The Occurrence of Beer Spoilage Lactic Acid Bacteria in Craft Beer Production

Beer is one of the world's most ancient and widely consumed fermented alcoholic beverages produced with water, malted cereal grains (generally barley and wheat), hops, and yeast. Beer is considered an unfavorable substrate of growth for many microorganisms, however, there are a limited number of bacteria and yeasts, which are capable of growth and may spoil beer especially if it is not pasteurized or sterile‐filtered as craft beer. The aim of this research study was to track beer spoilage lactic acid bacteria (LAB) inside a brewery and during the craft beer production process. To that end, indoor air and work surface samples, collected in the brewery under study, together with commercial active dry yeasts, exhausted yeasts, yeast pellet (obtained after mature beer centrifugation), and spoiled beers were analyzed through culture‐dependent methods and PCR‐DGGE in order to identify the contaminant LAB species and the source of contamination. Lactobacillus brevis was detected in a spoiled beer and in a commercial active dry yeast. Other LAB species and bacteria ascribed to Staphylococcus sp., Enterobaceriaceae, and Acetobacter sp. were found in the brewery. In conclusion, the PCR‐DGGE technique coupled with the culture‐dependent method was found to be a useful tool for identifying the beer spoilage bacteria and the source of contamination. The analyses carried out on raw materials, by‐products, final products, and the brewery were useful for implementing a sanitization plan to be adopted in the production plant.     

Investigation of Microbial Communities of Chinese Sourdoughs Using Culture‐Dependent and DGGE Approaches

Sourdough is typically characterized by the complex microbial communities mainly comprising of yeasts and lactic acid bacteria (LAB). The objective of this study was to explore the microbiota of Chinese traditional sourdoughs collected from different areas of China using culture‐dependent and denaturing gradient gel electrophoresis (DGGE) methods. A total of 131 yeasts, 2 molds, and 106 LAB strains were isolated and identified. Based on the culture‐dependent analysis, the populations of yeasts and LAB were at the level of 10⁵ to 10⁷ and 10⁶ to 10⁷ cfu/g, respectively. Similarly, the results of RT‐qPCR showed that the values of total yeasts and LAB populations were in the range of 10⁶ to 10⁷ and 10⁷ to 10⁸ copies/g, respectively. Using culture‐dependent method, a total of 7 yeasts, 2 molds and 7 LAB species were identified. Results showed that Saccharomyces cerevisiae and Lactobacillus plantarum were the predominant species among the yeasts and LAB microflora. Similarly, using PCR‐DGGE approach, 7 yeasts, 1 mold and 9 LAB species were detected. The yeast, S. cerevisiae, represented the predominant, while the yeast Candida tropicalis represented the subdominant species of the yeast community. Among the LAB community, Lactobacillus sanfranciscensis was the predominant species, while Lactococcus qarvieae, Enterococcus faecium, Lactobacillus delbrueckii and Enterococcus cecorum were among the less dominant species.     

Microbiological Analyses of Dry and Slurry Yeasts for Brewing

Classical microbiological methods in association with molecular methods (DNA amplification, Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) were used. These methods, developed to rapidly analyze microbial communities on the basis of sequence‐specific separation of DNA amplicons, allowed the detection of DNA differences in the amplicons tested and the identification of the strains analyzed by the comparison of unknown sequences with sequences of known species. TGGE allowed the comparison of the different Saccharomyces cerevisiae strains used in brewing while DGGE allowed the identification of lactic acid bacteria (LAB) in beer. These methods are a reliable tool for fast comparison of strains of Saccharomyces cerevisiae collected from different craft breweries where they were used as starters to check the presence of possible yeast contaminants in the brewing process and for rapid LAB identification.     

Diversity of Lactic Acid Bacteria Isolated from Brazilian Water Buffalo Mozzarella Cheese

The water buffalo mozzarella cheese is a typical Italian cheese which has been introduced in the thriving Brazilian market in the last 10 y, with good acceptance by its consumers. Lactic acid bacteria (LAB) play an important role in the technological and sensory quality of mozzarella cheese. In this study, the aim was to evaluate the diversity of the autochthones viable LAB isolated from water buffalo mozzarella cheese under storage. Samples were collected in 3 independent trials in a dairy industry located in the southeast region of Brazil, on the 28th day of storage, at 4 ºC. The LAB were characterized by Gram staining, catalase test, capacity to assimilate citrate, and production of CO₂ from glucose. The diversity of LAB was evaluated by RAPD‐PCR (randomly amplified polymorphic DNA‐polymerase chain reaction), 16S rRNA gene sequencing, and by Vitek 2 system. Twenty LAB strains were isolated and clustered into 12 different clusters, and identified as Streptococcus thermophilus, Enterococcus faecium, Enterococcus durans, Leuconostoc mesenteroides subsp. mesenteroides, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus. Enterococcus species were dominant and citrate‐positive. Only the strains of L. mesenteroides subsp. mesenteroides and L. fermentum produced CO₂ from glucose and were citrate‐positive, while L. casei was only citrate positive. This is the first report which elucidates the LAB diversity involved in Brazilian water buffalo mozzarella cheese. Furthermore, the results show that despite the absence of natural whey cultures as starters in production, the LAB species identified are the ones typically found in mozzarella cheese.     

Amino Acid Metabolism by Yeasts and Lactic Acid Bacteria during Bread Dough Fermentation

Metabolism of 22 free amino acids (AA) during fermentation(early and later steps) of wheat dough samples started with pure and associated cultures of yeast and lactic acid bacteria, and commercial compressed yeast (CY) were investigated. Unfermented and fermented straight doughs were studied byReversed-Phase HPLC of their dansyl derivatives. Used as starters were strains of Saccharomyces cervisiae, Lactobacillus brevis, Lactobacillus plantarum and Enterococcus faecium. Statistical data analysis indicated clustering of samples with yeast and samples without yeast (including uninoculated doughs) respectively, based on rate of metabolism of acidiC., basiC., aliphatic and aromatic A A. Differences in AA metabolism during the later fermentation step (DF2) categorized bacterial starters into groups according to balance between degree of assimilation(D F2<0) and exoproteolytic release(DF2>0) of amino acids.     

Bread making with Saccharomyces cerevisiae CEN.PK113‐5D expressing lipase A from Bacillus subtilis: leavening characterisation and aroma enhancement

Lipase A from Bacillus subtilis was expressed in the yeast Saccharomyces cerevisiae CEN.PK113‐5D strain as a cell wall‐immobilised enzyme. The recombinant yeast was used in bread making to test the effect of lipase A on the bread properties such as rheological and aromatic properties. The results were compared to the not transformed strain and the commercial baker's yeast. The recombinant strain resulted a good leavening agent comparable to the commercial baker's yeasts provided 1% sucrose was added to the dough. Whereas, during the leavening, the trend of the rheological properties (cohesivness, gumminess and rigidity) differed from the commercial and the nontransformed (NT) strains. Moreover, using of the recombinant strain enhanced total aroma which achieved in the baked product a value of 9 μg per g_product vs the 4 and 5 μg per g_product of the NT strain and the commercial baker's yeast, respectively. To our knowledge, this is the first time that a bacterial lipase is used in bread making.     

MICROBIOLOGICAL SHELF‐LIFE STUDIES ON COMMERCIALLY MANUFACTURED YEAST

ABSTRACT

During three replicate studies, cream, compressed and dry yeast samples were stored for 21 days at 4, 10, 25 and 37C. At 3‐day intervals, bacteria were quantified using standard plate counting procedures, and 1,044 predominant colonies isolated and characterized. The highest counts of aerobic bacteria and Enterococcus spp., up to 7 and 8 log colony‐forming units (cfu) per mL or g, were found when cream and compressed yeast samples were exposed to elevated temperatures (25 and 37C), while lower counts (4 and 6 log cfu/mL or g) were obtained from refrigerated samples (4C). At 10C, counts of aerobic bacteria and Enterococcus spp. increased from 4 to 7 log cfu/mL or g, highlighting the importance of temperature control during the storage and distribution of perishable cream and compressed yeast. Vacuum‐packaging of dry yeast reduced the growth of aerobic populations. Throughout storage, Lactobacilli dominated the bacterial populations in both cream and compressed yeast (45 to 78%), while Enterococcaceae predominated in vacuum‐packaged dry yeast (54 to 68%), suggesting they were primarily responsible for the spoilage of commercial yeast products.

PRACTICAL APPLICATIONS

In addition to producing high quality yeast for baking and brewing, it is an important objective for yeast manufacturers to increase product shelf life. Temperatures chosen for this shelf‐life study simulated various elevated temperatures of practical use for yeast processors and downstream consumers. For example, 4C (recommended refrigeration temperature), 10C (potential abuse temperature during transportation), 25C (wine‐making temperature) and 37C (temperatures often encountered in bakeries). Results from our study showed that storage temperature and time considerably influenced bacterial growth patterns associated with commercially manufactured fresh yeast. Bacterial spoilage and sensory deterioration were observed during lengthened storage periods, and especially at higher storage temperatures. Even when stored at 10C, cream and compressed yeast samples became bacteriologically and visually spoiled. It was therefore apparent that any break in the cold chain during processing, storage or distribution could affect the development of spoilage bacteria resulting in shortened product shelf life of commercially manufactured fresh yeast. Conversely, this study found vacuum‐packaged dry yeast to be the most bacteriologically stable manufactured product.     

CONTENTS OF CYTOCHROMES IN YEAST

The contents of cytochromes in yeast were determined quantitatively from the absorption spectra, using a solid cell paste of intact yeast. During the industrial production of baker's yeast, the contents of the cytochromes, particularly of cytochrome aa₃ at successive stages, increased gradually with increasing aeration. In semi-aerobically grown baker's yeast, the contents of cytochromes aa₃, b and c were 0·9, 2·9 and 2·9 × 10^?5 moles/litre of fresh yeast (total amount 6·7 × 10^?5 moles/litre), while in vigorously aerated commercial baker's yeast the respective values were 2·3, 4·8 and 5·2 × 10^?5 moles/litre (total amount 12·3 × 10^?5 moles/litre). In brewer's yeasts separated after the brewing process, the contents of cytochromes were markedly lower than in baker's yeast grown with limited aeration, whereas in top-fermenting yeast the total cytochrome content, aa₃ + b + c, was in some samples markedly higher, 7·1 × 10^?5 moles/litre, than in bottom-fermenting brewer's yeast, 2·4 × 10^?5 moles/litre. When brewer's bottom yeast was grown on a laboratory scale under increasing aeration, a maximum appeared in the cytochrome contents when aeration was moderate, and increased aeration inhibited the formation of cytochromes. The cytochrome contents in brewer's bottom yeast may exceed the amounts found in commercial baker's yeast. In addition to aeration, the type of metabolism influences the amounts of cytochromes in yeast.     

Populations and potential association of Saccharomyces cerevisiae with lactic acid bacteria in naturally fermented Korean rice wine

The microbial populations focused on predominant yeast species and lactic acid bacteria (LAB) in 15 commercial makgeolli brands, where a fungal starter nuruk was used were examined. Viable yeast counts were obtained on yeast potato dextrose (YPD) and MRS agar containing sodium azide. MRS-C (0.1% cycloheximide supplemented) was used for selective counts of LAB. Saccharomyces cerevisiae was found to be predominant in the 15 samples tested, with an average count of 4.6×10⁷ CFU/mL. Contrary to the earlier studies, Lactobacillus plantarum and Weissella cibaria were shown as predominant LAB species with an average count of 1.7×10⁷ CFU/mL. Surprisingly, as many as 7 log viable cells/mL were present at the ethyl alcohol concentration of 6–7%. The data from real-time PCR also indicated that the yeast populations remains almost constant during the refrigerated storage of 12 days, while that of LAB decrease slightly first 9 days and increase after then, despite the overall increase in acidity. Data from the differential microbial counts suggest that yeast S. cerevisiae might be associated with 2 LAB species, L. plantarum and W. cibaria, under ethyl alcohol stress during the turbid rice wine fermentation.     

Comparison of microbial communities between normal and swollen canned soy sauces using nested PCR‐denaturing gradient gel electrophoresis,HPLC and plate techniques

The microbial community of normal and swollen canned soy sauce was investigated using molecular biological method. The PCR‐denaturing gradient gel electrophoresis (DGGE) profiles showed that four lactic acid bacteria (LAB), including Lactobacillus acidipiscis, L. pobuzihii, L. piscium and another Lactobacillus sp., were involved in the swollen canned samples. Plate technique showed that three diverse species of Bacillus (B. subtilis, B. oleronius and B. flexus) were present in the swollen canned samples. However, much less bacterial contaminants were detected in the normal samples. According to the HPLC analysis, the lactic acid concentrations of the swollen canned samples were significantly higher than those in the normal samples. These results indicated that LAB can play a key role in contributing to the acidisation of the swollen canned soy sauce products. Our results confirmed the existence of Bacillus sp. and LAB in the packaged fermented soy sauce.     

Identification and molecular characterisation of lactobacilli isolated from traditional Koopeh cheese

This study was undertaken in order to phenotype and genotype wild‐type lactic acid bacteria isolated from Koopeh cheese of West Azerbaijan. Lactic acid bacteria (LAB) isolates were identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) and confirmed by phylogenetic analysis. Genotyping based on phylogenetic analysis of 16s rDNA gene revealed that LAB isolated from Koopeh cheese were mostly Lactobacillus plantarum as well as other species including Lactobacillus brevis, Entrococcus faecium and Enterococcus durans. It was concluded that a combined phenotypic and genotypic method could be used as a reliable technique for the identification and differentiation of LAB from dairy products.     

Dough-leavening by Zymomonas mobilis and Its Application to Breadmaking

Among five strains of Zymomonas mobilis strain Z6C had the highest leavening ability in doughs containing either glucose or sucrose. Z6C most efficiently leavened the dough when it contained 5% sucrose, and this leavening ability was twice as much as that of commercial compressed yeast. At the same concentrations of NaCl, the leavening ability of Z6C was reduced more than that of baker's yeast. When comparing baked goods made with yeast, those made with Z6C had similar specific volumes, but Z6C baked goods had slight acidic flavor. Chromatographic analysis of head-space gas of yeast-leavened bread showed that two chemicals appearing in Z6C leavened bread were missing.     

Characterization of the microbial community in three types of fermentation starters used for Chinese liquor production

This study examined and compared the microbial community in three typical fermentation starters (called as Daqu, Xiaoqu, and Fuqu in China) used for liquor production by analysing the 16S and 18S rRNA gene clone library. The results show that the microbial diversity in the three types of fermentation starters (JiuQu) differs significantly. The bacterial species in Daqu and Fuqu were mainly thermophilic or thermotolerant. In Daqu, the dominant bacterial species were Thermoactinomyces sanguinis (53.85%) and Pantoea agglomerans (19.23%), followed by uncultured bacteria (15.39%). The lactic acid bacterium Weissella cibaria (50%) and a member of Enterobacteriaceae, Enterobacter ludwigii (10%), were the dominant bacterial species in Xiaoqu. Low abundances of other bacteria, including Deinococcus radiodurans, Corynebacterium variabile and Acinetobacter baumannii, were reported for Xiaoqu. Enterococcus faecium, Clostridium beijerinckii and Bacillus cereus were observed in Fuqu and accounted for 46.67, 23.33 and 16.67% of the total bacteria identified, respectively. Fungal diversity was high in Daqu and consisted exclusively of thermophilic moulds, such as Aspergillus glaucus (62.5%), Thermomyces lanuginosus (12.5%) and Thermoascus crustaceus (12.5%). Only two fungal species were reported for Fuqu and Xiaoqu and both contained the mould Rhizopus oryzae. Saccharomyces cerevisiae and the non‐Saccharomyces yeast (Saccharomycopsis fibuligera) were also identified in Fuqu and Xiaoqu, respectively. This finding suggests that microbial community structure in JiuQu starters is the key factor to determine the variety of flavours. Copyright © 2015 The Institute of Brewing & Distilling     

Microbial diversity and stability during primary cultivation and subcultivation processes of Tibetan kefir

In this study, we have investigated the microbial diversity and stability in the Tibetan kefir during the primary cultivation and subcultivation processes via a combination of culture‐independent and culture‐dependent methods. According to the culture‐independent methods, the profiles of PCR‐DGGE (polymerase chain reaction–denaturing gradient gel electrophoresis) indicated that nine microbial species were predominant at different cultivation stages; seventy four isolates of seven predominant species were obtained and identified via the culture‐dependent methods. PCR‐DGGE further confirmed that Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus paracasei, Streptococcus thermophilus, Kazachstania unispora and Saccharomyces cerevisiae were the most dominant species in the Tibetan kefir during the process of primary cultivation, among which Lb. kefiri, Lb. paracasei and K. unispora showed relative strong stability during both the processes of primary cultivation and subcultivation. These findings suggested that some isolates of the three species possessed the potentiality of being used in the development of direct vat set (DVS) starters for the production of Tibetan kefir and the related products.     

Compressed Baker's Yeast: Mapping Patents on Post‐Fermentation Processes

In the baking industry, fresh yeast is generally available as a compressed paste containing about 70% moisture. While much literature has been published on its growth conditions in fermentation tanks, little information may be obtained on the final steps of baker's yeast production. In this review, 226 patents were found on the separation of yeast cells from spent growth media as well as its dewatering, forming, and packaging in the compressed form. The latter corresponded to 21% of 1096 unique families of patented inventions filed worldwide between 1787 and 2014 on the production of fresh baker's yeast. Patent specifications disclosed key technical information not available in scientific journals. Particularly between 1890 and 1960, highly specific equipment such as centrifugal separators, rotary vacuum filters, and extruding and cutting units were proposed. Compressed yeast may contain small amounts of processing aids, including water‐absorbing agents such as starch and salt, as well as plasticizing and whitening agents. Any variation in yeast solids and moisture contents will affect its gassing power and keeping properties. Disposal and proper treatment of very large volumes of spent growth media will remain a major challenge to the baker's yeast industry.     

Diversity of Lactic Acid Bacteria during Fermentation of a Traditional Chinese Fish Product,Chouguiyu (Stinky Mandarinfish)

Chouguiyu, or stinky mandarinfish, is a traditional Chinese fermented fish product made of mandarinfish by spontaneous fermentation at the anaerobic condition with low‐salt concentration. In order to get a primary understanding of the microbial community presenting in the Chouguiyu fermentation, 61 cultures of lactic acid bacteria (LAB) from various fermentation period were isolated using MRS agar plates and characterized based on a combination of phenotypic and genotypic approaches including amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA partial gene sequencing analysis. Eight distinct bacterial species belonging to 6 genera were identified in total. Among them, Lactobacillus sakei was the dominant species (63%) during the fermentation, which exhibited great variety in phenotypic tests but unique genotypic characters. Meanwhile, the other LAB species including Lactococcus (Lc.) garvieae, Lc. lactis, Lc. raffinolactis, Vagococcus sp., Enterococcus hermanniensis, Macrococcus caseolyticus as well as Streptococcus parauberis were also recovered from the different fermentation periods, especially at the initial point of the fermentation. This seems to be the 1st report investigating the LAB composition involved in Chouguiyu fermentation and the data obtained in this study may be valuable for selecting starter culture for Chouguiyu industrial‐scale production.     

The presence of Enterococcus, coliforms and E. coli in a commercial yeast manufacturing process

This study evaluated a typical commercial yeast manufacturing process for bacterial contamination. Product line samples of a commercial yeast manufacturing process and the corresponding seed yeast manufacturing process were obtained upstream from the final compressed and dry yeast products. All samples were analysed before (non-PI) and after preliminary incubation (PI) at 37 degrees C for 24 h. The PI procedure was incorporated for amplification of bacterial counts below the lower detection limit. Enterococcus, coliform and Escherichia coli counts were quantified by standard pour-plate techniques using selective media. Presence at all stages and progressive increases in counts of Enterococcus, coliforms and E. coli during processing in the commercial manufacturing operation suggested that the primary source of contamination of both compressed and dry yeast with these bacteria was the seed yeast manufacturing process and that contamination was amplified throughout the commercial yeast manufacturing process. This was confirmed by surveys of the seed yeast manufacturing process which indicated that contamination of the seed yeast with Enterococcus, coliforms and E. coli occurred during scale up of seed yeast biomass destined as inoculum for the commercial fermentation.     

Predominant yeasts in the sourdoughs collected from some parts of Turkey

In the present study, a total of eight sourdough samples were collected from three different bakeries at two different times in Turkey. Also, laboratory-scale sourdough production was conducted by daily back-slopping for 7 days. Microbiological and chemical properties of the sourdoughs were investigated. Yeast species in the sourdoughs were identified by subjecting all presumptive yeast cultures to internal transcribed spacer region amplification of the 5.8S rRNA gene, restriction fragment length polymorphism analysis using Hae III, Hha I, and Hinf I endonucleases, and sequence analysis of the D1/D2 domain of the 26S rDNA gene. A total of seven profiles were determined according to the restriction fragments. Totally, 148 yeast isolates were identified at the species level (≥400 bp , 99% identity) as Saccharomyces cerevisiae (106), Kazachstania bulderi (11), Pichia fermentans (nine), Pichia membranifaciens (eight), Kazachstania servazzii (seven), Kazachstania unispora (four), and Hanseniaspora valbyensis (three). Although collected sourdoughs were produced without using baker's yeast, S. cerevisiae was the most frequently isolated yeast species. This can be related to the contamination of the bakery environment with commercial baker's yeast during the production of other bakery products. The pH and acidity levels of the collected sourdough samples ranged from 3.71 to 3.96 and 6.78 to 23.93 mL 0.1 N NaOH/10 g dough, respectively. Mean values of the content of maltose + sucrose, glucose, fructose, lactic acid, and acetic acid were 2.43, 1.57, 2.67, 7.30, and 1.40 g/kg, respectively. Due to the artisan and region-dependent handling of the sourdough, different biochemical patterns were observed among the collected samples.     

Utilization of African grains for sourdough bread making

Abstract: Acha and Iburu flours were singly subjected to sourdough fermentation with previously selected autochthonous starters. Sourdoughs were used (30%, wt/wt) as aroma carriers and acidifiers during short time fermentation with the addition of baker's yeast. Acha and Iburu sourdough breads were compared to wheat sourdough bread started with the same strains and to breads made with the same formula but using baker's yeast alone. During Acha and Iburu sourdough fermentations, starter lactic acid bacteria reached almost the same cell density found in wheat sourdoughs. Acidification was more intense. Iburu sourdough bread had the highest total titratable acidity, the lowest pH, and contained the highest levels of free amino acids and phytase activity. The values of in vitro protein digestibility did not differ between Acha sourdough and wheat sourdough breads, while Iburu sourdough bread showed a slightly lower value. Acha and Iburu sourdough breads showed lower specific volume and higher density with respect to wheat sourdough breads. Nevertheless, Acha and Iburu sourdough breads were preferred for hardness and resilience. As shown by sensory analysis, Acha and especially Iburu sourdough breads were appreciated for color, acid taste and flavor, and overall acceptability. Practical Application: This study was aimed at evaluating the technological and nutritional properties of the African cereals Acha and Iburu. Sourdough fermentation and the use of selected starters increased the nutritional and sensory qualities and the potential application for bakery industry.