and quality of sour meat: Microbiology and metabolomics

The effects of low-salt fermentation on quality, volatile compounds, differential metabolites, and the microbial community structure of sour meat were studied. Low-salt fermentation could facilitate the production of volatile compounds, free amino acids, organic acids, and so forth and improve product safety. The results showed that the content of branched-chain amino acids such as valine, leucine, and isoleucine in the low-salt sour meat was respectively 1.50, 1.32, and 1.26 times higher than those in the traditional sour meat. Hexanal, Benzeneacetaldehyde, (E)-2-Heptenal, 1-Octen-3-ol, 1-Hexanol, 1-Butanol, 3-methyl-, and certain ethyl esters were identified as critical volatile flavour compounds for the low-salt sour meat. Lactobacillus, Pediococcus, Staphylococcus, Debaryomyces, Aspergillus, and Mucor were the main genera in the low-salt sour meat. And these microorganisms were closely associated with metabolites that included free amino acids, fatty acids, and nucleotides, which were crucial for the emergence of distinctive sour meat flavors as flavor precursors. In summary, low-salt fermentation can accelerate sour meat maturation, facilitate the oxidation and decomposition of protein and fat, and is more conducive to sour meat fermenting and distinct fermented flavor production.

Fermented shellfish condiments: A comprehensive review.

Fermented shellfish condiments are globally consumed especially among Asian countries. Condiments, commonly used as flavor enhancers, have unique sensory characteristics and are associated with umami and meaty aroma. The main reactions that occur during fermentation of shellfish include proteolysis by endogenous enzymes and microbial activities to produce peptides and amino acids. The actions of proteolytic enzymes and microorganisms (predominantly bacteria) are found to be largely responsible for the formation of taste and aroma compounds. This review elaborates different aspects of shellfish fermentation including classification, process, substrates, microbiota, changes in both physicochemical and biochemical components, alterations in nutritional composition, flavor characteristics and sensory profiles, and biological activities and their undesirable impacts on health. The characteristics of traditional shellfish production such as long duration and high salt concentration not only limit nutritional value but also inhibit the formation of toxic biogenic amines. In addition, this review article also covers novel bioprocesses such as low salt fermentation and use of novel starter cultures and/or novel enzymes to accelerate fermentation and produce shellfish condiments that are of better quality and safer for consumption. Practical Application: The review paper summarized the comprehensive information on shellfish fermentation to provide alternative strategies to produce shellfish comdiments that are of better quality and safer for consumption.

Growth of ɣ-Proteobacteria in Low Salt Cucumber Fermentation Is Prevented by Lactobacilli and the Cover Brine Ingredients.

ABSTRACTThis study investigated the ability of ɣ-proteobacteria, indigenous to fresh cucumber, to grow in the expressed fruit juice (CJM) and fermentation. It was hypothesized that fresh cucumbers can support prolific growth of ɣ-proteobacteria but that the cover brine composition and acid production by the competing lactobacilli in the fermentation of the fruit act as inhibitory agents. The ɣ-proteobacteria proliferated in CJM with an average maximum growth rate (μmax) of 0.3895 ± 0.0929 and doubling time (Td) of 1.885 ± 0.465/h. A significant difference was found between the ɣ-proteobacteria μmax and Td relative to Lactiplantibacillus pentosus LA0445 (0.2319 ± 0.019; 2.89/h) and Levilactobacillus brevis 7.2.43 (0.221 ± 0.015; 3.35/h) but not Lactiplantibacillus plantarum 3.2.8 (0.412 ± 0.119; 1.87/h). While inoculation level insignificantly altered the μmax and Td of the bacteria tested; it impacted the length of lag and stationary phases for the lactobacilli. Unlike the lactobacilli, the ɣ-proteobacteria were inhibited in CJM supplemented with a low salt fermentation cover brine containing calcium chloride, acetic acid and potassium sorbate. The ɣ-proteobacteria, P. agglomerans, was unable to proliferate in cucumber fermentations brined with calcium chloride at a pH of 6.0 ± 0.1 and the population of Enterobacteriaceae was outcompeted by the lactobacilli within 36 h. Together these observations demonstrate that the prolific growth of ɣ-proteobacteria in CJM is not replicated in cucumber fermentation. While the ɣ-proteobacteria growth rate is faster that most lactobacilli in CJM, their growth in cucumber fermentation is prevented by the cover brine and the acid produced by the indigenous lactobacilli. Thus, the lactobacilli indigenous to cucumber and cover brine composition influence the safety and quality of fermented cucumbers.IMPORTANCE While the abundance of specific ɣ-proteobacteria species varies among vegetable type, several harbor Enterobacteriaceae and Pseudomonadaceae that benefit the plant system. It is documented that such bacterial populations decrease in density early in vegetable fermentations. Consequently, it is assumed that they do not contribute to the quality of finished products. This study explored the viability of ɣ-proteobacteria in CJM, used as a model system, CJM supplemented with fermentation cover brine and cucumber fermentation, which are characterized by an extremely acidic endpoint pH (3.23 ± 0.17; n = 391). The data presented demonstrates that fresh cucumbers provide the nutrients needed by ɣ-proteobacteria to proliferate and reduce pH to 4.47 ± 0.12. However, ɣ-proteobacteria are unable to proliferate in cucumber fermentation. Control of ɣ-proteobacteria in fermentations depends on the cover brine constituents and the indigenous competing lactobacilli. This knowledge is of importance when developing guidelines for the safe fermentation of vegetables, particularly with low salt.

Potential improvement in the safety and quality of traditional fermented soybean products: A narrative review

Soybeans are one of the most significant crops in the world due to their nutritionally valuable. It has been converted to produce a variety of fermented soybean products. During conventional soybean fermentation, microorganisms are involved, biochemical reactions occur, and bioactive components are produced. With the rise of people's living standards, customers are paying more attention not only to the flavor and nutrition of fermented soybean meals, but also to their safety and quality. Mycotoxins, biogenic amine production, and high salt content are among the public health concerns associated with fermented soybean foods. This paper reviews the prevalent concerns about the safety and quality of fermented soybean foods, as well as potential improvements. Attempts and methods have been proposed to ensure the safety of the fermentation process and food quality. Official regulations, the employment of suitable microbes, the use of high-quality cultivars, and the administration of chemicals are all viable options for improving safety and quality. We conclude that implementing international food standards, guidelines, and codes of practice such as the Codex Alimentarius for fermented soybean products and the application of scientific novel methods (e.g., starter combination, high-pressure processing, or low-dose gamma irradiation, additive usage, low salt fermentation technique) are the potential solutions to mitigate the issues and improve the safety and quality of the products.

The Bacterial and Fungi Microbiota of Soy Sauce-Supplied Lactic Acid Bacteria Treated with High-Pressure Process

Background: Soy sauce is applied as a seasoning in daily life in East Asia. High amounts of salt in soy sauce can inhibit microbial growth in its production and preservation. However, the elevated salt content in food might increase the health risk. Low salt fermentation is rarely used in soil sauce production because of the potential harmful lactic acid bacteria growth. Therefore, dilution after high salt fermentation is commonly used to reduce the salt concentration. Methods: This study aims to treat the low salt fermented soy sauce with a high hydrostatic pressure process (HPP) to eliminate the harmful bacteria and to analyze the microbial community change compared with that in traditional high salt fermentation. Results: The results showed that the bacterial diversity was increased in low-salt and high-pressure (LS-HP)-treated soy sauce, though the bacterial abundance was decreased. Relative abundance in high-salt (HS), low-salt (LS), and LS-HP-treated soy sauce showed specific bacterial strains in the LS-HP group. Similarly, the fungal diversity was also increased in LS-HP-fermented soy sauce and the detected OTUs were increased. The fungi sensitive to salinity and pressure were indicated in our results. Conclusions: The present study suggests the enhanced bacterial and fungal diversity and different microbial community in HS, LS, and LS-HP-treated soy sauce, as well as the availability of LS and HPP treatment on soy sauce production.

Formation of γ-aminobutyric acid (GABA) during the natural lactic acid fermentation of cucumber

This study investigated γ-aminobutyric acid (GABA) formation during natural lactic acid fermentation of cucumbers. Two lots of cucumbers were fermented or acidified in duplicate in 4 % sodium chloride (NaCl) brine and analyzed for GABA content over 6 months storage. GABA was present in fresh and acidified pickling cucumbers at 0.83 ± 0.16 mM and 0.56 ± 0.30 mM, respectively. Additional GABA was generated in fermented cucumbers (1.21 ± 0.35 mM) and remained stable over time and during further processing. Brine salt content (2 % - 6 %) affected GABA formation with greatest GABA formation in cucumber brined in 2 % NaCl (1.38 ± 0.31 mM). Commercially available, fermented cucumber pickles packed in their original fermentation brines contained more GABA (1.32 ± 0.25 mM) than acidified (0.46 ± 0.26 mM) or fermented, desalted and re-packed products (0.49 ± 0.32 mM). This work demonstrated that low-salt fermentation enhances GABA content in fermented cucumber products prepared for direct consumption.

Effect of initial 5 days fermentation under low salt condition on the quality of soy sauce

A two-stage moromi fermentation method was employed to the soy sauce making in order to shorten the overall fermentation time. At the first stage, the koji was mixed with 10% brine solution and incubated at 40 °C for 5 days. The second fermentation stage was conducted under three different treatments, including reduced temperature from 40 °C to 30 °C, outdoor without temperature control, and indoor ambience temperature up to 3 months with the brine content increased from 10% to 18%. The increases of total nitrogen (TN) and amino nitrogen (AN) contents were mainly occurred at the first stage while maintain constant during the second stage. Both TN and AN values satisfied the requirement of first-grade soy sauce of Taiwan. Unlike TN and AN, the reducing sugar and total acid contents still increased with fermentation time during the second stages. Since most of the changes of these biochemical properties occurred within 5 days’ moromi fermentation, the use of high temperature and low salt fermentation condition in the first stage was very important to rapid fermentation process of soy sauce.

Improved fermentative production of the compatible solute ectoine by Corynebacterium glutamicum from glucose and alternative carbon sources

The cyclic amino acid ectoine is a compatible solute serving as a protective substance against osmotic stress. Ectoine finds various applications due to its moisturizing effect. To avoid the disadvantages of the prevailing so-called “bacterial milking ectoine production process” caused by the high salt concentration, low salt fermentation strategies are sought after. As l-lysine and ectoine biosynthesis share l-aspartate-semialdehyde as common precursor, l-lysine producing strains can be converted to ectoine producing strains. Corynebacterium glutamicum, which is used for l-lysine production in the million-ton-scale, was engineered for ectoine production by heterologous expression of the ectoine biosynthesis operon ectABC from Chromohalobacter salexigens. Derepression of glucose metabolism by deletion of the regulatory gene sugR and avoiding l-lactate formation by deletion of the lactate dehydrogenase gene ldhA increased ectoine productivity. In bioreactor fed-batch cultivations an ectoine titer of 22gL−1 and a volumetric productivity of 0.32gL−1h−1 were obtained. The ectoine yield of 0.16gg−1, to the best of our knowledge, exceeded previously reported yields. Moreover, ectoine production from the alternative carbon sources glycerol, glucosamine, xylose, arabinose, and soluble starch was achieved.

Biochemical, Nutritional, and Sensory Quality of the Low Salt Fermented Shrimp Paste

ABSTRACTChanges in the biochemical, nutritional, and sensory properties of the low salt fermented shrimp paste during fermentation were studied. The total volatile base nitrogen (TVB-N) content and total viable counts significantly increased. The pH value significantly decreased, while thiobarbituric acid (TBA) content significantly increased over 14 days. The lightness (L*) value significantly decreased during the initial 7 days of fermentation and significantly increased thereafter. The redness (a*) and yellowness (b*) values and astaxanthin content of the low salt fermented shrimp paste gradually increased during the 30 days of fermentation. C16:0, C18:0, C18:1n9t, C20:1n7c, C20:5n3, and C22:6n3 were the predominant fatty acids, while aspartate, glutamate, alanine, leucine, lysine, arginine, and proline were the predominant amino acids in the shrimp paste. N-containing compounds, such as pyrazine, comprised the majority of the volatile compounds. The results obtained from quantitative descriptive analysis showed that low salt fermentation can improve the flavor of the shrimp paste and is, therefore, a promising method to industrially prepare the shrimp paste.

Effects of indigenous yeasts on physicochemical and microbial properties of Korean soy sauce prepared by low-salt fermentation

This study deals with understanding the effects of salt reduction on both the physicochemical and microbiological properties of soy sauce fermentation and also the application of indigenous yeast starters to compensate for undesirable changes occurring in salt-reduced processes. Fermentation was tested in situ at a Korean commercial soy sauce processing unit. Salt reduction resulted in higher acidity as well as lower pH and contents of residual sugar and ethanol. Moreover, undesired flavor characteristics, due to a lack of distinctive compounds, was observed. In addition, putrefactive Staphylococcus and Enterococcus spp. were present only during salt-reduced fermentation. To control these adverse effects, a single or mixed culture of two indigenous yeasts, Torulaspora delbrueckii and Pichia guilliermondii, producing high ethanol and 3-methyl-1-butanol, respectively, were tested. Overall, all types of yeast applications inhibited undesirable bacterial growth despite salt reduction. Of the starter cultures tested, the mixed culture resulted in a balance of more complex and richer flavors with an identical flavor profile pattern to that obtained from high salt soy sauce. Hence, this strategy using functional yeast cultures offers a technological option to manufacture salt-reduced soy sauce while preserving its typical sensory characteristics without affecting safety.

저염 오징어 젓갈의 숙성 중 오징어 먹즙이 물성에 미치는 영향

저염 오징어 젓갈의 숙성 중 오징어 먹즙이 물성에 미치는 영향