Functional roles and engineering strategies to improve the industrial functionalities of lactic acid bacteria during food fermentation.

In order to improve the flavor profiles, food security, probiotic effects and shorten the fermentation period of traditional fermented foods, lactic acid bacteria (LAB) were often considered as the ideal candidate to participate in the fermentation process. In general, LAB strains possessed the ability to develop flavor compounds via carbohydrate metabolism, protein hydrolysis and amino acid metabolism, lipid hydrolysis and fatty acid metabolism. Based on the functional properties to inhibit spoilage microbes, foodborne pathogens and fungi, those species could improve the safety properties and prolong the shelf life of fermented products. Meanwhile, influence of LAB on texture and functionality of fermented food were also involved in this review. As for the adverse effect carried by environmental challenges during fermentation process, engineering strategies based on exogenous addition, cross protection, and metabolic engineering to improve the robustness and of LAB were also discussed in this review. Besides, this review also summarized the potential strategies including microbial co-culture and metabolic engineering for improvement of fermentation performance in LAB strains. The authors hope this review could contribute to provide an understanding and insight into improving the industrial functionalities of LAB.

Transcriptomic-metabolomic analysis reveals the effect of copper toxicity on fermentation properties in Saccharomyces cerevisiae.

Copper is one of the unavoidable heavy metals in wine production. In this study, the effects on fermentation performance and physiological metabolism of Saccharomyces cerevisiae under copper stress were investigated. EC1118 was the most copper-resistant among the six strains. The ethanol accumulation of EC1118 was 26.16-20 mg/L Cu2+, which was 1.90-3.15 times higher than that of other strains. The fermentation rate was significantly reduced by copper, and the inhibition was relieved after 4-10 days of adjustment. Metabolomic-transcriptomic analysis revealed that amino acid and nucleotide had the highest number of downregulated and upregulated differentially expressed metabolites, respectively. The metabolism of fructose and mannose was quickly affected, which then triggered the metabolism of galactose in copper stress. Pathways such as oxidative and organic acid metabolic processes were significantly affected in the early time, resulting in a significant decrease in the amount of carboxylic acids. The pathways related to protein synthesis and metabolism under copper stress, such as translation and peptide biosynthetic process, was also significantly affected. In conclusion, this study analyzed the metabolite-gene interaction network and molecular response during the alcohol fermentation of S. cerevisiae under copper stress, providing theoretical basis for addressing the influence of copper stress in wine production.

The gain effect of microbial consortia induced by adaptive domestication for efficient conversion of Chinese cabbage waste by anaerobic fermentation.

The resource-based treatment of Chinese cabbage waste by anaerobic fermentation can effectively mitigate air, soil, and groundwater pollution. However, the compatibility between fermentative microorganisms and the environment might be a crucial limiting factor for the resource recycling of Chinese cabbage waste. Therefore, the gain effect of microbial consortia (JMRS, JMRST, JMRSZ, JCCW, JCCWT and JCCWZ) induced by adaptive domestication for efficient conversion of Chinese cabbage waste by anaerobic fermentation were explored in this study. A total of 42 single subsamples with same weights were randomly divided into seven treatments: sterile deionized water (Control); anaerobic fermentation inoculated with JMRS (MRS); anaerobic fermentation inoculated with JMRST (MRST); anaerobic fermentation inoculated with JMRSZ (MRSZ); anaerobic fermentation inoculated with JCCW (CCW); anaerobic fermentation inoculated with JCCWT (CCWT); anaerobic fermentation inoculated with JCCWZ (CCWZ) and samples were taken on days 30 and 60 after anaerobic fermentation. The results exhibited that all the treatments contributed to high levels of lactic acid (178.77-201.79 g/kg dry matter) and low levels of ammonia-N (12.99-21.03 g/kg total nitrogen). Meanwhile, MRSZ enhanced (p < 0.05) acetic acid levels (1.53 g/kg dry matter) and resulted in the lowest yeast counts. Microbiologically, the addition of microbial consortia decreased the linear discriminant analysis (LDA) scores of Massilia and Stenotrophomonas maltophilia. Moreover, MRSZ enriched (p < 0.05) Lactobacillus hilgardii, and decreased (p < 0.05) the abundance of bacteria containing mobile elements and potentially pathogenic bacteria. In conclusion, JMRSZ improved the efficient conversion of Chinese cabbage waste for resource utilization.

Breeding of Saccharomyces cerevisiae with a High-Throughput Screening Strategy for Improvement of S-Adenosyl-L-Methionine Production.

S-adenosyl-l-methionine (SAM), a vital physiologically active substance in living organisms, is produced by fermentation over Saccharomyces cerevisiae. The main limitation in SAM production was the low biosynthesis ability of SAM in S. cerevisiae. The aim of this work is to breed an SAM-overproducing mutant through UV mutagenesis coupled with high-throughput selection. Firstly, a high-throughput screening method by rapid identification of positive colonies was conducted. White colonies on YND medium were selected as positive strains. Then, nystatin/sinefungin was chosen as a resistant agent in directed mutagenesis. After several cycles of mutagenesis, a stable mutant 616-19-5 was successfully obtained and exhibited higher SAM production (0.41 g/L vs 1.39 g/L). Furthermore, the transcript levels of the genes SAM2, ADO1, and CHO2 involved in SAM biosynthesis increased, while ergosterol biosynthesis genes in mutant 616-19-5 significantly decreased. Finally, building on the above work, S. cerevisiae 616-19-5 could produce 10.92 ± 0.2 g/L SAM in a 5-L fermenter after 96 h of fermentation, showing a 2.02-fold increase in the product yield compared with the parent strain. Paving the way of breeding SAM-overproducing strain has improved the good basis for SAM industrial production.

Anaerobic fermentation featuring wheat bran and rice bran realizes the clean transformation of Chinese cabbage waste into livestock feed.

Rapid aerobic decomposition and a high cost/benefit ratio restrain the transformation of Chinese cabbage waste into livestock feed. Herein, anaerobically co-fermenting Chinese cabbage waste with wheat bran and rice bran at different dry matter levels (250, 300, 350 g/kg fresh weight) was employed to achieve the effective and feasible clean transformation of Chinese cabbage waste, and the related microbiological mechanisms were revealed by high-throughput sequencing technology. The bran treatments caused an increase in pH value (4.75-77.25%) and free amino acid content (12.09-152.66%), but a reduction in lactic acid concentration (54.58-77.25%) and coliform bacteria counts (15.91-20.27%). In addition, the wheat bran treatment improved the levels of short-chain fatty acids, nonprotein nitrogen, water-soluble carbohydrates and antioxidant activity and reduced the ammonia nitrogen contents. In contrast, the rice bran treatment decreased the levels of acetic acid, water-soluble carbohydrates, nonprotein nitrogen, ammonia nitrogen, and antioxidant activities. Microbiologically, the bran treatments stimulated Pediococcus, Lactobacillus, Enterobacter, and Weissella but inhibited Lactococcus and Leuconostoc, which were the primary organic acid producers reflected by the redundancy analysis. In addition, Chinese cabbage waste fermented with wheat bran at 350 g/kg fresh weight or with rice bran at 300 g/kg fresh weight increased the scale and complexity of bacteriome, promoted commensalism or mutualism and upregulated the global metabolism pathways, including carbohydrate and amino acid metabolisms. Furthermore, the bran treatments resulted in an increase in bacterial communities that were facultatively anaerobic, biofilm-formed, Gram-negative, potentially pathogenic and stress-tolerant. Collectively, the bran treatments inhibited effluent formation and protein degradation and improved nutrient preservation but reduced organic acid production during the anaerobic fermentation, which is linked to the variations in the bacteriome, indicating that the constructed fermentation system should be further optimized.

Current advances for omics-guided process optimization of microbial manufacturing.

Currently, microbial manufacturing is widely used in various fields, such as food, medicine and energy, for its advantages of greenness and sustainable development. Process optimization is the committed step enabling the commercialization of microbial manufacturing products. However, the present optimization processes mainly rely on experience or trial-and-error method ignoring the intrinsic connection between cellular physiological requirement and production performance, so in many cases the productivity of microbial manufacturing could not been fully exploited at economically feasible cost. Recently, the rapid development of omics technologies facilitates the comprehensive analysis of microbial metabolism and fermentation performance from multi-levels of molecules, cells and microenvironment. The use of omics technologies makes the process optimization more explicit, boosting microbial manufacturing performance and bringing significant economic benefits and social value. In this paper, the traditional and omics technologies-guided process optimization of microbial manufacturing are systematically reviewed, and the future trend of process optimization is prospected.

New Saccharomyces cerevisiae-Kluyveromyces marxianus fusant shows enhanced alcoholic fermentation performance.

Kluyveromyces marxianus has a promising enological potential because of strong extracellular pectinase activity and the copious production of specific higher alcohols and esters. However, K. marxianus is unable to complete alcoholic fermentation on its own. For this reason it is only used in co- or sequential fermentation in conjunction with Saccharomyces spp. Here we applied the protoplast fusion technique to two commercial Saccharomyces cerevisiae strains (Lalvin EC1118® and Lalvin QA23®) and K. marxianus isolate IWBT Y885, to obtain fusants that possess pre-selected, enologically relevant features of K. marxianus, but with improved fermentation performance. After an extensive selection process, performed using non-auxotrophic markers, we identified one fusant, BF2020, that shows enhanced fermentation performance compared to the parental strain K. marxianus Y885, but that still exhibits strong pectinase activity and may be suitable for industrial production. The genome of the selected hybrid was further investigated and characterized by RAPD-PCR analysis and whole genome sequencing.

Recent advances in microbial ε-poly-L-lysine fermentation and its diverse applications.

The naturally occurring homo-polyamide biopolymer, ε-poly-L-lysine (ε-PL) consists of 25-35 L-lysine residues with amide linkages between α-carboxyl groups and ε-amino groups. ɛ-PL exhibits several useful properties because of its unusual structure, such as biodegradability, water solubility, no human toxicity, and broad-spectrum antibacterial activities; it is widely applied in the fields of food, medicine, clinical chemistry and electronics. However, current industrial production of ε-PL is only performed in a few countries. Based on an analysis of the physiological characteristics of ε-PL fermentation, current advances that enhance ε-PL fermentation, from strain improvement to product isolation are systematically reviewed, focusing on: (1) elucidating the metabolic pathway and regulatory mechanism of ε-PL synthesis; (2) enhancing biosynthetic performance through mutagenesis, fermentation optimization and metabolic engineering; and (3) understanding and improving the biological activity and functional properties of ε-PL. Finally, perspectives on engineering and exploiting ε-PL as a source material for the production of various advanced materials are also discussed, providing scientific guidelines for researchers to further improve the ε-PL fermentation process.

Isolation of Bacillus spp. with High Fibrinolytic Activity and Performance Evaluation in Fermented Douchi.

ABSTRACT: Fibrinolytic enzymes are effective and highly safe in treating cardiovascular and cerebrovascular diseases. Therefore, screening fibrinolytic enzyme-producing microbial strains with excellent fermentation performance is of great value to industrial applications. The fibrin plate method was used in screening strains with high yields of fibrinolytic enzymes from different fermented food products, and the screened strains were preliminarily identified using molecular biology. Then, the strains were used for solid-state fermentation of soybeans. Moreover, the fermentation product douchi was subjected to fibrinolytic activity measurement, sensory evaluation, and biogenic amine content determination. The fermentation performance of each strain was comprehensively evaluated through principal component analysis. Finally, the target strain was identified based on strain morphology, physiological and biochemical characteristics, 16S rDNA sequence, and phylogenetic analysis results. A total of 15 Bacillus species with high fibrinolysin activity were selected. Their fibrinolytic enzyme-producing activity levels were higher than 5,500 IU/g. Through molecular biology analysis, we found 4 strains of Bacillus subtilis, 10 strains of Bacillus amyloliquefaciens, and 1 strain of Bacillus velezensis. The principal component analysis results showed that SN-14 had the best fermentation performance and reduced the accumulation of histamine and total amine, the fibrinolytic activity of fermented douchi reached 5,920.5 ± 107.7 IU/g, and the sensory score was 4.6 ± 0.3 (out of 5 points). Finally, the combined results of physiological and biochemical analyses showed SN-14 was Bacillus velezensis. The high-yield fibrinolytic and excellent fermentation performance strain Bacillus velezensis SN-14 has potential industrial application.

Wheat gluten hydrolysates promotes fermentation performance of brewer's yeast in very high gravity worts.

The effects of wheat gluten hydrolysates (WGH) and their ethanol elution fractions obtained on XAD-16 resin on physiological activity and fermentation performance of brewer's yeast during very-high-gravity (VHG) worts fermentation were investigated. The results showed that the addition of WGH and their elution fractions in VHG worts significantly enhanced yeast biomass and viability, and further increased the fermentability, ethanol yield and productivity of yeast. Supplementation with 40% ethanol fraction exhibited the highest biomass (6.9 g/L dry cell), cell viability, fermentability (82.05%), ethanol titer (12.19%, v/v) and ethanol productivity during VHG worts fermentation. In addition, 40% ethanol fraction supplementation also caused the most consumption of amino acid and the highest accumulation of intracellular glycerol and trehalose, 15.39% of increase in cell-membrane integrity, 39.61% of enhancement in mitochondrial membrane potential (MMP), and 18.94% of reduction in intracellular reactive oxygen species (ROS) level in yeast under VHG conditions. Therefore, WGH supplementation was an efficient method to improve fermentation performance of brewer's yeast during VHG worts.

Two-Stage Semi-Continuous 2-Keto-Gluconic Acid (2KGA) Production by Pseudomonas plecoglossicida JUIM01 From Rice Starch Hydrolyzate.

A two-stage semi-continuous strategy for producing 2-keto-gluconic acid (2KGA) by Pseudomonas plecoglossicida JUIM01 from rice starch hydrolyzate (RSH) has been developed. The initial glucose concentration (140 g/L) was selected for first-stage fermentation due to its highest 2KGA productivity of 7.58 g/(L⋅h), cell weight of 3.91 g/L, and residual glucose concentration of 25.00 g/L. Followed by removing 70.0% (v/v) of the first-stage broth and feeding 400.0 g/L of glucose to the second-stage fermentor, a total of 50680.0 g glucose was consumed, and 50005.20 g 2KGA was obtained with a yield of 0.9867 g/g by P. plecoglossicida JUIM01 after a 3-cycle two-stage semi-continuous fermentation. Our results indicated that the developed two-stage semi-continuous fermentation could be industrially applied due to its high 2KGA concentration, 2KGA yield and operation efficiency.

A multiple-step strategy for screening Saccharomyces cerevisiae strains with improved acid tolerance and aroma profiles.

Acid tolerance and aroma profile are crucial factors for wine production in Saccharomyces cerevisiae. However, most wine yeasts to date fail to endure low-pH environments, therefore resulting in problems such as lengthened fermentation and poor flavor during acidic fruit wine production. In the present study, we established a multiple-step screening strategy, which was composed of atmospheric and room temperature plasma (ARTP), high-throughput screening (HTS), and laboratory adaptive evolution (ALE), to screen yeast strains for potential wine-producing with enhanced performances during low pH conditions. Importantly, we obtained the S. cerevisiae strain from the mutant library, ET008-c54, which displayed exhibited excellent performances in survival rate, fermentation time, aroma profile, and genetic stability. More specifically, the survival rate of ET008-c54 at low pH was increased by 10-fold, the fermentation time of greengage plum wine was shortened by about 70%, and the content of main aroma compounds was significantly increased by 52%. Collectively, we demonstrate the practical application of the screening platform designed for discovering mutant strains in winemaking technology.

Application of Protein Hydrolysates from Defatted Walnut Meal in High-Gravity Brewing to Improve Fermentation Performance of Lager Yeast.

Protein hydrolysates were prepared from an industrially defatted walnut meal (DWMPH) by enzymolysis employing Neutrase, Protamex, and Flavorzyme, respectively, with/without ultrasonic treatment. The effects of DWMPH supplementations on fermentation performance of lager yeast in high-gravity brewing were investigated. Results showed that ultrasonic-assisted enzymolysis simultaneous treatment (UAE) and ultrasonic pretreatment followed by enzymolysis (UPE) significantly increased degree of hydrolysis (DH) by 1.43 times and 0.71 times of traditional enzymolysis (TE) at least, respectively, Protamex treatment exhibited higher DH (13.3-32.8%) than Neutrase (9.2-25.3%) or Flavorzyme (11.8-28.7%). Compared with control, DWMPH supplementations prepared by UAE using Protamex (UAE-P), Neutrase (UAE-N), or Flavorzyme (UAE-F) significantly improved fermentation performance of lager yeast, especially for UAE-P with the highest major fractions of Mw < 1 kDa, increased wort fermentability and ethanol production by 15% and 17%, respectively, while UAE-F with the highest major fractions of Mw > 3 kDa obviously improved the foam stability of final beers. Furthermore, DWMPH supplementations significantly increased yeast growth and cell viability, promoted glycogen and trehalose accumulation, upregulated stress markers HSP12 and SSA3 expression in yeast cells, improved the formation of higher alcohols and esters, and increased the ratio of higher alcohol to ester indicating a better balanced taste of final beers.

Amino Acid Supplementations Enhance the Stress Resistance and Fermentation Performance of Lager Yeast During High Gravity Fermentation.

The effects of different wort gravity or ethanol concentration in initial wort on the fermentation performance of lager yeast and assimilation of free amino acids (FAAs) were studied. Results showed that compared with high wort gravity (24°P), high ethanol concentration (10%, v/v) decreased yeast growth, cell viability, and wort fermentability significantly. The assimilation of FAAs was changed dramatically by high ethanol toxicity, and positive correlations between the assimilation amounts of 10 FAAs (Asp, Ser, Gly, Arg, Tyr, Val, Met, Lys, Ile, and Leu) and fermentation performance (cell viability, fermentability, and ethanol production) were identified, especially for Arg and Lys exhibiting extremely significant positive correlations. Furthermore, confirmatory testing was carried out by supplementing 24°P worts with 10 FAAs of 0.5, 1, and 2 times of their standard concentrations, respectively. Results exhibited that 10 FAA supplementations improved physiological characteristics and fermentation performance of lager yeast significantly, especially for 1 times FAA supplementation increasing wort fermentability and ethanol yield by 6 and 17%, respectively, and upregulated the expression level of HSP12 and increased more intracellular trehalose accumulation in yeast cells, indicating that stronger protective function was stimulated in yeast cells. Therefore, it was suggested that these 10 FAAs could regulate yeast cells to adapt to high gravity environmental stresses.

Improvement of Multiple-Stress Tolerance and Ethanol Production in Yeast during Very-High-Gravity Fermentation by Supplementation of Wheat-Gluten Hydrolysates and Their Ultrafiltration Fractions.

The effects of wheat-gluten hydrolysates (WGH) and their ultrafiltration fractions on multiple-stress tolerance and ethanol production in yeast during very-high-gravity (VHG) fermentation were examined. The results showed that WGH and WHG-ultrafiltration-fraction supplementations could significantly enhance the growth and viability of yeast and further improve the tolerance of yeast to osmotic stress and ethanol stress. The addition of MW < 1 kDa fractions led to 51.08 and 21.70% enhancements in cell-membrane integrity, 30.74 and 10.43% decreases in intracellular ROS accumulation, and 34.18 and 26.16% increases in mitochondrial membrane potential (ΔΨm) in yeast under osmotic stress and ethanol stress, respectively. Moreover, WGH and WHG-ultrafiltration-fraction supplementations also improved the growth and ethanol production of yeast during VHG fermentation, and supplementation with the <1 kDa fraction resulted in a maximum biomass of 16.47 g/L dry cell and an ethanol content of 18.50% (v/v) after VHG fermentation.

Peptide (Lys-Leu) and amino acids (Lys and Leu) supplementations improve physiological activity and fermentation performance of brewer's yeast during very high-gravity (VHG) wort fermentation.

Lys and Leu were generally considered as the key amino acids for brewer's yeast during beer brewing. In the present study, peptide Lys-Leu and a free amino acid (FAA) mixture of Lys and Leu (Lys + Leu) were supplemented in 24 °P wort to examine their effects on physiological activity and fermentation performance of brewer's yeast during very high-gravity (VHG) wort fermentation. Results showed that although both peptide Lys-Leu and their FAA mixture supplementations could increase the growth and viability, intracellular trehalose and glycerol content, wort fermentability, and ethanol content for brewer's yeast during VHG wort fermentation, and peptide was better than their FAA mixture at promoting growth and fermentation for brewer's yeast when the same dose was kept. Moreover, peptide Lys-Leu supplementation significantly increased the assimilation of Asp, but decreased the assimilation of Gly, Ala, Val, (Cys)2, Ile, Leu, Tyr, Phe, Lys, Arg, and Pro. However, the FAA mixture supplementation only promoted the assimilation of Lys and Leu, while reduced the absorption of total amino acids to a greater extent. Thus, the peptide Lys-Leu was more effective than their FAA mixture on the improvement of physiological activity, fermentation performance, and nitrogen metabolism of brewer's yeast during VHG wort fermentation.

Comparative study on fermentation performance in the genome shuffled Candida versatilis and wild-type salt tolerant yeast strain.

BACKGROUND: The fermentation performance of a genome-shuffled strain of Candida versatilis S3-5, isolated for improved tolerance to salt, and wild-type (WT) strain were analysed. The fermentation parameters, such as growth, reducing sugar, ethanol, organic acids and volatile compounds, were detected during soy sauce fermentation process. RESULTS: The results showed that ethanol produced by the genome shuffled strain S3-5 was increasing at a faster rate and to a greater extent than WT. At the end of the fermentation, malic acid, citric acid and succinic acid formed in tricarboxylic acid cycle after S3-5 treatment elevated by 39.20%, 6.85% and 17.09% compared to WT, respectively. Moreover, flavour compounds such as phenethyl acetate, ethyl vanillate, ethyl acetate, isoamyl acetate, ethyl myristate, ethyl pentadecanoate, ethyl palmitate and phenylacetaldehyde produced by S3-5 were 2.26, 2.12, 2.87, 34.41, 6.32, 13.64, 2.23 and 78.85 times as compared to WT. CONCLUSIONS: S3-5 exhibited enhanced metabolic ability as compared to the wild-type strain, improved conversion of sugars to ethanol, metabolism of organic acid and formation of volatile compounds, especially esters, Moreover, S3-5 might be an ester-flavour type salt-tolerant yeast. © 2016 Society of Chemical Industry.

Fermentation performance of lager yeast in high gravity beer fermentations with different sugar supplementations.

The effects of glucose, sucrose and maltose supplementations on the fermentation performance and stress tolerance of lager yeast (Saccharomyces pastorianus) during high gravity (18°P) and very high gravity (24°P) fermentations were studied. Results showed that throughout 18°P wort fermentation, fermentation performance of lager yeast was significantly improved by glucose or sucrose supplementation, compared with maltose supplementation, especially for sucrose supplementation increasing wort fermentability and ethanol production by 6% and 8%, respectively. However, in the later stage of 24°P wort fermentation, fermentation performance of lager yeast was dramatically improved by maltose supplementation, which increased wort fermentability and ethanol production by 14% and 10%, respectively, compared with sucrose supplementation. Furthermore, higher HSP12 expression level and more intracellular trehalose accumulation in yeast cells were observed by maltose supplementation with increase of the wort gravity from 18°P to 24°P, indicating higher stress response of yeast cells. The excretion of Gly and Ala, and the absorption of Pro in the later stage of fermentation were promoted by maltose supplementation. In addition, with increase of the wort gravity from 18°P to 24°P, higher alcohols level was decreased with maltose supplementation, while esters formation was increased significantly with glucose supplementation. This study suggested that the choice of optimal fermentable sugars maintaining better fermentation performance of lager yeast should be based on not only strain specificity, but also wort gravity.

Effect of copper stress on growth characteristics and fermentation properties of Saccharomyces cerevisiae and the pathway of copper adsorption during wine fermentation.

The effect of copper stress on the fermentation performance of Saccharomyces cerevisiae and its copper adsorption pathway during alcoholic fermentation were investigated in this study. At the limits imposed by the regulations of the European Union and South African (⩽ 20 mg/l), copper had no effect on the cell growth of S. cerevisiae, but its fermentation performance was inhibited to a certain extent. Therefore, the regulated limit should be further reduced (⩽ 12.8 mg/l). Under 9.6-19.2 mg/l copper stress, S. cerevisiae could absorb copper; the copper removal ratio and the unit strain adsorption were 60-81% and 2.72-9.65 mg/g, respectively. S. cerevisiae has a non-biological adsorption of copper, but compared with biological (living yeast) adsorption, the non-biological adsorption was very low. The copper adsorption way of S. cerevisiae was primarily via biological (living yeast) adsorption, which was a two-step process.

The use of chitooligosaccharide in beer brewing for protection against beer-spoilage bacteria and its influence on beer performance.

OBJECTIVES: To identify a biological preservative that can protect beer from microbial contamination, which often results in the production of turbidity and off-flavor. RESULTS: The antimicrobial activity of a chitooligosaccharide against beer-spoilage bacteria and its effect on the fermentation performance of brewer's yeast was studied. Chitooligosaccharide with an average 2 kDa molecular weight was the best at inhibiting all tested beer-spoilage bacteria. The application of chitooligosaccharide in the brewing process did not influence the fermentation of brewer's yeast. The change in beer performance induced by the contamination of Lactobacillus brevis could be effectively controlled by application of chitooligosaccharide in the beer brewing process. CONCLUSION: The experimental data suggested that chitooligosaccharide should be an excellent preservative to inhibit beer-spoilage bacteria in the brewing process and in the end product.