Effective valorisation of distillery stillage by integrated production of lactic acid and high quality feed

Utilization of distillery stillage from bioethanol production for lactic acid and feed production was studied. The lactic acid fermentation of the stillage was performed by Lactobacillus rhamnosus ATCC 7469 and maximal lactic acid concentration of 50.18 g L^− 1, yield of 0.90 g g^− 1, productivity of 1.48 g L^− 1 h^− 1 and viable cell number of 5 × 10⁹ CFU mL^− 1 were achieved. Solid residues with biomass remains after lactic acid fermentation were assessed for animal consumption. The content of proteins and ash decreased in the residues after the fermentation, whilst the content of oil and nitrogen free extract was higher when compared to unfermented samples. The digestible (17480.64 kJ kg^− 1) and metabolisable (17389.08 kJ kg^− 1) energies as well as digestibility (966.95 g kg^− 1) of the fermentation residue were very high. The in vitro assessment of L. rhamnosus ATCC 7469 survival in simulated gastric conditions has shown high survival rate (87%). In addition, this bacterium has shown good antimicrobial activity against the most important pathogens and capability to produce exopolysaccharide on different sugars present in animal diet. After effective lactic acid fermentation, the residues could be recommended as a high quality feed for monogastric animals.     

Production of volatile aroma compounds by kefir starter cultures

Production of carbonyl compounds by single-strain cultures, kefir starter (Lactobacillus delbrueckii subsp. bulgaricus HP1+Lb. helveticus MP12+Lactococcus lactis subsp. lactis C15+Streptococcus thermophilus T15+Saccharomyces cerevisiae A13) and kefir grains during fermentation and storage of kefir was studied. The content of carbonyl compounds produced by kefir starter was greater than that produced by kefir grains. The maximum acetaldehyde concentration (18.3 μg g⁻¹) in kefir with starter culture was mainly due to the metabolic activity of Lb. delbrueckii subsp. bulgaricus HP1 isolated from kefir grains. The highest diacetyl production activity was recorded in the starter culture (1.87 μg g⁻¹) and the single-strain culture St. thermophilus T15 (1.62 μg g⁻¹), followed by Lb. helveticus MP12 (0.85 μg g⁻¹) and Lc. lactis subsp. lactis C15 (0.42 μg g⁻¹). The lactobacilli Lb. delbrueckii subsp. bulgaricus HP1 and Lb. helveticus MP12 produced acetone, which was not found in the cocci cultures. The presence of 2-butanone was related to the production ability of Lb. helveticus MP12. In comparison, Lc. lactis subsp. lactis C15 synthesized ethyl acetate more actively than the other single-strain cultures included in the starter. S. cerevisiae A13 produced ethanol and CO₂ in amounts (3975 μg g⁻¹; 1.80 g L⁻¹) that lent cultured kefir distinctive flavour and aroma characteristic of authentic kefir.     

Incorporation of microbial transglutaminase into non-fat yogurt production

This study investigated the physical, chemical and sensory characteristics of non-fat yogurts treated with microbial transglutaminase (MTGase) at varying concentrations from 0 to 0.5 g L⁻¹. Also, the effect of enzyme inactivation prior to fermentation on the selected properties of the yogurts was studied. Acid development rate was reduced with increasing MTGase doses. Cross-linking of milk proteins by MTGase had a growth-slowing effect on yogurt starter bacteria, which was more pronounced at higher concentrations. Physical properties of the yogurts were improved by MTGase throughout 21-day storage; on the contrary, the production of acetaldehyde was slowed down by increasing MTGase concentrations during the same period. Principal component analysis (PCA) and hierarchial cluster analysis (HCA) clearly differentiated the samples with added MTGase at lower (⩽0.3 g L⁻¹) and higher (0.4–0.5 g L⁻¹) concentrations regarding the physical and sensory properties. The physical and sensory properties of non-fat set yogurt could be improved by incorporating MTGase up to a level of 0.3 g L⁻¹.     

Effect of commercial adjunct cultures on proteolysis in low-fat Kefalograviera-type cheese

The effect of two commercially available adjunct cultures, LBC 80 (Lactobacillus casei subsp. rhamnosus) and CR-213 (containing Lactococcus lactis subsp. cremoris and Lc. lactis subsp. lactis) on the proteolysis in low-fat hard ewes’ milk cheese of Kefalograviera-type was investigated. Two controls, a full-fat cheese (306 g kg⁻¹ fat, 378 g kg⁻¹ moisture) and a low-fat cheese (97 g kg⁻¹ fat, 486 g kg⁻¹ moisture, made using a modified procedure), were also prepared. The effect of adjunct culture on proteolysis, as examined by polyacrylamide gel electrophoresis of cheese and water soluble cheese extracts, was marginal. The reverse-phase HPLC peptide profiles of the water soluble extracts from low-fat cheeses were similar although some quantitative differences were observed between low-fat control cheese and experimental cheeses. The fat content as reflected by the differences in peptide profiles affected the pattern of proteolysis. Proteolysis, as measured by the percentage of total nitrogen soluble in water or in 120 g L⁻¹ trichloroacetic acid, was significantly (P<0.05) affected by the addition of adjunct cultures. Furthermore, the adjunct cultures enhanced the production of low molecular mass nitrogenous compounds; the levels of total nitrogen, soluble in 50 g L⁻¹ phosphotungstic acid, and of free amino acids were significantly (P<0.05) higher in the low-fat experimental cheeses than in the low-fat control cheese.     

Non-volatile flavour components of Ganoderma tsugae

Ganoderma tsugae Murrill are currently popular and used in the formulation of nutraceuticals and as functional foods. The non-volatile components in the form of mature and baby fruit bodies (Ling chih), mycelia and fermentation filtrate from submerged culture were studied. Mycelia and filtrate contained significantly higher moisture contents (10.3% and 19.8%) and higher contents of carbohydrates, reducing sugars, crude ash and crude protein. Four forms of G. tsugae contained from 7.65% to 10.1% dry weight of total soluble sugars and polyols. Total free amino acid contents ranged from 2.50 to 149 mg g⁻¹ dry weight and in the descending order of filtrate, mycelia, baby Ling chih and Ling chih. Contents of monosodium glutamate-like components ranged from 0.16 to 26.0 mg g⁻¹ whereas, contents of sweet components ranged from 0.50 to 24.6 mg g⁻¹. The bitter components were predominant. Contents of total and flavour 5^′-nucleotides were high in filtrate (5.48 and 3.10 mg g⁻¹, respectively). The umami intensities were expected to be in the descending order of filtrate, mycelia, baby Ling chih and Ling chih.     

Purification of angiotensin I-converting enzyme inhibitory peptides and antihypertensive effect of milk produced by protease-facilitated lactic fermentation

Fresh low-fat milk was fermented with five mixed lactic acid bacteria for up to 30 h at 42 °C. A protease, prozyme 6, was added 5 h after the beginning of fermentation. The whey was separated from the fermented milk and freeze-dried. As the fermentation time extended to 30 h, soluble protein content increased from 30.9 to 195.9 mg g⁻¹, free amino acid content increased from 2.8 to 192.8 mg g⁻¹, peptide content increased from 6.4 to 402.8 mg g⁻¹ and γ-aminobutyric acid (GABA) increased from 0 to 80.6 mg 100 g⁻¹, while inhibition of angiotensin I-converting enzyme (ACE) increased as indicated by a decrease of IC₅₀ from 1.18 to 0.24 mg mL⁻¹, respectively. The amino acid sequences of two ACE inhibitory peptides were Gly–Thr–Trp and Gly–Val–Trp, of which the IC₅₀ values were 464.4 and 240.0 μm, respectively. The systolic blood pressure and diastolic blood pressure of spontaneously hypertensive rat (SHR) were reduced 22 and 21.5 mm Hg, respectively, after 8 weeks of oral administration of diluted whey (peptide concentration 5 mg mL⁻¹) from the 30 h fermentation.     

Fatty acid feedstock preparation and lactic acid production as integrated processes in mixed restaurant food and bakery wastes treatment

In this study, fatty acid feedstock preparation and lactic acid production as integrated processes in mixed restaurant food and bakery waste treatment were investigated. The treatment included the hydrolysis of waste using extracellular fungal enzymes in submerged fermentation. By hydrolysis, 0.27 g glucose, 4.7 mg free amino nitrogen (FAN) and 1.9 mg phosphate were recovered per gram dry waste material. After hydrolysis, a lipid-rich solid fraction, to be used as a source of fatty acids, remained. The nutrient-rich hydrolysate was used as medium in fed-batch cultures of the heterotrophic microalga Chlorella pyrenoidosa, which grew well at a rate of 1.4 day^− 1. In order to establish a cost- and water-efficient process, hydrolysis and algae cultivation were performed in recycled culture supernatant without any negative impacts on the fungal hydrolysis and growth of C. pyrenoidosa. The extraction of lipids from algal biomass and lipid-rich solids resulted in a saturated and unsaturated fatty acid-rich feedstock. Defatted waste derived solids and algal biomass were further tested successfully as nitrogen sources in lactic acid production using Bacillus coagulans. The outcomes of this study contribute to the establishment of a ‘green society’ by utilization of waste material in the production of chemicals, materials and fuels.     

Aluminium in foodstuffs

The aluminium content of a comprehensive food assortment typical of German nutritional habits was determined within the framework of market basket studies. Carried out in 1988 and 1991, a total of 128 items out of 12 groups of foodstuffs were included in this investigation. Aluminium content of the food assortment was low and comparable with literature data. Most investigated foodstuffs contained <5 μg Al g⁻¹ FM. Highest concentrations were determined in cocoa/cocoa products (33 μg g⁻¹), spices (145 μg g⁻¹) and black tea leaves (899 μg g⁻¹). In general, aluminium content of frequently consumed food, increased in the following order: beverages, food of animal origin, food of plant origin. With this low level of aluminium concentration in food, there is no danger of aluminium exposure in healthy persons.     

Characterization of bacteriocin ST8KF produced by a kefir isolate Lactobacillus plantarum ST8KF

Lactobacillus plantarum ST8KF, isolated from kefir, produced a 3.5 kDa bacteriocin (bacST8KF) active against Lb. casei, Lb. salivarius, Lb. curvatus and Listeria innocua. BacST8KF was sensitive to proteolytic enzymes, but stable between pH 2.0 and 10.0, and heat resistant (20 min at 121 °C). BacST8KF did not adsorb to the surface of the producer cell. Maximum activity (25,600 AU mL⁻¹) was recorded in MRS broth with glucose, in MRS broth with glucose replaced by sucrose, and in MRS broth with glucose, supplemented with KH₂PO₄ after 24 h at 30 °C. Tri-ammonium citrate and glycerol in excess of 5.0 g L⁻¹ repressed bacST8KF production. Production of bacST8KF increased from 800 AU mL⁻¹ after 3 h of fermentation in MRS broth at 30 °C to 12,800 AU mL⁻¹ after 9 h and to 51,200 AU mL⁻¹ after 27 h. These results suggest that bacST8KF may be a secondary metabolite and shows that its mode of activity is bacteriostatic.     

Improved astaxanthin production by Xanthophyllomyces dendrorhous growing on enzymatic wood hydrolysates containing glucose and cellobiose

Eucalyptus wood samples were treated with NaOH solutions and subjected to enzymatic hydrolysis with a commercial, β-glucosidase-deficient, cellulase complex or with a mixture of cellulase and β-glucosidase. Hydrolysates containing glucose or glucose and cellobiose were supplemented with KNO₃ and nutrients, and used as culture media for the proliferation of Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) ATCC 24228. For comparative purposes, similar experiments were carried out with media made from commercial sugars. All the bioconversion experiments were carried out in a batch fermenter. At selected fermentation times, the concentrations of substrate (or substrates), biomass and carotenoids were determined. Under the best conditions assayed (hydrolysates obtained with cellulases supplemented with an inorganic nitrogen source), biomass concentrations in the vicinity of 10 g litre⁻¹, with volumetric carotenoid concentration of 2.14 mg litre⁻¹, were reached after 74 h of fermentation. These results compare favourably with those reported for carotenoid production from enzymatic hydrolysates containing glucose as the sole carbon source..     

In-vitro mineral binding capacity of five fiber sources and their insoluble components for magnesium and calcium1

In-vitro binding of calcium (Ca) and magnesium (Mg) by total dietary fiber, hemicellulose A (HCL A), lignocellulose (LCL), cellulose (CL), and lignin (L) fractions isolated from rice bran (RB), wheat bran (WB), oat fiber (OF), apple fiber (AF) and tomato fiber (TF) was evaluated. At pH 6.8, significant amounts of Ca were bound by whole fibers, ranging from 800 μg g⁻¹ for RB to 10 097 μg g⁻¹ for TF. Mg bound by whole fibers varied from 496 μg g⁻¹ for OF to 2177 μg g⁻¹ for WB. Re-acid washing (pH<2.0) released 95–99% of the Ca and Mg bound to the fibers. Fibers with the highest endogenous Ca and Mg concentrations bound significantly (P<0.05) the highest amounts of the minerals studied. The Ca bound by HCL A varied from 9753 μg g⁻¹ for RB to 11 337 mg g⁻¹ for TF, whereas Mg bound varied from 1151 μg g⁻¹ for OF to 5626  μg g⁻¹ for TF hemicellulose fractions, respectively. Among the fiber components, Mg binding decreased in the order HCL A>LCL>L>CL, whereas Ca bound was in the order HCL A>LCL>CL>L. A relatively strong correlation was observed between the combined effects of protein content, hemicellulose, and lignin vs total Ca and Mg bound. 1998 Elsevier Science Ltd. All rights reserved     

Comparison of the effect of pulsed electric field or high voltage electrical discharge for the control of sweet white must fermentation process with the conventional addition of sulfur dioxide

The present work discusses the efficiency of pulsed electrical treatments for the inactivation of yeasts. The application of pulsed electric fields (PEFs) and high voltage electrical discharges (HVEDs) as alternatives to sulfites, which are used as anti-microbial to stop the fermentation of sweet white wine, was investigated. The influence of sulfite concentration (from 0 mg·L^− 1 to 500 mg·L^− 1), PEF (from 4 kV·cm^− 1 to 20 kV·cm^− 1; from 0.25 ms to 6 ms) and HVED (40 kV/cm; 1 ms or 4 ms) treatments on the inactivation of total yeasts and non-Saccharomyces yeasts was determined. The addition of SO₂ (250 mg·L^− 1) resulted in 8 log total yeast reduction. The maximum yeast inactivation obtained with PEF and HVED was respectively 3 and 4 logs. The use of SO₂, HVED and PEF allows decreasing the non-Saccharomyces yeast level by 7, 5 and 4 logs respectively. However, the wine browning was less pronounced for the samples treated by PEF in comparison with HVED and SO₂ treatments. PEF seems to be the most suitable alternative technique to sulfite addition.     

Synthesis of potentially-bioactive lactosyl-oligofructosides by a novel bi-enzymatic system using bacterial fructansucrases

Efficient enzymatic synthesis of lactosyl-oligofructosides (LFOS) with a degree of polymerization from 4 to 8 was achieved in the presence of sucrose:lactosucrose and sucrose:lactose mixtures by transfructosylation reaction. The main synthesized LFOS which consist of β-2,1-linked fructose to lactosucrose: β-d-galactopyranosyl-(1 → 4)-α-d-glucopyranosyl-[(1 → 2)-β-d-fructofuranosyl]n-(1 → 2)-β-d-fructofuranoside (where n refers to the number of transferred fructose moieties) was structurally characterized by nuclear magnetic resonance (NMR). The maximum formation of LFOS was 81% (in weight with respect to the initial amount of lactosucrose) and was obtained after 24 h of transfructosylation reaction based on sucrose:lactosucrose (250 g L^− 1 each) catalyzed by an inulosucrase from Lactobacillus gasseri DSM 20604 (IS). The production of LFOS in the presence of sucrose:lactose mixtures required a previous high-yield lactosucrose synthesis step catalyzed by using a levansucrase from Bacillus subtilis CECT 39 (LS) before the inulosucrase-catalyzed reaction. This novel one-pot bi-enzymatic system led to the synthesis of about 22% LFOS in weight, with respect to the initial amount of lactose (250 g L^− 1). The results revealed a high specificity for the substrate involved in the inulosucrase-catalyzed reaction given that, although lactosucrose (O-β-d-galactopyranosyl-(1 → 4)-O-α-d-glucopyranosyl-(1 → 2)-β-d-fructofuranoside) acted as a strong acceptor of β-2,1-linked fructose, lactose (β-d-galactopyranosyl-(1 → 4)-α-d-glucose) was found to be an extremely weak acceptor.     

Production of traditional Greek yoghurt using Lactobacillus strains with probiotic potential as starter adjuncts

Lactobacillus plantarum ACA-DC 146 and L. paracasei subsp. tolerans ACA-DC 4037 were examined for their potential application as adjuncts in the production of traditional Greek set-type yoghurt. Both strains displayed low milk acidification activity, while no inhibition was observed towards or from the yoghurt starters used. Yoghurt produced with L. paracasei subsp. tolerans ACA-DC 4037 exhibited the best sensory properties, with a rich traditional smooth taste, and the strain was selected for further trials. Yoghurt produced with this strain as an adjunct had good physicochemical properties. After 2 weeks of refrigerated storage, microbial loads (>7.0 log cfu g⁻¹) were in accordance with international recommendations and guidelines for probiotic and starter cultures in milk products. Increasing the microbial load further, using concentrated and encapsulated inocula (10–11 log cfu g⁻¹), gave yoghurt with long fermentation times and poor organoleptic properties.     

Antimicrobial agent detection in ewes’ milk by the microbial inhibitor test brilliant black reduction test—BRT AiM®

The detection limits of 24 antimicrobial agents were determined in ewes’ milk by one commercially available version of brilliant black reduction test, BRT Inhibitor Test with prediffusion AiM^® (BRT AiM^®). For each drug, eight concentrations were tested on 20 milk samples from individual ewes. The detection limits of the BRT AIM^® method were determined by means of logistic regression models: 6 μg kg⁻¹ amoxycillin, 6 μg kg⁻¹ ampicillin, 51 μg kg⁻¹ cloxacillin, 2 μg kg⁻¹ penicillin “G”, 230 μg kg⁻¹ cefadroxil, 1330 μg kg⁻¹ cephalosporin “C”; 270 μg kg⁻¹ cephalexin, 92 μg kg⁻¹ cefoperazone, 120 μg kg⁻¹ ceftiofur, 69 μg kg⁻¹ cefuroxime, 6000 μg kg⁻¹ streptomycin, 1200 μg kg⁻¹ gentamycin, 3700 μg kg⁻¹ neomycin, 630 μg kg⁻¹ erythromycin, 120 μg kg⁻¹ tylosin, 390 μg kg⁻¹ doxycycline, 5500 μg kg⁻¹ oxytetracycline, 6200 μg kg⁻¹ tetracycline, 5400 μg kg⁻¹ sulfadiazine, 3200 μg kg⁻¹ sulfamethoxazole, 6500 μg kg⁻¹ sulfamethoxypyridazine, 6200 μg kg⁻¹ sulfaquinoxaline, 22000 μg kg⁻¹ chloramphenicol and 4100 μg kg⁻¹ trimethoprim. The BRT AiM^® method presents detection limits for β-lactam antibiotics that are similar to those obtained as Maximum Residue Limits (MRLs) according to Regulation 2377/90 EEC as set out by the European Union. However, for other antimicrobial agents the estimated limits were higher than those of the EU-MRLs. It is therefore advisable to enhance the sensitivity of the method for the detection of the different antimicrobial groups or to develop a combined system of different microbiological inhibitor tests that would enable the detection of a greater number of antimicrobial agents.     

Influence of methylcellulose on dynamic rheology of dilute wheat gliadin solution in 50% (v/v) aqueous propanol

Influences of methylcellulose on dynamic rheological behaviors of 50 g L⁻¹ (dilute) wheat gliadins in 50% (v/v) aqueous propanol were investigated as a function of methylcellulose concentration C ranging from 5 to 20 g L⁻¹. The mixture solutions exhibit shear rate thinning that becomes pronounced with increasing C. Specific viscosities of the first and the second Newtonian plateau viscosities, as well as the steady-flow viscosity as a function of C, reveal that methylcellulose is in the semi-dilute unentangled regime at temperatures from 5 to 65 °C. The intermolecular interaction between methylcellulose and gliadins provides high flow resistance, improved shear thinning and steady-flow activation energy of the mixture solution in comparison with pure gliadins solution.     

Microbiological and chemical changes during the manufacture of Kefir made from cows’ milk,using a commercial starter culture

The counts of total aerobic mesophilic bacteria, lactic acid bacteria (in three different culture media, M17 agar, MSE agar, and Rogosa agar) and yeasts, and some biochemical parameters (levels of lactose, glucose, galactose, L(+)- and D(−)-lactic acids, ethanol, titratable acidity and pH) were determined during 196 h of fermentation in five batches of Kefir made from cows’ milk using a commercial starter culture. Lactococcus spp. predominated during the first 48 h of fermentation (∼8 log₁₀ cfu g⁻¹); Lactobacillus spp. became the predominant species after 48 h (∼8.5 log₁₀ cfu g⁻¹). During the first 24 h of fermentation, the lactose content decreased from a mean value of 4.92% (w/w) to 4.02% (w/w); the concentration of L(+)-lactic acid increased from 0.01% to 0.76% (w/w) and the pH decreased to 4.24 over the same period. After 24 h of fermentation, the changes in the levels of lactose and L(+)-lactic acid, and in pH, occurred more slowly. Neither glucose nor galactose were detected during fermentation. The production of ethanol was limited, reaching a mean final value of 0.018% (w/w).     

Effect of combinations of high-pressure treatment and bacteriocin-producing lactic acid bacteria on the survival of Listeria monocytogenes in raw milk cheese

The combined effect of high-pressure (HP) treatment and bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Listeria monocytogenes Scott A in cheeses made from raw milk that was inoculated with the pathogen at 4.80 log cfu mL⁻¹, a commercial starter and one of seven strains of BP-LAB was investigated. On day 3, the counts of L. monocytogenes were 7.03 log cfu g⁻¹ in a control cheese (without BP-LAB, not HP treated), 6.06–6.74 log cfu g⁻¹ in cheeses with BP-LAB, 6.13 log cfu g⁻¹ in a cheese without BP-LAB and treated on day 2 at 300 MPa, 2.01 log cfu g⁻¹ in a cheese without BP-LAB and treated on day 2 at 500 MPa, 3.83–5.43 log cfu g⁻¹ in cheeses with BP-LAB and treated on day 2 at 300 MPa, and 1.81 log cfu g⁻¹ or less in cheeses with BP-LAB and treated on day 2 at 500 MPa. HP treatment was more effective on day 51 than on day 2.     

Changing soybean isoflavone composition and concentrations under two different storage conditions over three years

Soybean in Asia have been consumed in various forms, including soymilk, tofu and fermented products such as miso, temph and sufu. It is popularly regarded as a healthy food, partly owing to the isoflavones contained in their seeds. The objective of this study was to evaluate the variation of isoflavone concentration in soybean (Glycine max (L.) Merrill) seeds under different storage conditions for long storage periods. Isoflavone concentrations varied from 699.7 to 2581.6 μg g⁻¹ with cropping year, and acetylglucoside groups and glycitein were only detected in small amounts or traces in the eight soybean cultivars. The Daweon cultivar showed a variation between storage at room (−2039.0 μg g⁻¹) and low temperature (−1822.0 μg g⁻¹) over three years, while the isoflavone concentration in the Hannam cultivar only varied slightly (room temperature: −91.6 μg g⁻¹, low temperature: −81.2 μg g⁻¹). With storage at room temperature, the acetylglucoside group (+7.2 μg g⁻¹) slightly increased the isoflavone concentration, while the other three groups decreased it. In particular, the malonylglucoside group (−519.0 μg g⁻¹) showed a severe decrease. In the Myeongjunamul cultivar, genistin (+105.0 μg g⁻¹) resulted in the highest increase, while malonylgenistin (−958.7 μg g⁻¹) resulted in the greatest decrease in the Daweon cultivar. With storage at low temperature, other than malonylglucoside (−438.1 g g⁻¹), the other three groups, aglycon (+11.4 μg g⁻¹), glucoside (+45.2 μg g⁻¹) and acetylglucoside (+12.8 μg g⁻¹), increased the isoflavone concentration. Genistin (+136.0 μg g⁻¹) in the Muhan cultivar showed the highest increase, and malonylgenistin (−833.4 μg g⁻¹) in the Daweon cultivar showed the greatest decrease for storage under low temperature for three years. The variation of isoflavone concentration was positively correlated with the two different storage conditions (r² = 0.33), and total isoflavones were correlated with the concentration of the malonylglucoside (r² = 0.88***) and glucoside (r² = 0.80***) groups. Our study suggests that it may be feasible to improve the preservation method of soybean isoflavones as high functional substances for longer storage periods.