Large‐scale submerged fermentation of Antrodia cinnamomea for anti‐hepatoma activity

BACKGROUND: Submerged cultivation of Antrodia cinnamomea was carried out for manufacturing the fermentation product with anti‐hepatoma activity. The fermentation process was optimized for different parameters at shake flask level to obtain products with high inhibition potency against Hep G2 hepatoma cells. Scale‐up of the fermentation process was then achieved from 250 mL shake flask to 5 L, 500 L and 5‐ton fermenters. RESULTS: Glucose and malt extract were found to be the best carbon and nitrogen sources, respectively. The initial pH of 5.0 and an operating temperature of 22 °C were the best for a product with lowest IC₅₀ value. A shorter cultivation time was required when the scale of fermentation increased from 5 L to 5 tons. The reducing sugar and solids contents of the broth filtrate were correlated exponentially with the IC₅₀ of the ethanolic extract of mycelium for hepatoma cells, and the level of ergosterol in the mycelium extract follows the same profile as the increase in Hep G2 cells inhibition. CONCLUSION: When Antrodia cinnamomea is cultured in a 5‐ton fermenter, 4 weeks are required for the fermentation product to reach the highest anti‐hepatoma activity. The solid and reducing sugar contents of the broth filtrate as well as the ergosterol content in the ethanol extract of mycelium can serve as the marker during fermentation for manufacturing product with anti‐hepatoma activity. Copyright © 2008 Society of Chemical Industry     

Ethanolic extracts of Antrodia cinnamomea mycelia fermented at varied times and scales have differential effects on hepatoma cells and normal primary hepatocytes

Mycelia of Antrodia cinnamomea (AC), an edible fungus native to Taiwan, were produced by submerged fermentation with various fermentation times in 250 mL, 5 and 500 L fermentors and were evaluated for the effect of fermentation products on the viabilities of Hep3B and HepG2 hepatoma cells and normal primary rat hepatocytes. The results showed that the ethanolic extracts of AC mycelia (from 250 mL fermentation for 8 wk and 5 and 500 L fermentations for 4 wk) possessed high antihepatoma activity. The IC(50) of ethanolic extract of AC mycelia fermented for 8 wk in a 250 mL fermentor against Hep3B and HepG2 cells were 82.9 and 54.2 microg/mL, respectively. Furthermore, the IC(50) for Hep3B and HepG2, treated with ethanolic extract of AC mycelia fermented for 4 wk in the 5 L fermentor were 48.7 and 3.8 microg/mL, respectively. Those treated with ethanolic extract of AC mycelia fermented for 4 wk in the 500 L fermentor were 36.9 and 3.1 microg/mL, respectively. No adverse effects of all samples on normal primary rat hepatocytes were observed.     

Screening of medium composition for the free radical‐scavenging properties by Antrodia cinnamomea

The effects of nutritional conditions on the scavenging capacity on superoxide anion radical and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) free radical of ethanolic extracts from filtrates of Antrodia cinnamomea were investigated in shake‐flask cultures. Culture medium significantly affected inhibition of superoxide anion generation and DPPH free radical scavenging activity. In contrast, both scavenging capabilities on superoxide anion radical and DPPH free radical were found to be greatly affected by varying the carbon source, the nitrogen source, the growth factors or the trace elements. The maximal inhibition of superoxide anion generation could be obtained when the culture medium compositions were: xylose, ammonium oxalate, nicotinic acid, NaH₂PO₄ and CuSO₄; while a maximal DPPH free radical scavenging activity could be achieved when the medium composition setting as: fructose, peptone, nicotinic acid, KH₂PO₄ and CaCl₂ (the scavenging effects on superoxide anion radical and DPPH free radical were increased to 96.3 ± 0.3% and 29.0 ± 1.0%, respectively). We proved that controlling the culturing conditions and modifying the composition of the medium can dramatically enhance the scavenging ability on superoxide anion radical and DPPH free radical of A. cinnamomea.     

Changes in volatile compound composition of Antrodia camphorata during solid state fermentation

BACKGROUND: Although the volatiles present in mushrooms and fungi have been investigated by many researchers, including Antrodia camphorata in submerged fermentation, there are few data available regarding changes in volatile compounds during fermentation. Our research has revealed that solid state fermentation of A. camphorata is highly odiferous compared with submerged cultures and the odor changed with increasing culture time. Therefore the aim of this study was to investigate the changes in volatile compound composition of A. camphorata during solid state fermentation. RESULTS: Altogether, 124 major volatile compounds were identified. The volatile compounds produced by A. camphorata during growth in solid state fermentation were quite different. Oct‐1‐en‐3‐ol, octan‐3‐one and methyl 2‐phenylacetate were predominant in exponential growth phase production, while the dominant volatiles produced in stationary phase were octan‐3‐one and methyl 2‐phenylacetate. In stationary phase, lactone compounds in A. camphorata, such as 5‐butyloxolan‐2‐one, 5‐heptyloxolan‐2‐one, 6‐heptyloxan‐2‐one, contributed greatly to peach and fruit‐like flavor. Terpene and terpene alcohol compounds, such as 1‐terpineol, L ‐linalool, T‐cadinol, (E, E)‐farnesol, β‐elemene, cis‐α‐bisabolene and α‐muurolene, made different contributions to herbal fresh aroma in A. camphorata. Nineteen volatile sesquiterpenes were detected from solid state fermentation of A. camphorata. The compounds 5‐n‐butyl‐5H‐furan‐2‐one, β‐ionone, (?)‐caryophyllene oxide, aromadendrene oxide, diepi‐α‐cedrene epoxide, β‐elemene, α‐selinene, α‐muurolene, azulene, germacrene D, γ‐cadinene and 2‐methylpyrazine have not hitherto been reported in A. camphorata. CONCLUSION: The preliminary results suggest that the aroma‐active compounds produced by A camphorata in solid state fermentation might serve as an important source of natural aroma compounds for the food and cosmetic industries or antibiotic activity compounds. The sesquiterpenes could be identified as possible taxonomic markers for A. camphorata. Copyright © 2011 Society of Chemical Industry     

不同发酵方式对樟芝真菌多糖产量的影响研究

通过深层发酵法,对比研究了连续、分批、补料分批3种培养方式对樟芝真菌菌体,胞内外多糖产量的影响,研究表明,补料分批相对于连续培养,菌体最大产量提高5.51%,胞内多糖最大产量提高11.25%。胞外多糖最大产量提高21.70%,补料分批培养优于连续培养和分批培养。     

Modulation of the nutritional value of lupine wholemeal and protein isolates using submerged and solid‐state fermentation with Pediococcus pentosaceus strains

The influence of different factors (submerged and/or solid‐state fermentation, pediococci strain, lupine variety and protein isolation process) on the protein digestibility, total phenolic compounds (TPC) content and radical scavenging activity of Lupinus luteus and angustifolius wholemeal and protein isolates was evaluated. As safety factor, biogenic amines (Bas) formation was analysed. The Pediococcus pentosaceus strains No. 8, No. 9 and No. 10 are suitable starters for lupine wholemeal fermentation and both applied processes (fermentation and protein isolation) increase protein digestibility (by 10%). Higher TPC content in fermented wholemeal can be obtained, compare to isolates. In SMF isolates by 58.3%, 68.2%, 95.6%, 76.6%, lower content of phenylethylamine, putrescine, cadaverine and histamine, respectively was found. The highest protein digestibility and the lowest BAs content using submerged fermentation with pediococcus No. 8 in lupine variety Vilnius protein isolates can be obtained and this technology for high‐quality lupine proteins preparation can be recommended.     

樟芝菌丝体的不同提取方法及其抗肿瘤活性的研究

采用不同的方法对樟芝菌丝体进行提取,使用高效液相(HPLC)对提取物进行了分析,选定乙醇为提取溶剂;并用不同浓度的乙醇在不同时间下提取樟芝菌丝体,确定95%乙醇超声波提取的三萜产量最高,在1g菌丝体中可以获得25.24mg的三萜;并对其进行体外抗肿瘤活性试验,结果表明,80%乙醇浸提18h,在50μg/mL时,体外对小鼠淋巴白血病细胞L1210和人肠癌细胞SW620都有很强的抑制作用。     

食用真菌工业深层发酵研究

本文报道了食用真菌工业深层发酵的研究结果。深层发酵的培养基要具备生长繁殖所需要的各种营养成份,具备形成适当大小菌丝球的环境因素,这种环境因素即是培养液的粘性拉力和生物物理上的剪切作用。食用真菌工业深层发酵的工艺程序与抗生素发酵基本相似,但不同食用菌对工艺条件的要求有些差异。香菇菌(Lentinus edodes)深层发酵72—96小时,每毫升发酵液的菌球数可达5300—6800,菌丝干鼋达3.0—3.45％。发酵液的pH值随发酵的进行而下降,pH值可作为发酵终点的判断依据。发酵后期氨基氮含量高达8-14mg/100ml。紫孢侧耳(Pleurotus sapidus)深层发酵48小时,发酵液的pH值基本上保持不变。菌丝于重达可达3.2％。本文还提出了工业深层发酵培养食用真菌的开发利用途径。     

Production of Agaricus brasiliensis mycelium from food industry residues as a source of antioxidants and essential fatty acids

Agaricus brasiliensis is a medicinal mushroom traditionally employed in prevention and treatment of cancer, used as immunostimulant and as a source of antioxidants. We investigated the chemical composition of the mycelium produced by submerged (SF) and solid‐state fermentation (SSF), using residues from food industry as substrates. After fermentation, antiradical activity and levels of antioxidants were enhanced, indicating that the micro‐organism produces these metabolites. For myceliated substrate (mushroom mycelia grown around and into the substrate particles) obtained by SSF, phenolics ranged from 18.57 to 70.46 mg g^?1 and flavonoids from 0.83 to 4.51 mg g^?1. For myceliated substrate obtained by SF, the variation was 27.19 to 66.99 mg g^?1 and 0.75 to 5.34 mg g^?1 for phenolics and flavonoids, respectively. The fatty acid profile determined by FT‐ICR MS and UPLC‐MS showed predominance of palmitic, linoleic, linolenic and oleic acids. Our findings indicated that mycelium has nutraceutical potential and can be incorporated in food supplements.     

Investigation of the use of ram horn hydrolysate for fungal protein production

Production of the fungus Aspergillus niger NRRL 330 was studied in submerged fermentation with ram horn hydrolysate (RHH) as substrate. The characteristics of RHH have been reported previously. The RHH was enriched by addition of glucose and KH₂PO₄. The effects of kinetic parameters on the biomass yield of the fungus were investigated. The optimal conditions for growth of A niger on RHH were initial pH 6.5, temperature 30 °C, fermentation time 96 h and agitation speed 150 rpm. Under these optimal conditions the initial carbohydrate content of RHH was reduced from 1.52 to 0.2% and the biomass yield was 8.9 g l^?1. The biomass contained about 48.1% protein, 5.2% fat and 9.4% ash (on a dry weight basis). The amino acid content of the biomass was compared with Food and Agricultural Organisation (FAO) and animal feed standards. The protein produced contained all the essential amino acids for animal feed, but the amounts of these amino acids were somewhat lower than those of FAO and soybean reference protein. However, the amino acid composition of the biomass was better than that of animal feed. The results with RHH were also compared with previously reported data on fungal mycelium grown on waste liquor substrate. In conclusion, it was found that RHH could be used as a substrate in the production of fungal protein for use as animal feed. Copyright © 2003 Society of Chemical Industry     

Proteolytic activity (aspartic endoproteinase and carboxypeptidase) of cocoa bean during fermentation

This study was carried out to examine the changes in cocoa aspartic endoproteinase and carboxypeptidase activities, mass pH and temperature during 144 h (6 days) of fermentation using shallow wooden boxes. The results showed that at 72 h of fermentation, the activity of aspartic endoproteinase was higher than the original value. However, at 96 h the carboxypeptidase activity was higher than its original activity. The study also found a high correlation 0·99 (P<0·05) between the mass temperature with the aspartic endoproteinase at 48 h of fermentation. No correlation was found between the mass pH and temperature with the carboxypeptidase during fermentation. The degradation of vicilin-class globulin was about 88·8% and that of albumin was 47·4% at the end of fermentation. © 1998 SCI.     

樟芝深层发酵多糖促进乳酸菌生长及增殖

目的 研究樟芝（Antrodia cinnamomea）深层发酵胞内多糖（Antrodia cinnamomea intracellular polysaccharide,AIPS）及胞外多糖（Antrodia cinnamomea extracellular polysaccharide, AEPS）对乳酸菌体外发酵生长及增殖的影响。方法 采用水提醇沉法和提取樟芝深层发酵AIPS及AIPS,采用96孔板法考察不同浓度AIPS及AEPS对4株乳酸菌生长或增殖的影响,采用平板菌落计数法考察AIPS及AEPS不同单糖组分对乳酸菌生长的影响。结果 AIPS对干酪乳杆菌LBC1-1菌株（LBC1-1）、植物乳杆菌LBP3-2菌株（LBP3-2）及鼠李糖乳杆菌LGG菌株（LGG）的生长均有明显促进作用,且主要表现为缩短迟滞期及增加最大生长量;AEPS对LBP3-2、LGG及鼠李糖乳杆菌LV-1菌株（LV-1）的生长有显著促进作用,且主要表现为提高最大生长速率及最大生长量;AIPS及AEPS促进乳酸菌生长不是因为提供了碳源营养物质,而是其中的某一种或几种多糖成分发挥了促进作用。结论 樟芝深层发酵多糖能显著促进乳酸生长和/或增殖,本研究为开发新型多功能益生元提供新思路及理论依据。     

α‐(1,4)‐Amylase,but not α‐ and β‐(1,3)‐glucanases,may be responsible for the impaired growth and morphogenesis of Paracoccidioides brasiliensis induced by N‐glycosylation inhibition

The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N‐glycosylation of proteins such as N‐acetyl‐β‐d ‐glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)‐mediated inhibition of N‐linked glycosylation on α‐ and β‐(1,3)‐glucanases and on α‐(1,4)‐amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 µg/ml TM to the fungal cultures did not interfere with either α‐ or β‐(1,3)‐glucanase production and secretion. Moreover, incubation with TM did not alter α‐ and β‐(1,3)‐glucanase activity in yeast and mycelium cell extracts. In contrast, α‐(1,4)‐amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N‐glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N‐glycans were essential for α‐(1,4)‐amylase activity involved in the production of malto‐oligosaccharides that act as primer molecules for the biosynthesis of α‐(1,3)‐glucan. Our results suggest that reduced fungal α‐(1,4)‐amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells. © 2013 The Authors. Yeast published by John Wiley & Sons Ltd.     

The Influence of Inoculum Level on Fermentation and Flavour Compounds of White Wines Made from cv. Emir

In this study, the influence of the addition of a commercial wine yeast (Saccharomyces cerevisiae) at inocula of 1 × 10⁴ to 1 × 10⁷ cells/ml in Emir must was investigated with a focus on yeast growth, fermentation rate, ethyl alcohol and flavour compound formation. Spontaneous fermentation without inoculation was also performed. Higher peak counts were observed with higher amounts of S. cerevisiae yeast. Addition of various amounts of yeast led to the earlier disappearance of non‐Saccharomyces yeasts. The fermentation rate was improved with higher amounts of yeast, but ethanol production was not affected. Concentrations of higher alcohols increased with increasing inoculum levels, especially inoculum sizes of 1 × 10⁶ cells/ml and 1 × 10⁷ cells/ml. The amount of ethyl acetate was reduced with increased inoculum levels.     

Improvement of flavour compound synthesis in a mixed culture system at high temperature by solid‐state fermentation

In order to enhance the synthesis of flavour compounds in solid‐state fermentation (SSF) at a high temperature, Bacillus subtilis XJ‐013 and Saccharomyces cerevisiae Z‐06 were used as a mixed culture with Monascus HQ‐3. The culture temperature was enhanced from 37°C to 56°C by the synergetic effect, and the synthesis of esterase was enhanced from 85.43 U/g to 129.65 U/g in the mixed culture system (over 50% higher than that of the culture using a single strain). This resulted in the synthesis of favourable flavour compounds in the solid‐state fermentation. These results signified that a mixed culture of Monascus and S. cerevisiae was favourable for enzyme production. The mixed culture of Monascus and B. subtilis resulted in a high culture temperature that promoted flavour compound synthesis in the solid‐state fermentation system dramatically. These results present a model to explain the synergetic effects between the fungus and the Bacillus in the solid‐state fermentation. Copyright © 2015 The Institute of Brewing & Distilling     

Characteristics and Novel Features of Thermostable α-Amylase Produced by Bacillus licheniformis M27 under Solid State Fermentation

The purified α-amylase from Bacillus licheniformis M27, produced under solid state fermentation technique, showed novel characteristics as compared to those reported by other workers for the purified α-amylases from B. licheniformis obtained by submerged fermentation process. Some of the novel features of the characteristics of the enzyme from B. licheniformis M27 include two peaks for pH optima at 6.5–7.0 and 8.5–9.0, gradual loss of activity to about 86% between pH 7.0–7.5 followed by rise to full activity between 7.5–8.5, temperature optimum at 85–90°C at pH 7.0 and 9.0, sharp fall in stability at acidic pH values and the thermostability response which is more similar to the enzyme from other species of Bacillus. The moleculuar weight of the enzyme was found to be 19,500 ± 500 and 56,000 ± 2,000 when determined by gel filtration and SDS PAGE, respectively. The activation energy is 20.4 times lower than that reported for the enzyme from another strain of B. licheniformis. It also showed differences in the contents of amino acids such as serine, proline and methionine.     

Optimization of Calvatia gigantea mycelia production from distillery wastewater

The focus of wastewater management has evolved from treatment technology into resource recovery, which enables one to minimize contaminants and to generate value‐added products. Calvatia gigantea is used not only as a source of food, but has also been used as a traditional Chinese medicine for thousands of years. In this study, the mycelial production of C. gigantea was studied under submerged fermentation conditions using non‐pretreated distilled wastewater from Chinese liquor production. The fermentation medium composition was optimized using response surface methodology involving a Box–Behnken design. Fermentation conditions were optimized using an orthogonal experimental design. The optimized medium composition was the non‐pretreated distilled wastewater of Chinese liquor supplemented with cornflour at 2.35 g/100 mL, (NH₄)₂SO₄ at 1.11 g/100 mL and CuSO₄ at 0.12 g/100 mL. The optimized fermentation conditions were a rotation speed of 150 rpm, an inoculum size of 10% (v/v), a fermentation temperature of 26 °C and a fermentation time 4.5 days. A maximum mycelial biomass yield of 2.75 g/100 mL was achieved using the optimized medium under the optimized conditions. Results from this study suggest that this is a feasible technology for the mycelial production of C. gigantea using the non‐pretreated distilled wastewater from Chinese liquor production. Copyright © 2015 The Institute of Brewing & Distilling     

Screening and Characterization of Lactobacillus sp. from the Water of Cassava’s Fermentation for Selection as Probiotics

The present study aimed to evaluate selected probiotic properties of Lactobacilli isolated from the water of submerged cassava fermentation. Following Lactobacilli isolation, isolates were screened for their antimicrobial activity. Acid and bile tolerances, bile salt hydrolase (Bsh) activity spectrum were assessed. Among the 113 isolates obtained, 16 showed a broad antimicrobial activity spectrum against the indicator microorganisms. From these 16, 12 were found acid and bile resistant. They hydrolyzed glycoconjugated or tauroconjugated bile salts. From the four bile Bsh genes screened, only Bsh-Lp1 was found in five isolates. They identified as Lactobacillus paraplantarum, Lactobacillus paracasei, Lactobacillus brevis, and Lactobacillus plantarum. Based on the principal component analysis, L. paracasei 62L, L. plantarum 85L and 86L were selected as the most promising strains. These results suggest that water from submerged cassava fermentation can be a source of Lactobacilli with high probiotic potential.     

Survival and Metabolic Activity of Microencapsulated Bifidobacterium longum in Stirred Yogurt

ABSTRACT: Live cells of Bifidobacterium longum, microencapsulated in K‐carrageenan, were added to stirred yogurt after fermentation (pH 4.6) and stored at 4.4 °C for 30 d. Cell enumeration indicated no decline of encapsulated cell number in yogurt samples, while there was significant reduction in nonencapsulated cell population (89.3% for B. longum B6 and 91.8% for B. longum ATCC 15708). Ion‐exchange high‐performance liquid chromatography showed comparable amounts of lactic and acetic acids in all samples, indicating little metabolic activity by bifidobacteria in experimental yogurts. Consumer sensory analysis of blackberry‐flavored yogurts revealed that samples containing encapsulated bifidobacteria had a grainy texture. Results suggested that microencapsulation protected bifidobacteria from the low pH of yogurt.