Efficient production of cellulolytic and xylanolytic enzymes by the rumen anaerobic fungus, Neocallimastix frontalis, in a repeated batch culture

In a batch culture of Neocallimastix frontalis in a medium (pH 6.8) containing 8 g.l(-1) cellulose, the concentrations of the fermentation products and the cellulolytic and xylanolytic enzymes did not increase in comparison with those of cultures in a medium containing 4 g.l(-1) cellulose. Therefore, kinetic studies were performed to determine the effect that products such as acetate, formate, lactate and ethanol have in inhibiting the growth. The reduction of the specific growth rate by the fermentation products could be expressed by using a noncompetitive inhibition model, and it was found that at low concentrations, acetate was the strongest inhibitor among the inhibitory products studied. In order to reduce the inhibition by the fermentation products, a repeated batch culture was carried out whereby none of the fatty acids exceeded 50 mM. In this repeated batch culture, xylanase, endoglucanase, beta-glucosidase, and avicelase were continuously produced in the medium during cultivation, and 18200, 4550, 3790 and 129 IU g.l(-1) of these enzymes, respectively, were produced up to 20 d of culture.     

Analysis of hemin effect on lactate reduction in Lactococcus lactis

Lactococcus lactis is a facultative anaerobic microorganism that produces lactate as the major product, and acetate and acetoin as by-products; some strains of this species produce an antimicrobial compound, nisin. Lactate has a strong inhibitory effect on L. lactis growth. On the other hand, hemin has a suppressive effect on lactate production during L. lactis growth under aerobic condition. To achieve the optimum effect of hemin on lactate amount reduction in L. lactis ATCC11454, cultures entailing various conditions were performed with and without hemin. In the culture with hemin, L. lactis growth and lactate reduction improved compared with those in the culture without hemin; that is, lactate production was suppressed by 1.8- and 1.3-fold under batch and fed-batch cultures, respectively. In microaerobic fed-batch culture with hemin, lactate production was sufficiently suppressed. This result suggests that microaerobic fed-batch culture could be applied to the maintenance of the low lactate amount. Under this condition, metabolic shift was observed from lactate to acetoin and acetate. However, no increase in nisin production was observed even though lactate production could significantly decrease in L. lactis ATCC11454.     

Competition mechanisms of lactic acid bacteria and bifidobacteria: Fermentative metabolism and colonization

Enzyme activities (α- and β-glucosidases, α- and β-galactosidases and β-fructofuranosidase) and organic acid production of four strains of lactic acid bacteria (LAB; Streptococcus thermophilus STY-31, Lactobacillus delbrueckii subsp. bulgaricus LBY-27, Lactobacillus casei LC-01 and Lactobacillus acidophilus LA-5) and Bifidobacterium lactis BB-12 were tested on milk and MRS fermentation broth with glucose, lactose or fructooligosaccharides (FOS) as carbon source. The highest β-galactosidase activity was found in L. acidophilus growing on milk. As compared to milk, α-glucosidase activity was increased with FOS by B. lactis, L. acidophilus and L. casei. The analysis of organic acids and short-chain fatty acids in the medium growth showed that lactate and acetate were the major fermentation metabolites produced by LAB and bifidobacteria, respectively. However, a metabolic shift towards more acetate and formate production, at the expense of lactate production, was observed during growth of L. casei on FOS. When grown on FOS as sole carbon source, L. acidophilus showed the highest production of lactate among the species tested. In addition, L. acidophilus demonstrated resistance to colonization against the intestinal pathogens Escherichia coli and Salmonella enterica in competition assays.     

Assessment of root uptake and systemic vine-transport of Salmonella enterica sv. Typhimurium by melon (Cucumis melo) during field production

Bifidobacterium adolescentis Int57 (Int57) and Propionibacterium freudenreichii subsp. shermanii ATCC 13673 (ATCC 13673) were grown either in coculture or as pure cultures in different media, such as cow's milk, soybean milk, and modified MRS medium. The viable cell counts of bacteria, changes in pH, concentrations of organic acids, and contents of various sugars were analyzed during incubation up to 7days. In soy milk, the survival of cocultured Int57 was six times higher than the monocultured cells, and ATCC 13673 cocultured with Int57 consumed 69.4% of lactic acid produced by Int57 at the end of fermentation. In cow's milk, coculture with ATCC 13673 increased the growth of Int57 from 24h until 120h by approximately tenfold and did not affect the survival of Int57 cells. After 96h of fermentation of modified MRS, the survival of ATCC 13673 cells cocultured with Int57 increased by 3.2- to 7.4-folds as compared with ATCC 13673 monoculture, whereas the growth of Int57 cells was unaffected. The growth and metabolic patterns of two strains during coculture showed noticeable differences between food grade media and laboratory media. The consumption of stachyose in soy milk during coculture of Int57 with ATCC 13673 was increased by more than twice compared with Int57 monoculture, and completed within 24h. The combinational use of Bifidobacterium and Propionibacterium could be applied to the development of fermented milk or soy milk products.     

Effects of fructooligosaccharide and whey protein concentrate on the viability of starter culture in reduced-fat probiotic yogurt during storage

ABSTRACT:  Viability of yogurt starter cultures and Bifidobacterium animalis was assessed during 28 d storage in reduced-fat yogurts containing 1.5% milk fat supplemented with 1.5% fructooligosaccharide or whey protein concentrate. These properties were examined in comparison with control yogurts containing 1.5% and 3% milk fat and no supplement. Although fructooligosaccharide improved the viability of Streptococcus thermophilus , Lactobacillus delbrueckii subs. bulgaricus, and Bifidobacterium animalis , the highest growth was obtained when milk was supplemented with whey protein concentrate in reduced-fat yogurt ( P < 0.05). Supplementation with 1.5% whey protein concentrate in reduced-fat yogurt increased the viable counts of S. thermophilus , L. delbrueckii subs. bulgaricus, and B. animalis by 1 log cycle in the 1st week of storage when compared to control sample. Similar improvement in the growth of both yogurt bacteria and B. animalis was also obtained in the full-fat yogurt containing 3% milk fat and no supplement. Addition of whey protein concentrate also resulted in the highest content of lactic and acetic acids ( P < 0.05). A gradual increase was obtained in organic acid contents during the storage.     

Effect of yogurt cultures on the survival of bifidobacteria in fermented milks

Three species of Bifidobacterium, Bif bifidum, Bif longum and Bif adolescentis were grown in reconstituted skim milk (12% total solids) either individually or in combination with one of three commercial yogurt cultures. Total colony counts were recorded for the bifidobacteria over a growth period of 24 hours and, for samples with the fermentation halted at pH 4.6, during storage at 5°C for up to 21 days. The results indicated that, while co-inoculation with the yogurt organisms tended to suppress the growth of the bifidobacteria, subsequent storage in the presence of the yogurt cultures did not lead to any significant decline in numbers.     

Acid production by bifidobacteria and yoghurt bacteria during fermentation and storage of milk

Three species of bifidobacteria, namelyBifidobacterium bifidum, Bifidobacterium longumandBifidobacterium adolescentiswere used in pure culture and in combination with yoghurt bacteria (B3 and SBI cultures) for the production of fermented milks. The number of bacteria during fermentation and the level of acid produced during fermentation and storage were assessed using Rogosa's modified selective agar and high performance liquid chromatography (HPLC). It was found that during fermentation all bifidobacteria exhibited growth uncoupled from acid production. Two of the species examined produced only low levels of acids when grown individually and onlyB. adolescentisproduced appreciable amounts. In mixed cultures, the level of acid was a reflection of the combination of yoghurt culture and species ofBifidobacterium, and this, observation suggests that there is a degree of influence between the cultures. During storage, the acid concentration remained quite stable in most samples. The prevention of post-production acidification that normally occurs during storage of yoghurt can be attributed to the presence of bifidobacteria, and it could be that acetic acid has a marginally inhibitory effect on theLactobacillusandStreptococcusspp.     

Effects of acetic acid and its assimilation in fed-batch cultures of recombinant Escherichia coli containing human-like collagen cDNA

The primary processing problem in recombinant Escherichia coli fermentation is the production of acetic acid, which can inhibit both cell growth and recombinant protein production. The ability of E. coli to assimilate acetate permits it to solve this problem in a rather creative manner. In this study, the effects of acetic acid assimilation through a glucose starvation period at different cell growth phases were investigated in fed-batch cultures of recombinant E. coli. Experimental results showed that the human-like collagen (HLC) production could be improved by introducing glucose starvation at the end of batch culture and pre-induction phase, while the glucose starvation at the induction phase resulted in a poor HLC productivity. The acetic acid assimilation was observed during all the glucose starvation periods. In addition, a systematic study for evaluating the effects of acetic acid was carried out by adding acetate into culture media at different cell growth phases and then employing a glucose starvation after several hours. It was found that obvious acetate inhibition on cell growth occurred in the batch culture phases while its inhibitory effect on HLC expression occurred only in the post-induction phase. The longer the elevated acetic acid concentration maintained, the stronger the inhibitory effects were. These results are of significance for optimizing and scaling-up fermentation processes.     

Extraction and Purification of Short-chain Fatty Acids from Fermented Reconstituted Skim Milk Supplemented with Inulin

Short-chain fatty acids (SCFAs) acetate, propionate, butyrate and lactate were determined in 12 % reconstituted skim milk (RSM) and RSM supplemented with inulin (RSMI). The fermentation was performed with Bifidobacterium animalis subsp. lactis (BB 12) and Lactobacillus rhamnosus LGG ATCC 53013. Fermentation culture activities produced substantial amounts of SCFAs, which were detected and quantitated using a HPLC-UV technique. Using HPLC-UV, we were able to detect low concentrations of lactate and SCFAs from fermented samples; lactate, acetate, propionate and butyrate were detected at 10.10, 12.06, 14.80 and 18.06 μg/mL, respectively. The retention time of all SCFAs and lactic acid were similar to the standard quality control (±0.05), and average recovery ranged between 89.73 and 91.03 %. The experimental conditions and sample preparation were applied to preparative HPLC to isolate and purify SCFAs with concentration range between 0.09 and 2.86 mg/mL. The purity of extracted SCFAs was confirmed using atmospheric pressure chemical ionization/mass spectrometry by determining the molecular masses of target purified compounds. The scaled up validated analytical HPLC-UV method will further enhance and improve the use of this approach to produce purified large-scale SCFAs.     

The relative effectiveness of pH control and heat treatment for enhancing biohydrogen gas production

Hydrogen gas can be recovered from the microbial fermentation of organic substrates at high concentrations when interspecies hydrogen transfer to methanogens is prevented. Two techniques that have been used to limit methanogenesis in mixed cultures are heat treatment, to remove nonsporeforming methanogens from an inoculum, and low pH during culture growth. We found that high hydrogen gas concentrations (57-72%) were produced in all tests and that heat treatment (HT) of the inoculum (pH 6.2 or 7.5) produced greater hydrogen yields than low pH (6.2) conditions with a nonheat-treated inoculum (NHT). Conversion efficiencies of glucose to hydrogen (based on a theoretical yield of 4 mol-H2/mol-glucose) were as follows: 24.2% (HT, pH = 6.2), 18.5% (HT, pH = 7.5), 14.9% (NHT, pH = 6.2), and 12.1% (NHT, pH = 7.5). The main products of glucose (3 g-COD/L) utilization (> or = 99%) in batch tests were acetate (3.4-24.1%), butyrate (6.4-29.4%), propionate (0.3-12.8%), ethanol (15.4-28.8%), and hydrogen (4.0-8.1%), with lesser amounts of acetone, propanol, and butanol (COD basis). Hydrogen gas phase concentrations in all batch cultures reached a maximum of 57-72% after 30 h but thereafter rapidly declined to nondetectable levels within 80 h. Separate experiments showed substantial hydrogen losses could occur via acetogenesis and that heat treatment did not prevent acetogenesis. Heat treatment consistently eliminated the production of measurable concentrations of methane. The disappearance of ethanol produced during hydrogen production was likely due to acetic acid production as thermodynamic calculations show that this reaction is spontaneous once hydrogen is depleted. Overall, these results show that low pH was, without heat treatment, sufficient to control hydrogen losses to methanogens in mixed batch cultures and suggest that methods will need to be found to limit acetogenesis in order to increase hydrogen gas yields by batch cultures.     

两歧双歧杆菌发酵橙汁酸豆奶的研究

本文尝试研究豆奶中两歧双歧杆菌的生长情况,以得到风味良好的发酵产品.以大豆与水1∶8的比例制取豆奶,添加一定量的橙汁,并加入两歧双歧杆菌进行发酵.考察了培养温度、橙汁添加量、接种量等因素对产品pH值和活菌教的影响,并在此基础上进行正交试验优化.正交试验结果表明,培养温度为39℃、接种量为5％、橙汁添加量为10％,18 ...     

Growth of Probiotic and Traditional Yogurt Cultures in Milk Supplemented with Whey Protein Hydrolysate

ABSTRACT: Growth of some probiotic bacteria was significantly improved in milk supplemented with whey protein hydrolysate (WPH). However, WPH had no effect on the growth of Lactobacillus delbrueckii ssp. bulgaricus 18, L. delbrueckii ssp. bulgaricus 10442, and Streptococcus thermophilus 1. When the probiotic bacteria were grown in combination with different yogurt cultures in milk, WPH caused significant increases in growth of Bifidobacterium longum S9, L. acidophilus O16, and L. acidophilus L-1. However, by day 28 of refrigerated storage, the populations of the probiotic cultures that had been grown in samples supplemented with WPH were similar or below those in the control samples.     

Development of a Chemically Defined Medium for Growth of Bifidobacterium animalis

ABSTRACT The growth of Bifidobacterium animalis was tested, under batch and continuous culture conditions, for dependence upon specific nitrogen compounds. The addition of nitrogen bases (adenine, xantine, and guanine) to the medium containing acid-hydrolyzed casein significantly affected the growth of B. animalis , whereas no significant growth was reported when those bases were added to the medium containing only free amino acids; B. animalis was characterized by a μmax of 0.16/h when growth took place in PROLAB medium containing lactose and nitrogenous bases. Our study is potentially important toward design of improved infant milk formulations for healthy newborns.     

Bifidobacterium longum Survival During Frozen and Refrigerated Storage as Related to pH during Growth

Four strains of Bifidobacterium longum were grown at pH 5.5, 6.0, 6.5 and 7.0 and evaluated for survival and bile tolerance during frozen and subsequent refrigerated storage in milk. There were no reductions in cell numbers following initial freezing. There were effects for strain, pH and storage for three of the four strains of B. longum du ring storage at 5°C in milk. Bifidobacterium longum S9 was more stable than other strains in that no losses occurred, regardless of pH during growth. Results were variable for strains II, III, and ATCC 15707 grown at the various pH levels. Bifidobacterium longum S9 did not lose bile resistance during refrigerated storage as the other three strains did.     

MANUFACTURE OF STARTERS: PRODUCTION OF FROZEN CONCENTRATED CHEESE STARTERS BY DIFFUSION CULTURE

Diffusion culture allows the production of concentrated cheese starter without centrifugation principally by removing inhibitory lactate from fermentations. Laboratory scale fermentation systems are described which remove lactate via a diffusion membrane. Single and mixed strain cultures were grown to concentrations of 10¹¹/ml in an enzyme digest of skimmed milk supplemented with yeast extract. The present stage system is capable of producing at least half the annual bulk starter requirements of a creamery processing 100,000 gal of milk/day. Concentrated cultures were harvested in hypodermic syringes and as frozen granules. Concentrates were tested for purity and activity and suitable ones used in a commercial cheesemaking trial which produced a high proportion of extra selected grade Cheddar cheese.     

EFFECT OF COLD STORAGE ON CULTURE VIABILITY AND SOME RHEOLOGICAL PROPERTIES OF FERMENTED MILK PREPARED WITH YOGURT AND PROBIOTIC BACTERIA

ABSTRACT

We examined the effect of storage time on culture viability and some rheological properties (yield stress, storage modulus, loss modulus, linear viscoelastic region, structural recuperation and firmness) of fermented milk made with Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus (LA) and Bifidobacterium animalis ssp. lactis in coculture with Streptococcus thermophilus (ST). Acidification profiles and factors that affect viability (postfermentation acidification, acidity and dissolved oxygen) were also studied during 35 days at 4C. Fermented milk prepared with a coculture of ST and Bifidobacterium lactis gave the most constant rheological behavior and the best cell viability during cold storage; it was superior to ST plus LA for probiotic fermented milk production.

PRACTICAL APPLICATIONS

Probiotic cultures should grow quickly in milk, provide adequate sensory and rheological properties to the product, and remain viable during storage. Commercially, it is very common to use yogurt starter culture (i.e. Streptococcus thermophilus[ST] and Lactobacillus delbrueckii ssp. bulgaricus) in combination with the probiotic bacteria in order to reduce fermentation time. However, LB tends to post acidify fermented milk, which reduces the viability of the probiotic bacteria; thus, it is recommended to use starter cultures devoid of this species. We found that the technological properties and the viability of the probiotic bacterium Bifidobacterium animalis ssp. lactis BL O4 in coculture with ST make it suitable for probiotic fermented milk production; it produces rheological characteristics similar to those of yogurt.     

Inhibition effects of furfural on aerobic batch cultivation of Saccharomyces cerevisiae growing on ethanol and/or acetic acid

Physiological effects of furfural on Saccharomyces cerevisiae growing on ethanol (15 g.l(-1)) or acetate (20 g.l(-1)) as the carbon and energy source were investigated. Furfural (4 g.l(-1)), which was added during the exponential growth phase in batch cultures, was found to strongly inhibit cell growth on both carbon sources. No biomass formation occurred in the presence of furfural. However, furfural was in both cases converted to furfuryl alcohol and furoic acid, and growth resumed after complete conversion of furfural. During growth on ethanol, a rapid initial conversion of furfural to furfuryl alcohol was observed during the first few minutes after the addition of furfural, after which the conversion rate decreased to approximately 0.15 g.g(-1).h(-1) for the remaining conversion time. Acetaldehyde accumulated in the medium during the first few hours of conversion. Interestingly, addition of acetate after furfural addition resulted in an increased conversion rate of furfural and a higher carbon dioxide evolution rate, but no growth was observed until after complete conversion of furfural. Furfural addition to cells growing on acetate as the sole carbon source induced no formation of acetaldehyde, and the furfural conversion rate was lower than that on ethanol. The relationship between inhibition effects of furfural and NADH consumption is discussed.     

Influence of combinations of Lactobacillus lactis and potassium sorbate on growth of psychrotrophs in raw milk

Cells of Lactobacillus lactis from frozen concentrated cultures were added (1 X 10(8)/ml) to refrigerated raw milk to determine their effect on growth of psychrotrophs during storage of the milk at 5 or 7 degrees C. Some strains of Lactobacillus lactis were significantly inhibitory toward the psychrotrophs in refrigerated raw milk at both temperatures. Two selected strains of Lactobacillus lactis were added along with potassium sorbate (.1 and .2%) to refrigerated raw milk stored at 5 degrees C to determine their combined effect on growth of the psychrotrophs. The combinations of cells of lactobacilli and sorbate were more inhibitory than either lactobacilli or sorbate alone. The measurement of hydrogen peroxide produced by strains of Lactobacillus lactis indicated that the strains most antagonistic toward psychrotrophs also were the more active producers of hydrogen peroxide. Addition of sorbate had no apparent effect on hydrogen peroxide production by the cultures during refrigerated storage.     

Efficient hydrogen production from acetate through isolated Rhodobacter sphaeroides

Photosynthetic bacteria produce hydrogen from lactate and acetate that are products of hydrogen producing bacteria in the dark. Thus, their coculture is a promising method for hydrogen production. However, the hydrogen production yield from acetate of Rhodobacter sphaeroides RV, which has been shown to possess the highest yield and hydrogen production rate, is low as compared to that from lactate. Photosynthetic bacteria that produce hydrogen from acetate as well as lactate were screened from lakes and swamps in the Tokyo and Chiba areas in Japan. Seventy-six strains of photosynthetic bacteria were obtained and the analysis of their 16S rRNA gene sequences revealed that they belong to R. sphaeroides. Among the isolated bacteria, R. sphaeroides HJ produced the highest amount of hydrogen from acetate and lactate. The HJ strain produced a 2300 ± 93 ml/L-broth of hydrogen from 75 mM acetate consumed during for 120 h of fermentation. The amount of hydrogen and the yield from acetate were 1.9 and 2.1 times higher, respectively, than those of R. sphaeroides RV. The amount and yield of hydrogen, produced by R. sphaeroides HJ from lactate were similar to those produced by R. sphaeroides RV. Since the amount and yield of produced hydrogen by the HJ strain were similar regardless of the substrate (acetate or lactate), its metabolic pathway could have a key to increasing hydrogen production from acetate.     

Bacteriocin production by Enterococcus faecium FAIR-E 198 in view of its application as adjunct starter in Greek Feta cheese making

Bacteriocin production by Enterococcus faecium FAIR-E 198, isolated from Greek Feta cheese, was studied in batch fermentations, under conditions simulating Feta cheese preparation. Maximum enterocin activity and growth rate was obtained in de Man-Rogosa-Sharpe (MRS) broth at 37 degrees C with controlled pH 6.5. The enterocin was produced throughout the growth phase of the microorganism, showing primary metabolite kinetics with a peak activity during the mid-exponential phase. The use of skimmed milk as substrate revealed low enterocin activity. When fermentations were performed in skimmed milk in the presence of rennet, CaCl2, and a mixed starter culture, no enterocin activity was observed, although the examined strain grew well under the above conditions. Finally, when E. faecium FAIR-E 198 was applied as adjunct starter in Feta cheese making, no enterocin activity was detected throughout ripening. Results obtained underline the frequently underestimated finding that in vitro production by novel bacteriocinogenic starter or co-cultures is no guarantee for in situ efficiency. It was concluded that the complex food environment thoroughly interferes with bacteriocin production levels.