Acidification Power Research Articles

Optimization of cryoprotectant formulation to enhance the viability of Lactobacillus brevis ED25: Determination of storage stability and acidification kinetics in sourdough

In this study, various kinds of cryoprotectant (skim milk, lactose, and sucrose) formulations were tested to enhance the survival of Lactobacillus brevis ED25 after freezing and freeze-drying. A Box–Behnken experimental design was used to optimize cryoprotective medium and the highest cell survival was observed with the 17.28% skim milk, 2.12% lactose, and 10% sucrose cryoprotectant as the optimum condition. The structural and physicochemical characteristics of freeze-dried powder were acceptable for application with regards to particle surface morphology, moisture and water activity (Aw), glass transition temperature (Tg), Fourier transform infrared spectra, X-ray structure, and also storage stability under the refrigeration and room temperature conditions. Accelerated storage test based on Arrhenius equation could be used to predict the freeze-dried bacterial shelf life but only with a certain degree of predictability for long-term storage. The acidification kinetics of fresh and stored culture in sourdough fermentation was also described on the basis of the Gompertz equation. Practical applications Freezing and storage are crucial factors for the viability and acidification power of starter culture. Therefore, various types and concentrations of cryoprotectants have been used to preserve the microorganisms. L. brevis ED25 has been a good potential for the manufacture of industrial sourdoughs and this research has aimed to investigate long-term protective effects of optimum cryoprotectant formulations on the viability of bacteria and also determine the acidification power in sourdough. The results showed the potential value of freeze-dried L. brevis ED25 culture for commercialization.

Test acidifikační síly (AP) a příbuzné metody pro stanovení a předpověď fermentační aktivity průmyslových mikroorganismů.

Je podán stručný přehled metod používaných pro stanovení vitality a fermentační aktivity mikroorganismů (kvasinek, bakterií) používaných v pivovarském, pekařském a mlékárenském průmyslu, se zvláštním zaměřením na metody založené na protonových a/nebo iontových poměrech a rovnováhách mezi buňkami a okolím. Většina těchto metod je založena na měření rychlosti výstupu protonů z buněk (acidifikační síle) stanovené za různých podmínek, a na důležitých buněčných charakteristikách s ní spojených, jako je vnitrobuněčné pH.

Selection of Saccharomyces pastorianus variants with improved fermentation performance under very high gravity wort conditions

Saccharomyces pastorianus FBY0095 was mutated and variants were selected for efficient very high gravity brewing using 15% (w/v) maltose and 15% (w/v) ethanol. Two useful variants were obtained of which one (L6) had growth, wort consumption and ethanol production rates of 0.036, 1.13 and 0.49gl(-1)h(-1), respectively. The corresponding results for the wild type were 0.028, 0.98 and 0.4gl(-1)h(-1), respectively. The vitality of the variant (expressed as acidification power) was 2.5 while that of the wild type was 2.3. There was also an obvious improvement on flavor of resulting beer when using L6 and the other variant.

Development of Co-Culture Sourdough Systems for Improving Bread Quality and Delaying Staling

Six different blends of lactic acid bacterial cultures (Lactobacillus plantarum or Pediococcus cerevisiae) combined with Saccharomyces cerevisiae were used as co-culture for preparing sourdoughs. These prefermented sourdoughs were applied for producing pan breads. Acidification power, leavening ability, and microbial counts were conducted at different sour fermentation intervals. The highest acidification power in sourdoughs were recorded when 2% wd/wd (dry weight of cells/dry weight of flour) of either L. plantarum or P. cerevisiae was used in combination with 2% wd/wd S. cerevisiae. The leavening ability of sourdoughs was significantly (p < 0.05) enhanced by increasing the addition percentage of the studied LAB. According to staling rate, physical, and sensory characteristics, the best two treatments were obtained when the co-culture of 2% wd/wd S. cerevisiae with 4% wd/wd L. plantarum or 4% wd/wd P. cerevisiae were used. These two treatments were successfully applied in production of the flat bread. According to staling rate, physical and sensory characteristics, sourdough prefermented with 2% wd/wd S. cerevisiae combined with 4% wd/wd L. plantarum was the best treatment for preparing of flat bread. Overall the co-culture of L. plantarum and with S. cerevisiae might be a useful tool for preparing sourdough starter that was most effective for improving sensory and physical properties of both pan and flat bread. In addition it can help extend the shelf life by delaying bread staling and inhibiting mold spoilage.

Net effect of wort osmotic pressure on fermentation course, yeast vitality, beer flavor, and haze

The net effect of increased wort osmolarity on fermentation time, bottom yeast vitality and sedimentation, beer flavor compounds, and haze was determined in fermentations with 12 degrees all-malt wort supplemented with sorbitol to reach osmolarity equal to 16 degrees and 20 degrees. Three pitchings were performed in 12 degrees/12 degrees/12 degrees, 16 degrees/16 degrees/12 degrees, and 20 degrees/20 degrees/12 degrees worts. Fermentations in 16 degrees and 20 degrees worts decreased yeast vitality measured as acidification power (AP) by a maximum of 10%, lowered yeast proliferation, and increased fermentation time. Repitching aggravated these effects. The 3rd "back to normal" pitching into 12 degrees wort restored the yeast AP and reproductive abilities while the extended fermentation time remained. Yeast sedimentation in 16 degrees and 20 degrees worts was delayed but increased about two times at fermentation end relative to that in 12 degrees wort. Third "back-to-normal" pitching abolished the delay in sedimentation and reduced its extent, which became nearly equal in all variants. Beer brewed at increased osmolarity was characterized by increased levels of diacetyl and pentanedione and lower levels of dimethylsulfide and acetaldehyde. Esters and higher alcohols displayed small variations irrespective of wort osmolarity or repitching. Increased wort osmolarity had no appreciable effect on the haze of green beer and accelerated beer clarification during maturation. In all variants, chill haze increased with repitching.

Yeast vitality determination based on intracellular NAD(P)H fluorescence measurement during aerobic-anaerobic transition

This work presents a yeast-cell vitality-assessment method based on on-line intracellular fluorescence measurement. The intracellular NAD(P)H fluorescence of a cell suspension is recorded during transition from aerobic to anaerobic conditions and the output signal is evaluated as a measure of yeast vitality (quality). This fluorescence method showed a highly satisfactory correlation with even low dead cell numbers where the acidification power test could not be applied.

A new method of optical detection of yeast acidification power

We describe here a newly developed method for a contact-free optical pH measurement in yeast suspensions supplemented with glucose, and containing the pH sensitive triphenylmethane dye bromocresol green. It is suitable for performing the acidification power test (based on measuring the rate of pH drop of yeast suspension caused by active extrusion of acidity from cells after glucose addition) used for assessing yeast vitality in fermentation industries. Using this methodology we monitored the pH in yeast suspensions in the course of acidification in the pH range of 3.5-5.3. Optical pH measurement allows simultaneous testing of several samples, minimizes the sample volume, simplifies sample handling and reduces the hands-on time in sample processing.

Optimised Acidification Power Test of Yeast Vitality and its Use in Brewing Practice

The optimised acidification power test (APT) of brewer's yeast quality includes storing the yeast slurry at 2°C under beer (AP remains constant for up to 6 days), a 15 min sample equilibration to room temperature, decantation, and washing by triple centrifugation in deionised water. The final yeast pellet keeps its AP for up to 6 h at room temperature under water and thus the APT does not need to be performed immediately after yeast collection. The correct AP value (maximum acidification produced by given yeast) is determined at 25 ± 0.1°C in a 15 mL sample containing ≥5% glucose and ≥1.5 g yeast wet weight. The cell concentration is conveniently measured as absorbance (A660). Cell flocculation and/or sedimentation that can distort APT results can be prevented by stirring the sample at ≥200 rpm. The AP of yeast of different generations used to pitch brewery fermentations in cylindroconical tanks had a very low correlation with the wort half-attenuation time (T1/2) due to large scatter, while each yeast generation separately showed a clear T1/2-AP relationship. The lowest AP of yeast cropped from cylindroconical tanks was displayed by the first cropped fraction. Post-cropping cooling had no effect on AP. Variations in pitching yeast vitality and their effect on the outcome of a brewery fermentation can be masked by variations in pitching rate, wort composition, ambient conditions in the cylindroconical tanks and other factors.

Factors affecting the outcome of the acidification power test of yeast quality: Critical reappraisal

Brewery bottom yeast strain 95 from the Pilsner Urquell propagation unit was used to reappraise the efficiency of the acidification power (AP) test consisting in determining the spontaneous (oxygen-induced) and glucose-induced medium acidification caused by yeast and lactic acid bacteria under standard conditions, and used widely for assessing and predicting the vitality of industrial strains. AP was evaluated in yeast stored for different periods of time (0-28 d) at 4 degrees C, at different temperatures before and during the test (0-55 degrees C), and at different concentrations of cells and glucose and different cells-to-glucose ratios. All these factors had a strong effect on acidification kinetics and the AP value. By contrast, the duration of the lag period between yeast collection and the test (0-6 h) had no perceptible effect on the AP value. The best results were achieved at saturation concentrations of cells (> 10 g pressed yeast or approximately 14 g yeast slurry per 100 mL) and glucose (approximately 3 %) and at 25 degrees C. Since an exact evaluation of acidification characteristics depends strongly on the kinetics of the process, the AP test should include monitoring the time course of the acidification.

Significance of microbial transglutaminase on the sensory, mechanical and crumb grain pattern of enzyme supplemented fresh pan breads

The impact of microbial transglutaminase (TGM) on the sensory, mechanical and crumb grain pattern of fresh pan breads has been investigated in white and wholemeal flour sponge dough samples supplemented with amylolytic-(NMYL) and non-amylolytic (PTP) enzymes. Assessment of bread performance has been carried out by physico-chemical measurements, texture profile analysis, sensory evaluation, digital image analysis and multivariate data handling. Improving effects of TGM addition were prominent when added to low extraction rate flours (increased cohesiveness by 11%, volume, aroma intensity by 31%, typical taste and crumb to cell ratio by 25% and decreased cell number by 17%). For white and whole flours, NMYL addition to TGM breads led to 16% softer breads. For the pool of enzyme supplemented samples tested, it was observed that the higher the specific volume of breads, the lower the acidification power, the better the mechanical behaviour with high cohesiveness and low hardness values and the higher the sensory scores for visual and textural attributes.

Development of Bifidobacterium lactis Bb 12 on β‐(2,6)‐Linked Fructan‐Containing Substrate

Abstractβ‐(2,1)‐linked fructan of plant origin (inulin) and the related oligosaccharides (FOS) as non‐digestible carbohydrates, i.e., potent prebiotics, can stimulate the growth of various probiotic lactic acid bacteria, including a number of bifidobacteria strains. The related β‐(2,6)‐linked fructans of microbial origin (levan and FOS), however, have scarcely been investigated in this respect. Therefore, the bifidogenic properties of various fructans, i.e., inulin, levan, fructooligosaccharides (FOS) and fructan syrup (FS), were tested as glucose substitutes in MRS media and were compared concerning their effect on the commercial strain Bifidobacterium lactis Bb 12. Although glucose was the preferred substrate for growth and biomass formation, FS exhibited a comparable cell growth (8.4 × 107 counts/mL and 1.0 × 107 counts/mL, respectively) and acidification power (84 °T and 74 °T, respectively) during 48 h of fermentation, as well as an increase in lactic acid and decrease in acetic acid formation. Bifidobacterium lactis Bb 12 did not utilize inulin as a sole carbon source as judged from the 60 % decrease in cell count and the insignificant (0.1 pH unit) acidification of the growth medium, whereas levan provided a noticeable increase in cell count and acidification (0.4 pH units) during 48 h of fermentation. FOS preparation appeared to be a satisfactory carbon source for this strain, but lower acidification power (56 °T) and cell counts were observed as compared to glucose‐ or FOS‐containing media (2.6 % and 22 %, respectively). The products obtained under conditions of mild lactic acid hydrolysis of levan (37 °C, pH 3.3, 24 h) enhanced the cell count (7–10 %) and acidification power (by a factor of 2.7) of Bifidobacterium lactis Bb 12.

Impact of Attachment Immobilization on Yeast Physiology and Fermentation Performance

A novel bioreactor with immobilization matrices was constructed to study physiological differences between immobilized and free cells and to compare the fermentation behavior of the immobilized-cell system with that of a free-cell system. Although the yeast sample released from the immobilization matrix (immobilized cells) showed relatively low activity of glucose utilization according to the acidification power test, fermentation in the immobilized-cell system was completed 20 hr earlier than in the free-cell system. In addition, flavor-active compounds, such as fusel alcohols and esters, were produced at higher levels (23% more fusel alcohols and 22% more esters) in the immobilized-cell system compared with the free-cell system. Whole-cell immobilization induced a reduction of cellular fatty acid contents, as well as an increase of fatty acid saturation percentages. These alterations of cellular fatty acid compositions in the immobilized cells had negative effects on cellular metabolic activities and stress resistance. It is suggested that cell attachment on solid surfaces, mass transfer limitations within the immobilization matrix, or both are responsible for the modifications of cellular fatty acid contents in the immobilized yeast.

Rapid assessment of cell viability of Lactobacillus delbrueckii subsp. bulgaricus by measurement of intracellular pH in individual cells using fluorescence ratio imaging microscopy

The aim of this study was to investigate if the measurement of intracellular pH (pH i) of individual cells by fluorescence ratio imaging microscopy (FRIM) could be utilized as a rapid method for determining the bacterial viability, using Lactobacillus delbrueckii subsp. bulgaricus as a model organism. Five different standardized cultures with equal cell densities but varying viability were prepared on a trial-to-trial basis by combining aliquots of frozen and lyophilized cells with a 50-fold difference in viability, determined by the ability to form colonies on solid growth media. The acidification of milk and Acidification Power Test were used to determine the activity of these cultures. As expected, the cultures containing a higher proportion of viable cells acidified milk faster and performed better in the Acidification Power Test. All cells were fluorescent after staining with carboxyfluorescein diacetate succinimidyl ester but frozen cells reached higher fluorescence intensities than lyophilized cells. The number of strongly fluorescent cells determined by flow cytometry exceeded the number of viable cells determined by CFU. Analysis of pH i of individual cells by FRIM at an extracellular pH of 6.0 revealed two populations of cells with an average pH i of 6.9±0.1 and 6.1±0.1. As the number of cells maintaining a pH-gradient of 0.9±0.1 correlated well with CFU, we suggest that FRIM can be used as a rapid method for the determination of viability of L. delbrueckii subsp. bulgaricus. Measurement of pH i on a single cell basis is expected to provide accurate prediction of the fermentation performance in a wider range of industrial fermentation.

Yeast vitality during cider fermentation: assessment by energy metabolism.

In an apple juice-based medium, an ethanol-tolerant Australian wine-yeast used for cider manufacture produced more than 10% ethanol over a 5 week period. Growth of the inoculum (10(6) organisms ml(-1)) occurred to a population of 3.1 x 10(7) ml(-1) during the first few days; at the end of the fermentation only 5 x 10(5) yeasts ml(-1) could be recovered as colony-forming units on plates. Respiratory and fermentative activities were measured by mass spectrometric measurements (O2 consumption and CO2 and ethanol production) of washed yeast suspensions taken from the cider fermentation at intervals. Both endogenous and glucose-supported energy-yielding metabolism declined, especially during the first 20 days. Levels of adenine nucleotides also showed decreases after day 1, as did adenylate energy charge, although in a prolonged (16.5 week) fermentation the lowest value calculated was 0.55. AMP was released into the medium. 31P-NMR spectra showed that by comparison with aerobically grown yeast, that from the later stages of the cider fermentation showed little polyphosphate. However, as previously concluded from studies of 'acidification power' and fluorescent oxonol dye exclusion (Dinsdale et al., 1995), repitching of yeast indicated little loss of viability despite considerable loss of vitality.

Use of Methylene Violet Staining Procedures to Determine Yeast Viability and Vitality

Beer quality is strongly influenced by the biochemical performance of the yeast during fermentation; therefore, brewers require consistent yeast quality. Thus, predicting the capacity of pitching yeast to perform well during fermentations is a necessity. Brewing yeast quality is usually described in terms of its “viability” or “vitality.” Yeast viability was determined using cell proliferation or plate counts and citrate methylene blue and citrate methylene violet staining techniques. Yeast vitality (yeast activity) may also represent the physiological state of the viable cell population. Four techniques were used to assess the relative activity of the yeast cultures, including the acidification power test, safranin-O methylene blue double staining, alkaline methylene blue, and alkaline methylene violet. The relative sensitivity and response of each method were examined using ale, lager, and cider yeasts in various physiological states. It is suggested that methylene violet staining may provide an effective alternative to the more traditional methylene blue stains.

VITALITY OF CIDER YEAST GROWN MICRO-AEROBICALLY WITH ADDED ETHANOL, BUTAN-J-OL ORISO-BUTANOL

Vitality and viability of an alcohol-tolerant wine yeast, used in cider production, were assessed after exposure to alkanol(s) during growth. Criteria employed were: methylene blue reduction, ability to form colonies on yeast extract-peptone-glucose agar medium, glucose driven proton efflux rates (“acidification power”), fermentative (CO2 output) rates and adenylate energy charge values. We also monitored the maintenance of transmembrane electrochemical potential across the plasma membrane as measured by flow cytometry and by scanning confocal laser microscopy of oxonol dye exclusion. Growth rates were diminished by a third by 7.5% (v/v) added ethanol, 1% butan-1-ol or 1.4% iso-butanol. Exposure to 10% (v/v) ethanol gave 16% loss of “viability”, as measured by methylene blue reduction, during the first 20 h of growth. For 1% butan-1-ol, 50% loss of “viability” occurred over 40 h, whereas a similar effect of iso-butanol took 55 h. Adenylate charge values were high (<0.8) in growing cultures, remained high in early stationary phase but declined to 0.4 after 115 h. These values were hardly affected by 5 or 7.5% (v/v) ethanol whereas 10% or 15% (v/v) ethanol gave values of 0.58 and 0.16 after only 5 h exposure. 1% butan-1-ol or iso-butanol decreased adenylate charge values to a greater extent than 10% (v/v) ethanol, with the straight chain alcohol the more potent. Oxonol exclusion indicated that the vast majority of colls with greatly diminished vitality have maintained the plasma membrane potential values required to retain viability, despite extensive exposure to alkanol(s). Thus loss of ability to reduce methylene blue indicates diminished vitality but is not a reliable index of loss of viability. “Acidification power” was a more sensitive indicator of vitality than adenylate charge values. When mixtures of C2 + C4 alcohols were employed effects were generally additive rather than synergistic.

THE EFFECTS OF ETHANOL, HEXAN-1-OL, AND 2-PHENYLETHANOL ON CIDER YEAST GROWTH, VIABILITY, AND ENERGY STATUS; SYNERGISTIC INHIBITION

An ethanol-tolerant strain of Saccharomyces cerevisiae, used for cider manufacture, when grown aerobically in the presence of either 1.7 M ethanol (10%, v/v), 3.35 mM hexan-1-ol (0.05%), or 25 mM 2-phenylethanol (0.25%), showed decreased growth rates (about 60%, 60% and 75%) and diminished viability. The normal changes in medium pH (increased acidity over the first 24 h from pH 6.0 to 5.4 followed by a decreased acidity to pH 7.8 by 70 h) were altered in the presence of an added alcohol. “Acidification power” (glucose-driven pH change) was also markedly reduced by growth with the alcohols from a value of 1.77 for control cultures, to 0.5, 1.35 or 0.6 respectively after 48 h. Adenylate energy charge values were decreased from 0.8 to 0, 0.3 and 0.2 respectively after 115 h; control cultures without an added alcohol showed a final value of 0.4. Ethanol plus either hexan-l-ol or 2-phenylethanol gave much more pronounced decreases in viability, medium pH changes during growth, acidification power, and adenylate energy change values than predicted by summation of individual effects. It is concluded that synergy between ethanol and the higher alkanol or aryl-alcohol occurs to produce membrane-associated lesions with deleterious effects during cider fermentation.

Titrated Acidification Power: A Simple and Sensitive Method to Measure Yeast Vitality and its Relation to Other Vitality Measurements

The acidification power (AP) test is an interesting method to evaluate the vitality of pitching yeast. However, this method lacks sensitivity at high AP values. We have elaborated a titrated acidif...

Titrated Acidification Power: A Simple and Sensitive Method to Measure Yeast Vitality and Its Relation to Other Vitality Measurements

The acidification power (AP) test is an interesting method to evaluate the vitality of pitching yeast. However, this method lacks sensitivity at high AP values. We have elaborated a titrated acidification power test that is better suited to the evaluation of highly vital yeast and compared this test with the AP and conductivity measurement during yeast storage experiments.

YEAST VITALITY DURING CIDER FERMENTATION: TWO APPROACHES TO THE MEASUREMENT OF MEMBRANE POTENTIAL

Simulated cider fermentations were carried out using a monoculture of a cider strain of Saccharomyces cerevisiae in a sterile apple juice-based medium. The membrane potential of the yeast was assessed in two different ways: firstly by using the fluorescent dyes oxonol and rhodamine 123 and visualising the yeast with flow cytometry and fluorescent microscopy, and secondly by using the acidification power test. These techniques were used on yeast at various stages of a simulated cider fermentation and also on freshly grown yeast (3 days after inoculation) after the addition of an alcohol mixture similar to that accumulating in mature cider (10% ethanol, 200 ppm propan-1-ol, 32 ppm butan-1-ol, 70 ppm butan-2-ol, 190 ppm amyl alcohol, 17 ppm hexan-1-ol and 260 ppm 2-phenyl ethanol). Little change was observed to the plasma membrane transmembrane potential as indicated by oxonol exclusion over a 4 week fermentation period, but rhodamine 123 staining which indicates mitochondrial membrane potential showed a drastic decline during the first 3 days. The total acidification power index remained almost constant with fermentation age, but the individual components of the index showed considerable changes.