响应面法优化啤酒有害菌检测培养基

为了提高啤酒有害菌的检出率和检测的灵敏性,采用Plackett-Burman设计法和响应面分析法(Response Surface Analysis)对CNFF-A培养基组分和配比进行优化。先用Plackett-Burman设计从7个因子中筛选出对菌落数有显著影响的因素,再用最陡爬坡试验及Box-Behnken设计进一步优化。结果表明,蜂蜜、叶酸和乙酸钠是影响菌落数的显著因素,优化后的培养基配方为:蜂蜜0.1g/L、牛肉浸粉7.0g/L、酵母浸出物5.0g/L、精氨酸1.0g/L、叶酸2.0g/L和乙酸钠1.5g/L。此条件下,培养基对啤酒有害菌的检出率大大提高,且检测时间缩短了24h。     

An improved plate culture procedure for the rapid detection of beer‐spoilage lactic acid bacteria

Lactic acid bacteria (LAB) are the most frequently encountered beer‐spoilage bacteria, and they can render beer undrinkable owing to the production of lactic acid, diacetyl and turbidity. Three beer‐spoilage strains, 2011–6, 2011–8 and 2011–11, were isolated from finished beers. Based on the 16S rRNA sequence analysis, these three isolates were identified as Lactobacillus acetotolerans. Only the horA homologue was detected in these strains, while the horC homologue was not detected. In addition, an improved plate culture method for the rapid detection of beer‐spoilage LAB by the addition of catalase was evaluated. Supplementation with catalase enhanced the growth and colony sizes of the spoilage LAB investigated. These beer‐spoilage bacteria, including some slowly growing strains, were detected within five days of incubation using the modified method. Taken together, the modified procedure could be a rapid countermeasure against beer‐spoilage LAB, and it compared favourably with the conventional plate culture method. Copyright © 2014 The Institute of Brewing & Distilling     

The impact of different hop compounds on the growth of selected beer spoilage bacteria in beer

Beer spoiling lactic acid bacteria are a major reason for quality complaints in breweries around the world. Spoilage by a variety of these bacteria can result in haze, sediment, slime, off-flavours and acidity. As these bacteria occur frequently in the brewing environment, using certain hop products that inhibit the growth of these spoilers could be a solution to prevent problems. To investigate the impact of seven different hop compounds (α-acids, iso-α-acids, tetrahydro-iso-α-acids, rho-iso-α-acids, xanthohumol, iso-xanthohumol and humulinones) on the growth of six major beer spoilage bacteria (Lactobacillus brevis. L. backi, L. coryniformis, L. lindneri, L. buchneri, Pediococcus damnosous), two concentrations (10 and 25 mg/L) of each hop substance were added to unhopped beer. The potential growth of the spoilage bacteria was investigated over 56 consecutive days. A comparison of the results shows a strong inhibition of growth of all spoilage bacteria at 25 mg/L of tetrahydro-iso-α-acids closely followed by α-acids as the second most inhibitory substance. The results showed a high resistance of L. brevis to all hop compounds as well as an inhibition of L. coryniformis and L. buchneri at low concentrations of most hop components. In comparison with the control sample, L. lindneri showed increased growth in the presence of some hop compounds (rho-iso-α-acids, xanthohumol, iso-xanthohumol, humulinones). © 2020 The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

Detection of culturable and viable but non‐culturable cells of beer spoilage lactic acid bacteria by combined use of propidium monoazide and horA‐specific polymerase chain reaction

Current methods of detecting beer spoilage lactic acid bacteria (LAB) are time‐consuming and do not differentiate between viable and non‐viable bacteria. In this study, a combination of the conventional polymerase chain reaction (PCR) and propidium monoazide (PMA) pretreatment has been described to circumvent the disadvantages. The horA‐specific PMA‐PCR described here identifies beer spoilage LAB based not on their identity, but on the presence of a gene that is shown to be highly correlated with the ability of LAB to grow in beer. The results suggest that the use of 20 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable, but putatively non‐culturable (VPNC) Lactobacillus acetotolerans. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. acetotolerans cells was 1.5 µg/mL. The detection limit of established PMA‐PCR assays was found to be 100 VPNC cells/reaction for the horA gene. Furthermore, the horA‐specific PMA‐PCR assays were subjected to 18 reference strains, representing 100% specificity with no false positive amplification observed. In conclusion, the use of horA‐specific PMA‐PCR allows for a substantial reduction in the time required for the detection of potential beer spoilage LAB and efficiently discriminates between live and dead cells. Copyright © 2016 The Institute of Brewing & Distilling     

High‐pressure homogenization: a non‐thermal process applied for inactivation of spoilage microorganisms in beer

The inactivation of spoilage microorganisms in beer using high‐pressure homogenization (HPH) was studied with the aim of evaluating the possibility of changing the conventional pasteurization process using this particular process. The homogenization pressure required for the inactivation of lactic acid bacteria, acetic bacteria and yeasts was investigated. For the most resistant microorganisms, the pressure inactivation kinetics and the effects of multiple process passes, initial temperature of the beer and the CO₂ concentration were studied. The results indicated that Lactobacillus delbrueckii was the most resistant microorganism tested, requiring 250 MPa to reach a six decimal reduction. Additionally, results showed that L. delbrueckii inactivation followed a second‐order kinetic process. A multi‐pass process and the use of a high initial beer temperature increased inactivation by HPH with L. delbrueckii, allowing the use of 150 MPa to achieve a five log cycle of inactivation. In contrast, a high CO₂ concentration reduced the efficacy of the HPH process. The results that were obtained are useful for high‐pressure homogenization applications in breweries and help to elucidate the effect of this new technology in a beverage that is both alcoholic and carbonated. Copyright © 2013 The Institute of Brewing & Distilling     

Investigation of beer‐spoilage ability of Dekkera/Brettanomyces yeasts and development of multiplex PCR method for beer‐spoilage yeasts

There have been many beer‐spoilage incidents caused by wild yeasts. Saccharomyces cerevisiae, Dekkera anomala and D. bruxellensis have been recognized as beer‐spoilage yeasts in the brewing industry. In contrast, the beer spoilage ability of Brettanomyces custersianus has not been well characterized, although this species was isolated from beer. In this study, the beer‐spoilage ability of currently described Dekkera/Brettanomyces yeast species was investigated. As a consequence, D. anomala, D. bruxellensis and B. custersianus were shown to grow in commercial beers. On the other hand, the remaining two Brettanomyces species, B. naardenensis and B. nanus, did not grow in beer. These results indicate that B. custersianus should be recognized as a beer‐spoilage species, in addition to S. cerevisiae, D. anomala, and D. bruxellensis. Therefore we developed multiplex polymerase chain reaction (PCR) for the simultaneous detection and identification of B. custersianus and the other beer‐spoilage yeast species. For this purpose, PCR primers were designed in the internal transcribed spacer region or 26S rDNA, and each PCR product was made in different sizes to easily discriminate the species from electrophoretic results. Specificity, reactivity and sensitivity of the designed primers were evaluated. As a result, the developed multiplex PCR method was shown to have high specificity and reactivity, and therefore was considered as an effective tool to identify beer‐spoilage yeast species. This tool can contribute to microbiological quality assurance in breweries. Copyright © 2015 The Institute of Brewing & Distilling     

Development of a real‐time multiplex PCR system for the quantitative detection of Chinese GM rice

BACKGROUND: Rice is one of the most important staple food crops for more than half the global population, who rely on it for as much as 80% of their diet. By one estimate, the world population is projected to grow to approximately 11 billion people by the year 2050. So it is a formidable task to meet the future demand. For this reason, breeders make a great deal of effort to produce new rice varieties with traits such as higher yield, improved nutritional content and better resistance to disease and pests, via transgenic biotechnological protocols. Dozens of transgenic rice lines have been developed since the first transgenic rice plant production in the late 1980s. With the rapid approach of transgenic rice commercialisation, it is becoming necessary to develop techniques capable of detecting and quantifying genetically modified (GM) rice. RESULT: Here we describe a method in which transgenic DNA is quantified by amplifying part of the 35S‐CaMV promoter and standardising it against an amplified portion of an endogenous single copy, rice specific gene encoding sucrose phosphate synthase. Both reactions are performed simultaneously in a single tube. Standard calibration curves were developed by diluting DNA extracted from a blend of non‐transgenic (c.v., Nipponbare) and 5% KMD2 transgenic rice. The method was tested for the quantification of the five GM rice events, including KMD2, Wan 21A, GC‐1, H1597 and TR4, which contain the 35S‐CaMV promoter. The coefficient of variation varied from 3.15% to 12.84%, which is up to acceptance criterion over the dynamic range of the method. CONCLUSION: In this study, we successfully applied a multiplex real‐time PCR assay to GM rice, which employed SPS as the endogenous reference gene and the gene regulation element 35S‐CaMV promoter as a GMO marker. The detection limit and limit of quantification is sufficient to comply with all relevant regulations in the EU and worldwide. The detection system could be applied in routine analysis for the quantification of GM rice in food materials, such as instant rice, unpolished rice, rice flours, biscuit powders, and starch. It may prove useful with regard to a robust screening technique of broad utility as transgenic rice enters global commodity markets. Copyright © 2009 Society of Chemical Industry     

Detecting adulteration of Dendrobium officinale by real‐time PCR coupled with ARMS

The high commercial value of ‘Tiepi Fengdou’ presents the constant risk of adulteration with cheaper Dendrobium products. Therefore, a novel method, combining SYBR Green II‐based real‐time PCR (rt‐PCR) with amplification refractory mutation system (ARMS), was established. By performing the diagnostic rt‐PCR assay, a 109‐bp fragment was specifically amplified from the internal transcribed spacer region of D. officinale. The difference between the average cycle threshold (C_t) value of ‘Tiepi Fengdou’ (20.48) and the mean of those of all reference Dendrobium products (31.83) was statistically significant (P < 0.001). The results indicate that the established system can be able to rapidly and unequivocally discriminate ‘Tiepi Fengdou’ from its adulterants.     

Combined effects of matrix and gene marker on the real‐time PCR detection of wheat

DNA‐based methods have been advanced as excellent alternatives for the detection and quantification of gluten‐containing cereals. In this study, it was intended to evaluate the effect of three distinct food matrices on the performance three TaqMan real‐time PCR approaches targeting α2‐gliadin, agglutinin isolectin (Tri a 18) and thioredoxin h(Tri a 25) genes of wheat. The results of wheat flour detection in soya bean, maize and rice model matrices evidenced that sensitivity was considerably affected by both the food matrix and the target gene. Rice matrix allowed the highest sensitivity, in opposition to soya bean. The α2‐gliadin method enabled the best sensitivity, especially when combined with rice matrix (5 mg kg^?1), followed by maize (10 mg kg^?1) and soya bean (50 mg kg^?1). These findings suggest that food matrix effects need to be carefully evaluated when developing real‐time PCR assays for wheat detection/quantification, but without compromising their great effectiveness as tools to monitor gluten‐containing cereals.     

Effect of bacteriocin‐producing Pediococcus acidilactici K10 on beer fermentation

Beer is generally considered to be a beverage that has high microbiological stability. However, some undesirable lactic acid bacteria (LAB) can grow in beer and consequently spoil this beverage. In this study, bacteriocin‐producing Pediococcus acidilactici K10 was used as a means of bio‐acidifying the mash and reducing the spoilage LAB content of the beer. The K10 strain had antimicrobial activity against two beer spoilage LAB strains in wort and did not grow in a beer environment. The K10 strain was inoculated before the mashing step. The effect of K10 as a starter culture was investigated and compared with a control. As a result, filtration time was shortened by 17 min, alcohol content was increased by 137%, foam stability was increased by 156%, bitterness was increased by two bitterness units and there was a significant difference (p < 0.05) in aromatic and sour odour. The feasibility of using bacteriocin‐producing LAB strain in beer brewing is envisaged. Copyright © 2016 The Institute of Brewing & Distilling     

Screening and construct‐specific detection methods of transgenic Huafan No 1 tomato by conventional and real‐time PCR

Genetically modified (GM) tomatoes have been approved for commercialization in many countries since the first GM tomato FLAVR SAVR was permitted for planting in 1994. In China, GM tomato Huafan No 1 with a character of long shelf‐life was the first GM plant which was approved for commercialization in 1996. To meet the requirement of the GM tomatoes labeling policy that has been actualized in China since 2001, screening and construct‐specific PCR detection methods for detecting the universal elements transformed into tomato, such as cauliflower mosaic virus 35S (CaMV35s) promoter and the nopaline synthase (NOS) terminator of Agrobacterium tumefaciens, and the specifically inserted heterologous DNA sequence between CaMV35s promoter and anti‐sense ethylene‐forming enzyme (EFE) gene were set up. To make the detection methods normative, a novel single copy tomato gene LAT52 was also used as an endogenous reference gene in the PCR detection systems. The limit of detection of screening and construct specific detection methods for Huafan No 1 was 68 haploid genome copies in conventional PCR detection, and three copies in TaqMan real‐time PCR detection. The limit of quantitation of screening quantitative PCR assays for Huafan No 1 was three copies and was 25 copies for construct‐specific quantitative PCR. Two samples with known Huafan No 1 tomato content were detected using the established conventional and real‐time PCR systems, and these results also indicated that the established Huafan No 1 screening and construct‐specific PCR detection systems were reliable, sensitive and accurate. Copyright © 2005 Society of Chemical Industry     

Development of a real‐time PCR for quantification of the family Syntrophomonadaceae in pit mud from Chinese Luzhou‐flavour liquor distilleries

Syntrophomonadaceae was previously identified to be the predominant bacteria in Chinese Luzhou‐flavour liquor pit mud. In this work, a real‐time quantitative PCR assay was first employed to monitor the variation of the family Syntrophomonadaceae in the pit mud. The specific primers SynF and SynR were designed based on the 16S rDNA gene. A single specific fragment of about 161 bp was amplified in each positive sample, while no signal or a high cycle threshold (Cq) value (>36) was detected in the negative controls. The correlation coefficient (R²) and amplification efficiency (E) of the standard curve were 0.9997 and 106%, respectively. In addition, the sensitivity was 6.0 × 10² copies/μL and the mean recovery rate was 103%. All of these results indicate that the method is rapid, specific, sensitive and accurate. The quantitative results provide further understanding of the time differences and spatial differences in the family Syntrophomonadaceae in pit mud. Copyright © 2015 The Institute of Brewing & Distilling     

Identification of pork genome in commercial meat extracts for Halal authentication by SYBR green I real‐time PCR

The identification of pork DNA in meat extracts is very important for Halal authentication and Muslim consumers demand protection from falsely labelled meat products. A pig‐specific SYBR green I real‐time PCR assay has been developed to address this issue. Using specific primers for pig mitochondrial DNA, successful amplification has been obtained by DNA extracted from control meat samples. With SYBR green I real‐time PCR, the specificity of the amplification was showed by Tm value. Detection limit of the real‐time PCR was down to 0.1 ng of porcine DNA. An appropriate linearity was obtained by construction of a standard curve based on Ct value and different concentrations of porcine DNA. By conventional PCR, no amplification was shown by porcine DNA less than 0.1 ng. The established method was conducted on commercially available meat extracts for detection and quantification of porcine DNA. The results showed the SYBR green I real‐time PCR could be considered a robust method for Halal authentication of meat extracts.     

食品中腐败酵母的实时荧光PCR鉴定

为研究食品中腐败酵母实时荧光PCR快速鉴定方法,设计和筛选出了可用于腐败酵母鉴定的多条探针和引物,并建立了针对酿酒酵母、鲁氏接合酵母、斯巴达克毕赤酵母和布鲁塞尔德克酵母等4种腐败酵母菌的实时荧光PCR鉴定方法。用文中建立的方法对从糕点、蜂蜜、饮料等市售食品中分离出的60余株酵母菌进行了鉴定,发现其中4株为酿酒酵母,21株为鲁氏酵母,其他为与上述4种不同的酵母。以实时荧光PCR方法鉴定酵母,全过程仅需约3 h,与常用的生化鉴定方法相比,简化了鉴定步骤,提高了鉴定准确性,缩短了鉴定时间。