黄酒发酵过程中微生物筛选及菌株产香分析

对古越龙山机械发酵黄酒的醪液中的微生物进行纯培养分离以及生物鉴定,并分析分离微生物在黄酒模拟液中的生长情况及挥发性物质的变化。结果表明,从发酵醪液当中共分离出53株微生物,其中有33株细菌、16株霉菌以及4株酵母。通过对分离微生物在黄酒模拟液中的生长情况监测发现,细菌中Pantoea agglomerans L211、Clostridium tyrobutyricum L311以及Lactobacillus helveticus M41生长较好,其中Lactobacillus helveticus M41为乳酸菌;分离酵母菌中菌株Saccharomyces cerevisiae Y7生长情况优于其他分离酵母,霉菌除菌株CO1和MY4外,生长趋势较为一致,菌株Lichtheimia ramosa C122和Lichtheimia corymbifera P181生长情况要稍优于其他霉菌。实验利用GC-MS测定菌株产风味情况,共检测出142种风味物质;发现除酵母产香外,细菌中菌株Clostridium tyrobutyricum L311产挥发性风味物质总含量最高,其次是菌株Bacillus ginsengisoli L15;在真菌中,霉菌Penicillium expansum C01产总挥发性风味物质要优于其他霉菌。     

Volatile odour substances produced by microflora

Literature indicates a broad contribution of microorganisms to the odours occurring in foods. Own research on volatile odour compounds produced by moulds, bacteria and yeasts growing on cereal grains hase been concentrated to 1-octen-3-ol, especially, which was found to be produced in great amounts by certain moulds as well as by mushrooms both growing wild and cultivated. In the bacteria strains studied about 50 volatile compounds have been identified. In most of the bacterial cultures developed on cereal grain acetoin was the predominant substance. Practical applications are proposed and discussed. On one side a flavour concentrate may be produced from mould cultures yielding fungal odour. Otherwise the main microbial metabolites could be used as indices of the growth rate of individual groups of the microflora on cereal grains and other foodstuffs.     

Influence of volatile compounds produced by yeasts predominant during processing of Coffea arabica in East Africa on growth and ochratoxin A (OTA) production by Aspergillus ochraceus

The effects of volatile compounds produced during coffee processing by Pichia anomala, P. kluyveri and Hanseniaspora uvarum on growth of Aspergillus ochraceus and production of ochratoxin A (OTA) were studied. On malt extract agar (MEA) and on coffee agar (CA), exposure of A. ochraceus to the gaseous phase of malt yeast glucose peptone (MYGP) plates inoculated with P. anomala, P. kluyveri and H. uvarum inhibited fungal growth, with the two Pichia spp. showing the strongest effect. The main esters and alcohols produced by the three yeasts were ethyl acetate, isobutyl acetate, 2-phenyl ethyl acetate, ethyl propionate and isoamyl alcohol. The individual esters and alcohols were found to affect fungal growth. The most effective compound in inhibiting fungal growth was 2-phenyl ethyl acetate; which at 48 microg/l headspace completely inhibited growth of A. ochraceus. Exposure of A. ochraceus to the gaseous phase of MYGP plates inoculated with P. anomala, P. kluyveri and H. uvarum prevented production of OTA. On CA medium, only the headspace of P. anomala and P. kluyveri prevented OTA production. Furthermore, when A. ochraceus was exposed to the headspace of the individual volatile compounds, 2-phenyl ethyl acetate was the most effective in preventing OTA production. Prevention of OTA seems to be due to reduction of fungal biomass.     

Identification of fungal contamination and determination of mycotoxigenic molds by micellar electrokinetic capillary chromatography in smoked paprika

The purpose of this work was to analyze the fungal contamination in smoked and unsmoked paprika processed from different cultivars of pepper and to investigate the ability of these and other mycotoxigenic molds to grow and synthesize mycotoxins in smoked paprika. Eighteen mycotoxins were evaluated using micellar electrokinetic capillary chromatography. No relevant differences were found in fungal contamination between smoked and unsmoked paprika. The number of yeasts obtained was low, ranging from 0.4 to 3.29 log CFU g(-1); most of the yeast strains were identified as Cryptococcus spp. followed by Candida spp. All mold counts were <4 log CFU g(-1). Aspergillus, Cladosporium, Penicillium, and Fusarium were the predominant hyphomycete genera. Six mycotoxins were identified in the extracts of several strains isolated from paprika and incubated on malt extract agar. Penicillium expansum followed by Penicillium citrinum and Penicillium raistrickii were the dominant mycotoxigenic fungi isolated. Most of themycotoxin-producing fungi produced detectable amounts of mycotoxins when grown on paprika agar.     

草莓致腐霉菌的鉴定及其产毒能力研究

目的调查草莓表面主要致腐真菌,并研究其是否产生毒素。方法对草莓真菌进行分离,综合利用菌落形态学观察、显微生殖观察、真菌ITS序列系统发育学分析鉴定真菌;利用高效液相色谱法检测腐烂草莓及真菌发酵液中赭曲霉毒素A(ochratoxin A,OTA)的含量。结果 4种毛霉属真菌和1种青霉属真菌可引起草莓腐烂,经分子鉴定分别为易脆毛霉菌、卷枝毛霉菌、多枝毛霉菌、根毛霉菌和草酸青霉菌。腐烂的草莓和毛霉发酵液检测不出OTA,但青霉菌发酵液中检测出OAT毒素存在。结论真菌是致草莓腐烂的主要诱因,毛霉菌在腐烂过程中占主导地位,但产毒素的青霉菌与其共生,应加强草莓及制品的风险监测。     

Effects of the volatile organic compounds produced by Enterococcus spp. strains isolated from maize grain silos on Fusarium verticillioides growth and fumonisin B1 production

Fusarium verticillioides is a phytopathogenic fungus that can contaminate maize grain silos and result in important losses in the post-harvest product. The objective of this work was to investigate the effects of volatile organic compounds produced by four lactic acid bacterial strains isolated from maize grain silos on F. verticillioides M3125 growth and fumonisin B₁ (FB₁) production. The bacterial isolates 55 and 49 were identified as Enterococcus faecium and M4A and M4G as Enterococcus casseliflavus. The fungal growth was inhibited by 33.33% by the volatiles released by the M4A strain and by approximately 10% by the volatiles emitted by the 55 and 49 strains. The volatiles produced by the M4A strain also significantly reduced (88.75%) FB₁ biosynthesis. The gas chromatography–mass spectrometer analysis identified 21 volatile organic compounds, with diacetyl, acetic acid and acetoin being the main volatiles emitted by the four bacterial strains. Acetoin was the volatile produced in the highest proportion by the four strains, with M4A generating the highest proportion of diacetyl (35.11%). Diacetyl and acetic acid completely inhibited fungal growth at concentrations of 0.3 and 1 mM, respectively, while acetoin promoted fungal growth. Only acetoin significantly reduced FB₁ production. These results showed that diacetyl was the main compound involved in fungal inhibition, while the effect on FB₁ production could have been due to the combination of the volatile organic compounds produced by the M4A strain. In conclusion, the volatiles emitted by the E. casseliflavus M4A strain could be a promising tool for the biocontrol of F. verticillioides in storage maize grain silos.     

Antifungal activity of Mexican oregano (Lippia berlandieri Shauer)

Antifungal and sensorial properties of spices have been recognized for years. The antifungal compounds are products of the plant's secondary metabolism, and the action of those compounds could be used to inhibit the growth of spoilage and pathogenic microorganisms in food. Mexican oregano (Lippia berlandieri) grows wildly in the desert zone of Mexico and is usually added to regional foods. The goal of this study was to evaluate the antifungal activity of Mexican oregano versus food-contaminant fungi. Fungi were isolated from spoiled fruit and vegetables and identified according to morphological characteristics. The antifungal activity of oregano was evaluated by radial growth measurement on potato dextrose agar added with dried oregano (0.25 to 4.0%). The essential oil antifungal activity of oregano was also evaluated by the diffusion well test. Twenty-one fungal strains were isolated, which included Penicillium, Geotrichum, Aspergillus, and Bipolaris. In seven of the 21 strains, no inhibitory effect was observed at either concentration of oregano. An increase in growth at the lower or higher concentrations of oregano, when compared to the control, was observed in two fungal strains; in 12 strains, a strong inhibitory effect of oregano was evident. The oregano essential oil was inhibitory to all fungal strains, but there were differences in the extent of the effect. Although the antifungal effect of oregano is strongly established, there was a differential effect with the fungal strains studied. Besides pathogenic fungi and bacteria, microbial spoilage flora should be considered when the addition of spices for food preservation is proposed.     

Production of volatile compounds by Rhizopus oligosporus during soybean and barley tempeh fermentation

Rhizopus oligosporus Saito can ferment soybeans or cereal grains to tempeh, a sliceable cake with improved nutritional properties. Volatiles produced by different R. oligosporus strains grown on malt extract agar (MEA), barley and soybean were investigated. The effect of co-cultivation with Lactobacillus plantarum on the production of volatiles was also studied. Volatile compounds were collected in situ by headspace diffusion and identified by GC-MS. The ten R. oligosporus strains that had different colony morphologies on MEA produced very similar volatile profiles, except for slight variations among the minor volatile compounds (e.g. sesquiterpenes). Likewise, practically no differences in volatile profiles were observed between three of the strains grown on soybeans. In contrast, the R. oligosporus volatile profile on soybean was different from that on barley from the same strain. Co-cultivation with L. plantarum did not influence volatile production by R. oligosporus. The dominant compounds produced on all three substrates were ethanol, acetone, ethyl acetate, 2-butanone, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-methyl-1-butanol. Acetaldehyde and 2-methyl-propanal were also produced on MEA and barley, while 2-pentanone, methyl acetate, 2-butanol and 3-methyl-3-buten-1-ol were observed on soybeans. Ethanol, 2-methyl-1-butanol and 3-methyl-1-butanol were the most abundant volatile compounds produced on MEA and barley, while 2-butanone was the dominant volatile metabolite on soybean. The mushroom odour compounds, 3-octanone and 1-octen-3-ol, were only detected from soybean and soybean tempeh.     

Volatile fungal metabolites and their relation to the spoilage of agricultural commodities

Abstract

Spoilage of agricultural commodities, mainly grains, due to fungal deterioration is a significant source of their loss. Fungi invading stored commodities influence their quality causing decrease in germination, dry matter loss, heating, and off‐odor formation. They can also produce mycotoxins ‐ metabolites harmful to animals and humans. Volatile metabolites produced by fungi can be characteristic markers of fungal presence. This paper reviews proposed methods for fungal contamination assessment based on volatile metabolites, together with literature on volatile metabolites produced by fungi belonging mainly to three genera: Aspergillus, Penicillium, and Fusarium being the most important in grains and other agricultural commodities spoilage.     

山东省部分地区2018年收获玉米的真菌污染状况研究

目的 对山东省部分地区2018年产玉米表面污染真菌进行调查。方法 在马铃薯葡萄糖培养基(potato dextrose agar, PDA)上通过稀释涂布的传统分离培养方法获得真菌菌株, 利用传统的形态学与分子生物学技术相结合的方法对真菌种类鉴定。结果 共获得真菌菌株169株, 经形态学鉴定隶属于8属: 镰刀菌属(Fusarium), 青霉属(Penicillum), 曲霉属(Aspergillus), 交链孢属(Alternaria), 枝顶孢属(Acremonium), 梗霉属(Lichtheimia), 枝孢属(Cladosporium)、篮状菌属(Talaromyces)。其中, 镰刀菌(Fusarium)、青霉(Penicillum)、曲霉(Aspergillus)分离获得的数量最多, 镰刀菌种类是引起玉米穗腐病的主要病原菌, 其污染主要来自于田间。随玉米仓储时间延长, 优势真菌改变为青霉属和曲霉属。结论 山东地区玉米真菌污染初期以田间污染为主, 储藏过程中优势菌群受环境影响发生改变; 需要根据真菌污染状况改进提升传统果穗囤藏技术。     

Emission patterns of wound volatile compounds following injury of ripe strawberry fruit

Volatile compounds emitted from plants following wounding influence the growth of fungal pathogens, including Botrytis cinerea (grey mould), which causes serious economic loss of strawberry fruit. In the present study the patterns of volatile compound emission from ripe strawberry fruit, comprising store‐purchased fruit and two known cultivars, were determined following injury, using solid phase microextraction analysis to sample a stream of air continuously flowing over the fruit. The principal compounds produced from all fruits comprised C₆ aldehydes, alcohols and acetate and butyrate esters derived from the lipoxygenase–hydroperoxide lyase pathway. There was an initial burst of production of aldehydes, including t‐2‐hexenal, following wounding and then a gradual decline, which in one cultivar continued for 3 h. t‐2‐Hexenal is of special interest, since it has been shown to inhibit or promote Botrytis growth in vitro depending on concentration. The acetate esters t‐2‐hexenyl acetate, c‐3‐hexenyl acetate and hexyl acetate were major wound compounds released by the fruit and were produced for 3 h after wounding. The production of relatively large quantities of wound‐derived esters is markedly different from the patterns observed for most vegetative tissue. Knowledge of the wound compounds produced by strawberry fruit and their profiles of emission after injury provides a basis for studies of the potential roles of these compounds in pathogen development on fruit. © 2003 Society of Chemical Industry     

The effect of substrate on mycotoxin production of selected Penicillium strains

Analytical methods are presented for detecting simultaneously 11 fungal metabolites (aflatoxins B1, B2, G1 and G2, citrinin, cyclopiazonic acid, mycophenolic acid, ochratoxin A, penicillic acid, penitrem A and roquefortine C) on different matrices. The methods were applied to determine the mycotoxins produced by different Penicillium crustosum, Penicillium nordicum and Penicillium verrucosum strains on yeast extract sucrose (YES) agar and cheese and bread analogues and are based on high-performance liquid chromatography (HPLC) and photodiode array detection (PDA). The growth substrate had a distinctive effect on the mycotoxin production ability of the fungi examined. The P. crustosum strains produced roquefortine C on all the substrates, with the highest amounts being detected on the cheese analogue. Penitrem A was synthesised on the cheese analogue only. The strains of P. verrucosum produced exclusively citrinin on YES, but both ochratoxin A and citrinin were detected in considerable amounts on the bread analogue. On the bread, toxin profiles varied significantly between the individual P. verrucosum strains. The cheese analogue was not favourable for the mycotoxin production of this species. The growth substrate had the least effect on the toxin production of the P. nordicum strains, which synthesised ochratoxin A in moderate amounts on all three media.     

Antifungal effect of dairy propionibacteria--contribution of organic acids

Large amounts of food and feed are lost every year due to spoilage by moulds and yeasts. Biopreservation, i.e. the use of microorganisms as preservatives instead of chemicals, has gained increased interest. Lactic acid bacteria and propionibacteria might be particularly useful due to their important role in many food fermentations. Knowledge of the antifungal effects of the organic acids produced by these bacteria is necessary to understand their inhibitory activity. We evaluated the antifungal activity of the type strains of five dairy propionibacteria, Propionibacterium acidipropionici, P. jensenii, P. thoenii, P. freudenreichii subsp. freudenreichii and P. freudenreichii subsp. shermanii against eight food- and feedborne moulds and yeasts. A dual culture system assayed the inhibitory activity on three different agar media, sodium lactate (SL), de Man Rogosa Sharp (MRS) and MRS without acetate (MRS-ac). The amounts of organic acids produced during growth of propionibacteria in liquid SL, MRS and MRS-ac were also determined. The minimal inhibitory concentration (MIC) values of propionic, acetic and lactic acid were established for all fungi at pH 3, 5 and 7. Propionic acid, followed by acetic acid, was the most potent antifungal acid. Inhibition at pH 7 generally required concentrations above 500 mM for all three acids, at pH 5 the MIC values for propionic and acetic acids were 20-120 mM and above 500 mM for lactic acid. At pH 3, the MIC values were, with one exception, below 10 mM for both propionic and acetic acid and above 160 mM for lactic acid. The yeast Pichia anomala was the fungus most resistant to organic acids. The propionibacteria exhibited a pronounced species variation in antifungal activity on MRS (+/-acetate) agar, with P. thoenii being the most potent. Four of the five propionibacteria species produced more propionic and acetic acid in liquid SL medium than in MRS (+/-acetate) broth. However, when SL agar was used as the growth medium, none of the propionibacteria inhibited fungal growth.     

Physicochemical and volatile profile alterations in pasteurized and frozen strawberry pulp during storage

This study evaluated the physicochemical characteristics and volatile profile of strawberry pulp subjected to factors, pasteurization (unpasteurized and pasteurized), freezing method (static air and forced air), and their interactions, during 12 months. Strawberry fruit were washed, sanitized, and pulped. The pulp was packaged, pasteurized, and frozen (0, 2, 4, 6, and 12 months). We concluded that pasteurization alters the strawberry pulp color. The impact of pasteurization and freezing method on the strawberry pulp pH, titratable acidity, and soluble solids variables is negligible. We tentatively identified 13 volatile compounds in fresh fruit and pulp, ethyl acetate, ethyl hexanoate, and linalool being the volatiles with the highest area percentage in the two products. The esters were predominant in both the fruit and strawberry pulp. Time is the most determining factor in modifying the strawberry pulp volatile profile, having an isolated effect on the increase of ethanol, ethyl butanoate, and linalool.

Practical applications

The strawberry is very used worldwide as raw material to different products, due to its color, flavor, and aroma. Thus, obtaining further information about the changes caused by processing and storage is of great importance for the food industry. This study shows the effect of simultaneous application of different processing techniques (pasteurization and freezing methods) on physicochemical variables and volatile profile of strawberry pulp over prolonged storage. Significant and unpublished results involving color, pH, acidity, soluble solids, and volatile compounds of strawberry frozen pulp over storage period are presented and may be applied for food industry to keep the pulp quality.     

Naturally occurring phenols: a detoxification strategy for fumonisin B1

Phenolic compounds from plants offer a means for both the prevention and detoxification of mycotoxins that affect human health. This research investigates the control of fungal growth and toxin production by Fusarium verticillioides with plant phenolic compounds, namely chlorophorin, iroko and maakianin, benzoic acid, caffeic acid, ferulic acid, and vanillic acid. Inhibition by these compounds of fungal growth was determined by the agar overlay method and their effect on fumonisin B₁ (FB₁) production was determined by high-performance liquid chromatography. Chlorophorin was the most effective compound in inhibiting fungal growth, followed by iroko, maakianin, vanillic acid and caffeic acid. Chlorophorin also was the most effective compound in reducing toxin production (94% reduction), followed by caffeic acid, ferulic acid, vanillic acid and iroko, which reduced FB₁ levels by 90-91%. The widespread occurrence of fumonisins world-wide and the lack of adequate prevention of fumonisins require 'biologically safe' alternatives to prevent the transfer of fungi and their health hazardous toxins into our daily foods and environment.     

产香真菌M6-5的鉴定及其挥发性物质对库尔勒香梨果实采后黑斑病的抑制效果

通过形态学和分子生物学（ITS序列和tef1序列）鉴定产香真菌M6-5,采用平板对扣法测定不同生长时期的M6-5菌株对库尔勒香梨黑斑病菌链格孢菌（Alternaria alternata）的抑菌活性,采用顶空固相微萃取和气相色谱-串联质谱法检测分析其挥发性物质,以纯品形式验证并确定有效抑菌成分;使用干燥器分析M6-5菌株产生的挥发性物质对库尔勒香梨果实采后黑斑病的抑制效果。结果表明：菌株M6-5为猬木霉（Trichoderma erinaceum）,生长第5天的菌株M6-5产生的挥发性物质对链格孢菌的抑制率达到最大值,为（82.48±1.17）%,且持续期为20 d;从生长第5天的M6-5菌株中共检测出65 个组分,主要为6-戊基-2H-吡喃-2-酮、3-羟基丁酸乙酯、苯乙醇、2-烯丙基呋喃、棕榈酸甲酯等,分别占挥发性物质总量的40.12%、6.49%、4.38%、4.13%和3.48%。在挥发性物质纯品含量为714.28 μL/L时,6-戊基-2H-吡喃-2-酮、3-羟基丁酸乙酯和苯乙醇对链格孢菌的抑制率分别为（21.27±0.60）%、（41.43±2.36）%、（50.23±1.07）%。M6-5菌株产生的挥发性物质能够显著降低库尔勒香梨果实黑斑病的病斑直径,比对照组降低了42.46%。本研究可为库尔勒香梨采后黑斑病的防治提供一定参考。     

海洋真菌产酶能力的研究

从大连丽娇湾和金石滩采集获得海水、海藻等样品,采用PDA培养基分离海洋真菌,并对产酶真菌进行筛选及鉴定,及其产酶能力进行初步研究。结果显示,共筛选得到真菌32株,产酶菌株18株,其中有1株同时产4种酶,3株同时产3种酶,8株同时产2种酶。初步鉴定18株产酶菌分布于4个属,其中优势属为曲霉属（Aspergillus sp.）和青霉属（Penicillium sp.）,各6株,均占总筛选产酶菌株数的33.33%,其次为酵母属（Saccharomyces sp.）,共4株,占总筛选产酶菌株数的22.22%。11株产植酸酶、10株产纤维素酶、6株产淀粉酶、5株产蛋白酶、3株产脂肪酶。     

剁椒山笋后熟过程中微生物与挥发性风味物质的相关性分析

采用高通量测序技术和顶空固相微萃取-气相色谱-质谱联用技术测定剁椒山笋后熟过程中微生物多样性和挥发性风味物质的变化,并分析了两者之间的潜在相关性。结果表明：刺盘孢属（Colletotrichum）、青霉菌属（Penicillium）、黄单胞菌属（Xanthomonas）、欧文氏菌属（Erwinia）、劳尔氏菌属（Ralstonia）、假单胞菌属（Pseudomonas）、片球菌属（Pediococcus）和肠杆菌属（Enterobacter）在剁椒山笋后熟贮藏过程中占据主导优势。在发酵过程中共检测到71种挥发性化合物,总含量从406.57μg/kg上升至900.08μg/kg。通过主成分分析得出剁椒山笋样品在后熟初期与末期分离明显。由正交偏最小二乘判别分析变量投影重要性大于1筛选出28种重要代谢物。Pearson相关性分析表明,细菌对风味的影响大于真菌,且优势菌与多数风味物质呈正相关。本研究将为发酵辣椒制品后熟贮藏过程中风味品质的改善提供理论基础。     

Contribution of enterococci to the volatile profile of slightly-fermented sausages

The volatile profiles of slightly-fermented sausages inoculated with eight different enterococci strains were determined and compared with a fermented sausage manufactured without any inoculation. The volatile profile was determined by solid-phase microextraction (SPME) and gas chromatography coupled to mass spectrometry (GC/MS). A total of 121 volatile and semi-volatile compounds were identified in the samples of the final products (day 21). Terpenes from spices added during manufacture of dry-fermented sausages were the main group of compounds in the volatile profile. Other groups included alcohols (18% of total volatile compounds), aldehydes (16%), alkanes and alkenes (12%), and ketones (10%). These are derived from lipids, proteins or carbohydrates and formed during fermentation and ripening. In terms of global volatile composition, the batches inoculated with enterococci strains were similar to those obtained for non-inoculated batch. However, some quantitative differences in the volatile profiles of different batches were found, mainly in the compounds formed from amino acid catabolism (i.e.: propanol, isoamyl), from free fatty acid oxidation (i.e.: hexanal, hexanol), or from carbohydrate fermentation (i.e.: 2-butanone, acetoin). In comparison with non-inoculated sausages, some batches were more resistant to oxidation; they contained fewer volatile compounds arising from lipid oxidation. The use of different selected enterococci strains did not alter the volatile profile of slightly-fermented sausages. The enterococci strains provide similar volatile compounds to those usually reported in the literature.