Variation Among Japanese Miso Breweries in Indoor Microbiomes is Mainly Ascribed to Variation in Type of Indoor Surface.

Miso is a microbially-fermented soybean food. The miso brewery indoor microbiome contributes to miso fermentation. Japanese breweries are not climate-controlled, so indoor spaces are strongly affected by the prevailing climate. Because climate influences microorganism distribution, our first hypothesis is that latitude, as a proxy for climate, is a major determinant of brewery indoor microbiome structure. Breweries vary in interior surface materials and in the way operations (steaming, processing, fermenting) are apportioned among rooms. Therefore, our second hypothesis is that more variability in indoor microbiomes exists among breweries than can be ascribed to a latitudinal gradient. Most miso produced today is inoculated with commercial microbial strains to standardize fermentation. If commercial strains outcompete indigenous microbes for membership in the indoor microbiome, this practice may homogenize indoor microbiomes among regions or breweries. Therefore, our third hypothesis is that inoculant fungal species dominate indoor fungal communities and make it impossible to distinguish communities among breweries or across their latitudinal gradient. We tested these hypotheses by sampling indoor surfaces in several breweries across a latitudinal gradient in Japan. We found that latitude had a significant but relatively small impact on indoor fungal and bacterial communities, that the effect of brewery was large relative to latitude, and that inoculant fungi made such small contributions to the indoor microbiome that distinctions among breweries and along the latitudinal gradient remained apparent. Recently, the Japanese Ministry of Agriculture, Forestry and Fisheries specified fungal inoculants to standardize miso production. However, this may not be possible so long as the indoor microbiome remains uncontrolled.

Assay Analysis of Tannase from Lactobacillus plantarum.

Tannin, which is an astringent taste in the mouth, is a polyphenol compound contained in some plants. Tannin causes denaturation of proteins of the tongue or oral mucosa. Tannase, a hydrolase that cleaves carboxylic ester bonds specifically, is used in many industrial fields. Some tannase (tannin acyl hydrolase, EC3.1.1.20) is used widely to prevent or reduce creaming of some foods and beverages. Because some tannins are formed of insoluble salts combined with protein, they reduce creaming such as the white hazing of iced tea. Moreover, they can clarify beverages such as fruit juices during wine and beer production. Tannase is produced by microorganisms under conditions with tannic acid present, mainly from plants. Tannase characteristics differ according to its microorganism of origin. Therefore, it is important to study the microbes used as lactic acid bacteria (LAB), evaluate new methods of tannase assay, and apply them in food or other industries. In this chapter, assay of tannase in LAB is demonstrated using methyl gallate as substrate, with color development by rhodanine and potassium hydroxide solution, using a spectrophotometer. Actual data of high tannase-producing LAB, Lactobacillus plantarum, and enzyme characteristics in optimum conditions are presented in this chapter.

Hydroxylation of Fatty Acids by Lactic Acid Bacteria.

Hydroxy fatty acids (HFAs) are fatty acids with hydroxyl functional groups attached to the main chain. HFAs are used in widely diverse industrial applications, healthy functional foods, artificial food flavorings, and alcoholic beverages. A lactic acid bacterium (LAB), Lactobacillus sakei, hydroxylates oleic acid. Furthermore, the hydroxyl fatty acid was identified by GC-MS as 10-hydroxystearic acid. The Lactobacillus sakei hydroxylated more than 90% of the oleic acid in the medium at 15 °C after 30-48 h. The hydroxyl enzyme needs a coenzyme for an electron donor as NADPH. The enzyme is useful for assay with monitoring NADPH concentration used an A340 device. The hydroxylate fatty acids are converted by LAB lactonize aroma lactone from commercial yeast strains, which can be detected directly by scent. Commercial beer brewing yeast T-58 produced the highest concentration of aroma lactone from hydroxyl fatty acids. Furthermore, the aroma lactone is identified by GC-MS as gamma-dodecalactone. The ratio of conversion is 87%. These results suggest that the lactonization conversion system is useful to hydroxylate fatty acids for alcoholic beverages.

Assaying D-Alanine Racemase in Lactic Acid Bacteria Using NADH Oxidoreduction Enzymic System.

A notable characteristic of amino acids is their optical isomerism, existing as L-form and D-form. Proteins are composed exclusively of L-form amino acids. However, recently, it is reported that D-alanine is evaluated particularly highly in terms of sensory evaluation. D-body amino acids convert L-body amino acid proteolysis from a substrate such as foods during fermentation of lactic acid bacteria. This chapter presents a description of methods used for D-alanine racemase assays in the solution producing by lactic acid bacteria (LAB) using D-amino acid oxidase and lactic acid dehydrogenase via a NADH oxidoreduction system.

Eliminating Lipopolysaccharide (LPS) Using Lactic Acid Bacteria (LAB).

Gram-negative bacteria such as Escherichia coli, among intestinal bacteria, have lipopolysaccharide (LPS), which induces inflammation of human intestines. However, lactic acid bacteria (LAB) can improve human intraintestinal conditions. One reason is that ingestion of LAB prevents bacterial diarrhea. This chapter describes a method of LPS elimination using lactic acid bacteria (LAB). First, the LPS concentration is assayed using an LPS assay kit with the limulus cascade reaction made by limulus amebocyte lysate. Some LABs, four bacillus strains and one coccus strain, have LPS-elimination activity. Particularly, the coccus strain Pediococcus pentosaceus eliminates LPS to 43%. The cells fractionate and eliminate four fractions: the extracellular fraction, cell membrane fraction, cytoplasm fraction, and cell wall fraction. Only the cell wall digesting fraction eliminates LPS to 45%. Results confirm that the LAB eliminates all LPS having O-antigen under a low-sugar medium condition at temperatures of 15-30 °C. This method can be used for assay of LPS elimination by LABs exactly and easily for the probiotics field.

Separation of Lactic Acid Bacteria Using Blue-Native (BN) Polyacrylamide Gel Electrophoresis (PAGE) and Analyses of Lipopolysaccharide-Elimination Protein.

Some gram-negative bacteria such as Escherichia coli and Salmonella spp. among intestinal bacteria might induce inflammation of human intestines. However, lactic acid bacteria (LAB) display anti-inflammatory activity, which improves the intestinal environment. Particularly, the cell surface protein on Pediococcus pentosaceus exhibits high LPS elimination activity, which is expected to provide anti-inflammatory activity in the intestines. This chapter describes that surface proteins are separable using Blue-Native PAGE, which relies upon the theory that protein binds with Coomassie brilliant blue to produce a negative charge for easy separation.

Isolation of Lactic Acid Bacteria Eliminating Trimethylamine (TMA) for Application to Fishery Processing.

Fishy odor of fish flesh (meat) presents a severe problem for marine production. The main cause of fishy odor is trimethylamine (TMA), which increases during storage. It is produced from trimethylamine oxide (TMAO), an osmosis-regulating substance in fish cells that functions by a reduction reaction. Bacterial growth in fish meat increases TMA. Its odor reduces the commercial value of the meat. Technologies for its regulation and elimination are desired. This chapter presents a description of the use of lactic acid to eliminate TMA. The lactic acid is producible safely by bacteria during food processing using picric acid-toluene.A method of eliminating TMA was demonstrated using Lactobacillus plantarum H78. Furthermore, an assay method was explained for reducing TMA in fish meat by fermenting the H78 strain.

Assaying D-Amino Acid in Japanese Sake Using L-Amino Acid Derivatizing Agent.

The D-amino acids of D-alanine, D-glutamic acid, and D-aspartic acid increase tasting evaluation scores of Sake, a Japanese traditional alcohol beverage. Sake is brewed using seed mash for growth of brewing yeast without growth of contaminating microorganisms. Kimoto is brewed using lactic acid bacteria growth to decrease pH. Sake brewed using the Kimoto method also has a rich taste and a higher tasting evaluation score than Sake brewed using the Sokujyo Syubo (Moto) method, which adds lactic acid instead of using lactic acid bacteria growth. D-alanine, D-glutamic acid, and D-aspartic acid in Sake have the function of increasing tasting evaluation scores. They are converted by enzymes in lactic acid bacteria respectively as alanine racemase (EC 5.1.1.1), glutamate racemase (EC 5.1.1.3), and aspartate racemase (EC 5.1.1.13) in Kimoto Mash. Herein, simultaneous assay methods for D-alanine, D-glutamic acid, and D-aspartic acid are explained. Sample solutions adjusted to alkalinity are derivatized by an L-FDLA solution only for L-amino acid. Results demonstrate that, for D-alanine, D-glutamic acid, and D-aspartic acid, this method can assay them easily using no expensive or specialized equipment.

Characteristics of alanine racemase in Lactobacillus sakeiZH-2 strain.

Some d-amino acid functions for food production are widely known: d-alanine improves sensory evaluations of sake, beer, and fermented foods. Therefore, for the application of d-amino acids, alanine racemase (ALRase) in Lactobacillus sakei ZH-2, which has strong racemization, was analyzed using molecular biological methods. It had been hypothesized that ALRase coding DNA, alr, in ZH-2 strain differs from those of other Lactobacillus sakei strains. However, complete genome sequencing by the National Center for Biotechnology (NCBI) revealed the amino acid sequence of alr in ZH-2 strain to have homology of 99.4% similarity with the alr in Lactobacillus sakei 23K strain. However, it is considered that the sequence of alr was a unique amino acid sequence in the lactic acid bacteria group. DNA "alr" of ZH-2 strain has a 1140 bp DNA base with 41 kDa molecular mass. Its molecular mass was inferred as approximately 38.0 kDa using SDS-PAGE. Its optimum conditions are pH 9.0 at 30-40°C, showing stability at pH 9.0-10.0 and 4-40°C. Its cofactor is pyridoxal phosphate. Its activity is activated more by copper and zinc ions than by the lack of a metal ion. Additionally, its K m is 1.32 × 10-3 (mol), with V max of 4.27 × 10-5 (µmol-1 min-1). ALRase reacted against alanine most strongly in other substrates such as amino acids. The enzyme against serine was found to have 40% activity against alanine. The enzyme converted up to 54.5% of d-alanine from l-alanine ZH-2 strain.

Evaluating estrogenic activity of isoflavones in miso using yeast two-hybrid method.

Estrogenic activity in miso was evaluated without in vivo animal experimentation using in vitro method with yeast in a two-hybrid method because of its similarities with human cells. First, the recombinant yeast containing genes of human estrogen receptor (hER) was prepared for modeling human cells. Subsequently, standard solutions of 17ß-estradiol and isoflavone (1.0 × 10-12 - 1.0 × 10-6 ) were assayed using the yeast. Their yeast produces ß-glucosidase according to the concentrations of their solutions. Therefore, the estrogenic activity can be evaluated using recombinant yeast for the yeast two-hybrid method. Results show that 17ß-estradiol has affinity to bind with Y187-αα. Genistein has affinity to bind with Y187-ßß. Daidzein, genistein, and glycitein in miso were 2.0-2.2 times the average concentrations of miso. Particularly, Mame miso had the highest concentration of isoflavones among all miso samples. Isoflavone in miso samples showed estrogenic activity against Y187-ßß. Mame miso had particularly high activity (1.97 U/OD660 1.0) against Y187-ßß modeling hERßß. Finally, the interaction of the human estrogen receptors was analyzed with 17ß-estradiol and isoflavones using Y187 strains. Isoflavone inhibited the estrogenic activity of 17ß-estradiol using Y187-αα. However, the estrogenic activity of 17ß-estradiol against Y187-αß and Y187-ßß, which model hER-αß and hER-ßß, was activated by isoflavone. Results showed genistein as the antagonist of estrogenic activity within 17ß-estradiol against hERαα. However, it is an agonist of the activity within 17ß-estradiol against hERαß and hERßß. The yeast two-hybrid method has some potential for assessing the estrogenic activity of isoflavone in foods as a human model. PRACTICAL APPLICATION: Today, isoflavones in foods must be evaluated using in vivo methods such as animal experimentation because the estrogenic activities of isoflavones are agonist or antagonist with 17ß-estradiol against estrogen receptors. Because animal experimentation is time-consuming and expensive, isoflavones in foods can be evaluated using yeast, a eukaryote that has similarity to human cells, while obviating in vivo methods. The yeast two-hybrid method is useful to assay the estrogenic activity of isoflavones in foods.

Lipopolysaccharide neutralizing protein in Miso, Japanese fermented soybean paste.

Miso, a fermented paste made from soybeans, is used traditionally for seasoning of food. It has been a protein and nitrogen source since ancient times in Japan because of its high nutritional value. Furthermore, it has important health functions such as the estrogen-like activity of isoflavones, anti-oxidation, and angiotensin-converting-enzyme inhibition activity. Moreover, it has activity for neutralization of lipopolysaccharide (LPS) from Escherichia coli. Nevertheless, the mechanisms of that activity remain unclear. For this study, we purified and identified the proteins responsible for LPS-neutralization. After proteins were isolated from a miso extract using Blue native polyacrylamide gel electrophoresis, a protein found at 10 to 30 kDa on the polyacrylamide gel was identified using nano LC-MS/MS as 2S albumin in soybean (Glycine max). The protein had two LPS binding motifs: SKWQHQ (22 amino acid residues) and EKQKKKMEKE (131 amino acid residues). The protein in miso was found to have LPS neutralization activity, as assayed by prostaglandin D2 (PGD2 ) production from macrophage cells. The PGD2 production by macrophage cells was inhibited by LPS-neutralizing protein (LNP) from miso. Particularly, 50 mg/mL of LNP solution and LPS (10 µg/mL) inhibited production of PGD2 from the cells. The data were inferred as significantly different (P < 0.05) from statistical analyses by analysis of variance testing and Tukey tests. The 2S albumin in soybean is LNP, an LPS-neutralizing protein, produced in miso. PRACTICAL APPLICATION: A protein from miso fermented soy paste neutralizes an Escherichia coli intestinal bacterial product, lipopolysaccharide (LPS), which causes intestinal inflammation. Miso and its protein are anticipated for use as a probiotic agent to prevent intestinal inflammation in humans and domestic animals. Miso is useful not only as a seasoning for food, but also as a health-functional food because it is an LPS-neutralizing agent.

Screening the Lactic Acid Bacteria converting Hydroxy Fatty Acid from Unsaturated Fatty Acid.

Hydroxyl fatty acids (HFAs) are used in widely diverse industrial applications, healthy functional foods, artificial food flavorings, and alcoholic beverages. A lactic acid bacterium (LAB), Lactobacillus sakei, hydroxylates oleic acid. Furthermore, the hydroxyl fatty acid was identified by GC-MS as 10-hydroxystearic acid. The Lactobacillus sakei hydroxylated more than 90% of the oleic acid in the medium at 15 °C after 30-48 h. The hydroxyl enzyme needs a coenzyme for an electron donor as NADPH. The enzyme is useful for assay with monitoring NADPH concentration used an A340 device. The hydroxylate fatty acids converted by LAB lactonize aroma lactone from commercial yeast strains, which can be detected directly by scent. Commercial beer brewing yeast T-58 produced the highest concentration of aroma lactone from hydroxyl fatty acids. Furthermore, the aroma lactone is identified by GC-MS as gamma-dodecalactone. The ratio of conversion is 87%. These results suggest that the lactonization conversion system is useful to hydroxylate fatty acids for alcoholic beverages.

Assaying D-Body Amino Acids as D-Alanine and Amino Acid Racemase (AARase) Activity Using NADH Oxidoreduction Enzymic System.

Fermented foods produced using edible microorganisms have been consumed worldwide since ancient times. Microorganisms produce some healthy functional substances. Recently, many people have been attracted to the notion that D-body amino acids have healthy functional effects. D-body amino acids convert L-body amino acid proteolysis from a substrate such as foods during fermentation. This chapter presents a description of methods used for D-alanine assays in the solution as medium and AARase assays in lactic acid bacteria (LAB) cells for isolation of high AARase-containing strains using D-amino acid oxidase and lactic acid dehydrogenase via a NADH oxidoreduction system.

Isolation of Lactic Acid Bacteria Eliminating Trimethylamine (TMA) for Application to Fishery Processing.

Fishy odor of fish flesh (meat) presents a severe problem for marine production. The main cause of fishy odor is trimethylamine (TMA), which increases during storage. It is produced from trimethylamine oxide (TMAO), an osmosis-regulating substance in fish cells that functions by a reduction reaction. Bacterial growth in fish meat increases TMA. Its odor reduces the commercial value of the meat. Technologies for its regulation and elimination are desired. This chapter presents a description of the use of lactic acid to eliminate TMA. The lactic acid is producible safely by bacteria during food processing using picric acid-toluene.A method of eliminating TMA was demonstrated using Lactobacillus plantarum H78. Furthermore, an assay method was explained for reducing TMA in fish meat by fermenting the H78 strain.

Cloning and Expression of Lipopolysaccharide Elimination Protein (LEP) in Lactic Acid Bacteria.

Lipopolysaccharide (LPS) is related to human inflammation. Therefore, in the probiotics research field, controlling the mechanisms of LPS neutralization and elimination of inflammation of human intestines are important. This chapter presents a description of the identification of LPS elimination protein (LEP) in lactic acid bacteria (LAB), cloning of its protein, and its expression. First, LEP is extracted from the LAB cell wall digestion fraction using Blue Native PAGE. Then LEP is identified by the elimination activity of LPS on gel pieces. Results show that the LEP is an approx. 200 kDa protein part of heat shock protein in lactic acid bacteria. After sequencing amino acids of LEP, LEP cloning is done using a Brevibacillus sp. expression system without a general transformation system but with Gram-negative Escherichia coli having LPS. Results presented in this chapter demonstrate the elimination activity of recombinant LEP.

Eliminating Lipopolysaccharide (LPS) Using Lactic Acid Bacteria (LAB) and a Fraction of its LPS-Elimination Protein.

Lactic acid bacteria (LAB) can improve human intraintestinal conditions. One reason is that ingestion of LAB prevents bacterial diarrhea. Furthermore, inflammation of human intestines can be caused by a lipopolysaccharide (LPS) component in the cell walls of gram-negative bacteria. This chapter describes a method of LPS elimination using lactic acid bacteria (LAB). First, the LPS concentration is assayed using an LPS assay kit with the limulus cascade reaction made by limulus amebocyte lysate. Some LABs, four bacillus strains and one coccus strain, have LPS-elimination activity. Particularly, the coccus strain Pediococcus pentosaceus eliminates LPS to 43%. The cells fractionate and eliminate four fractions: the extracellular fraction, cell membrane fraction, cytoplasm fraction, and cell wall fraction. Only the cell wall digesting fraction eliminates LPS to 45%. Results confirm that the LAB eliminates all LPS having O-antigen under a low-sugar medium condition at temperatures of 15-30 °C. This method can be used for assay of LPS elimination by LABs exactly and easily for the probiotics field.

Neutralization of Lipopolysaccharide by Heat Shock Protein in Pediococcus pentosaceus AK-23.

About 1000 species of bacteria are present in the human intestine. Some Gram-negative bacteria such as Escherichia coli or Salmonella spp. among intestinal bacteria have lipopolysaccharide (LPS), which might induce inflammation of human intestines. Actually, LPS, especially its lipid A constituent, is toxic. Small amounts of LPS in bacteria cause inflammation of mucosa and other tissues in humans. Such bacteria may be regulated by beneficial lactic acid bacteria to maintain human health. Many lactic acid bacteria show cancer prevention activity and anti-inflammatory activity in intestines. Recently, Pediococcus pentosaceus AK-23 was isolated from fermentative vegetable pickles for neutralization of LPS. For this study, a protein for LPS neutralization was purified partly from P. pentosaceus AK-23. For this study, a protein for LPS neutralization was purified partly from P. pentosaceus AK-23, by ultrafiltration using a 300 kDa membrane and a 100 kDa membrane after cell wall digestion by lysozyme. Gel running blue native electrophoresis revealed the existence of a 217 kDa protein. The band of the protein having the ability to bind LPS on the gel was analyzed for amino acid homology. As the result, it is revealed as part of a subunit of heat shock protein (HSP). Furthermore, it displayed LPS binding or hydrophobic motifs. The protein neutralized LPS to release fatty acid as myristic acid and glucose from polysaccharide. These findings suggest that HSP in P. pentosaceus AK-23 neutralizes LPS to decompose it compising fatty acid and polysaccharide.

Isolation of Endotoxin Eliminating Lactic Acid Bacteria and a Property of Endotoxin Eliminating Protein.

Recently, many scholars have reported lactic acid bacteria (LAB) functions, such as anticancer activity and anti-inflammatory activity for intestines. To decrease inflammatory substances such as endotoxins, LAB consumed safely with meals were isolated from food and food ingredients. First, LAB were isolated as 168 strains of bacillus LAB (49 strain) and coccus LAB (119 strains) from food ingredients and fermented foods such as rice, rice bran, malt, grains, miso soy paste, and some pickles. Their LAB (168 strains) were cultivated in medium containing endotoxin from Escherichia coli O18 LPS at 15 and 30 °C for 64 h to identify endotoxin-eliminating LAB. Consequently, the AK-23 strain was screened as an endotoxin-eliminating LAB strain. The strain decreased endotoxin in YP medium without sugar at 30 °C for 64 h until 9% of endotoxin. The strain was identified as Pediococcus pentosaceus according to morphological characteristics such as its cell shape, physiological characteristics related to its fermentation type, assimilation of sugars, pH tolerance, optimum growth temperature, and molecular biological characteristics as its homology to 16S rRNA. To investigate the location of the endotoxin-eliminating substance, 4 fractions were separated from AK-23 cells as extracellular, cell wall digestion, cytoplasm, and cell membrane fractions. The endotoxin-decreasing substance, located on a cell wall, was identified as a 217 kDa protein.