Mixed-strain Starter Cultures Research Articles

Process for improving the quality of direct acidified Cottage cheese

The effects of plain and fermented curd dressing ripened by single (Lactococcus lactis subsp. lactis biovar. diacetylactis) as well as mixed-strain starter cultures (L. lactis subsp. lactis; L. lactis subsp. cremoris; L. lactis subsp. lactis biovar. diacetylactis:: 1:1:1), different levels of fat (18–24%) in curd dressing and inoculation rate (1–5%) on direct acidified Cottage cheese were observed. Ripened curd dressing containing 22% fat and mixed-strain starter cultures at 3% imparted a pleasant acidic note, delicate overtones of diacetyl, improved the body and texture, visual appearance and thereby enhanced the overall quality of the product.

Effect of Mixed Strain Starter Culture on Rheological Properties of Wheat Dough and Quality of Steamed Bread

AbstractIn this study, a mixed strain (MS) starter culture was prepared by fed batch fermentation method and its effect on wheat dough and steamed bread quality was investigated. The MS had higher leavening ability and more viable yeast counts than traditional mixed strain (TMS) starter culture made by static fermentation method. A slight decrease was observed in the extensibility of dough fermented by yeast, TMS and MS as resting time increased. The extensibility leavened by MS was higher than that by yeast but similar to TMS during the overall fermenting process. No significant differences could be observed between the resistance to extension of dough fermented by MS and yeast at all three tested times. The specific volume of the steamed bread prepared by MS was higher than that made by TMS, and MS significantly increased the sensory score of steamed bread when compared with yeast.Practical ApplicationsMixed strain (MS) starter culture was prepared by rice wine fed batch fermentation method and had higher leavening ability than TMS starter culture. Application of MS in steamed bread making could improve dough and steamed bread properties on specific volume and sensory quality. The research provided useful information for production of steamed bread.

Development of quantitative PCR and metagenomics-based approaches for strain quantification of a defined mixed-strain starter culture

Although the strain composition of mixed cultures may hugely affect production of various fermented foods, such as e.g. cheese, tools for investigating it have so far been limited.In this study, two new approaches for quantification of seven Lactococcus lactis subsp. cremoris strains (S1–S7) in a defined mixed-strain starter culture were developed and verified.By mapping NGS reads from 47 sequenced L. lactis strains to de novo assembly contigs of the seven strains, two strain-specific sequence regions (SEQ1 and SEQ2) were identified for each strain for qPCR primer design (A1 and A2). The qPCR assays amplified their strain-specific sequence region target efficiently. Additionally, high reproducibility was obtained in a validation sample containing equal amounts of the seven strains, and assay-to-assay coefficients of variance (CVs) for six (i.e. S1, S2, S4–S7) of the seven strains correlated to the inter-plate CVs. Hence, at least for six strains, the qPCR assay design approach was successful.The metagenomics-based approach quantified the seven strains based on average coverage of SEQ1 and SEQ2 by mapping sequencing reads from the validation sample to the strain-specific sequence regions. Average coverages of the SEQ1 and SEQ2 in the metagenomics data showed CVs of ≤17.3% for six strains (i.e. S1–S4, S6, S7). Thus, the metagenomics-based quantification approach was considered successful for six strains, regardless of the strain-specific sequence region used.When comparing qPCR- and metagenomics-based quantifications of the validation sample, the identified strain-specific sequence regions were considered suitable and applicable for quantification at a strain level of defined mixed-strain starter cultures.

Characterisation of Gouda cheeses based on sensory, analytical and high-field 1H nuclear magnetic resonance spectroscopy determinations: Effect of adjunct cultures and brine composition on sodium-reduced Gouda cheese

This study aimed at the interception of flavour deviations of salt-reduced Gouda-type cheeses, salted in sodium-reduced brines (NaCl + KCl brine) compared with a reference brine (NaCl brine), by the addition of adjunct strains of Lactobacillus helveticus and Lactobacillus paracasei, next to the common mixed-strain starter culture for Gouda. A multiphasic analysis of carbohydrates, organic acids, amino acids, and volatile flavour compounds was performed. After 56 days of ripening, the sensory characteristics of the cheeses were evaluated. Trained taste panel analysis determined some significant differences (P < 0.05) in saltiness, bitterness, texture and preference. The selected chemical fingerprint techniques, combined with multivariate data analysis, could be used to characterise Gouda cheeses that varied in ripening time and to which adjunct cultures were added. The variations were related to proteolysis and metabolic processes during ripening. There were no significant differences in the chemometric results between the reference NaCl-brined cheeses and the NaCl + KCl-brined cheeses.

Kinetic Analysis of Strains of Lactic Acid Bacteria and Acetic Acid Bacteria in Cocoa Pulp Simulation Media toward Development of a Starter Culture for Cocoa Bean Fermentation

The composition of cocoa pulp simulation media (PSM) was optimized with species-specific strains of lactic acid bacteria (PSM-LAB) and acetic acid bacteria (PSM-AAB). Also, laboratory fermentations were carried out in PSM to investigate growth and metabolite production of strains of Lactobacillus plantarum and Lactobacillus fermentum and of Acetobacter pasteurianus isolated from Ghanaian cocoa bean heap fermentations, in view of the development of a defined starter culture. In a first step, a selection of strains was made out of a pool of strains of these LAB and AAB species, obtained from previous studies, based on their fermentation kinetics in PSM. Also, various concentrations of citric acid in the presence of glucose and/or fructose (PSM-LAB) and of lactic acid in the presence of ethanol (PSM-AAB) were tested. These data could explain the competitiveness of particular cocoa-specific strains, namely, L. plantarum 80 (homolactic and acid tolerant), L. fermentum 222 (heterolactic, citric acid fermenting, mannitol producing, and less acid tolerant), and A. pasteurianus 386B (ethanol and lactic acid oxidizing, acetic acid overoxidizing, acid tolerant, and moderately heat tolerant), during the natural cocoa bean fermentation process. For instance, it turned out that the capacity to use citric acid, which was exhibited by L. fermentum 222, is of the utmost importance. Also, the formation of mannitol was dependent not only on the LAB strain but also on environmental conditions. A mixture of L. plantarum 80, L. fermentum 222, and A. pasteurianus 386B can now be considered a mixed-strain starter culture for better controlled and more reliable cocoa bean fermentation processes.

Time‐resolved genetic responses of Lactococcus lactis to a dairy environment

Lactococcus lactis is one of main bacterial species found in mixed dairy starter cultures for the production of semi-hard cheese. Despite the appreciation that mixed cultures are essential for the eventual properties of the manufactured cheese the vast majority of studies on L. lactis were carried out in laboratory media with a pure culture. In this study we applied an advanced recombinant in vivo expression technology (R-IVET) assay in combination with a high-throughput cheese-manufacturing protocol for the identification and subsequent validation of promoter sequences specifically induced during the manufacturing and ripening of cheese. The system allowed gene expression measurements in an undisturbed product environment without the use of antibiotics and in combination with a mixed strain starter culture. The utilization of bacterial luciferase as reporter enabled the real-time monitoring of gene expression in cheese for up to 200 h after the cheese-manufacturing process was initiated. The results revealed a number of genes that were clearly induced in cheese such as cysD, bcaP, dppA, hisC, gltA, rpsE, purL, amtB as well as a number of hypothetical genes, pseudogenes and notably genetic elements located on the non-coding strand of annotated open reading frames. Furthermore genes that are likely to be involved in interactions with bacteria used in the mixed strain starter culture were identified.

Use of response surface methodology to investigate the effects of processing conditions on sourdough wheat bread quality

Response surface methodology was used to investigate the influence of three factors, sourdough fermentation time, proof time and amount of yeast addition on the quality of sourdough wheat bread. Each predictor variable was tested at five levels. Sourdough fermentation times were 5, 11, 20, 29 and 35 h, yeast addition rates were 0.05, 0.75, 1.77, 2.80 and 3.50% (flour weight basis) and proof times were 16, 40, 75, 110 and 134 min. The performance of two different starter culture types, a mixed strain starter culture called Bocker Reinzucht–Sauerteig Weizen and a single strain starter culture of Lactobacillus brevis, was compared by separately completing the experimental design for each. Independently non-acidified control bread was prepared. A range of loaf quality parameters was determined including pH, total titratable acidity, loaf height, specific volume, crumb mean cell area and crumb hardness. Overall breads with better specific volume values were achieved with the use of sourdough relative to the control. Results indicated that maximum loaf specific volume was achieved when L. brevis sourdough was used particularly when it was used in conjunction with a high rate of yeast. Given a lower rate of yeast addition however, the mixed strain starter culture yielded better bread.

Starter culture development for improving the flavour of Proosdij-type cheese

The use of the additional mesophilic strain B851, which has specific flavour forming abilities, was tested for improving the flavour development of a Proosdij-type cheese made with a combination of an acidifying mesophilic and an adjunct thermophilic culture. This strain was selected because of its ability to produce the branched chain aldehyde, 3-methylbutanal, which is a key flavour compound in Proosdij cheese. In order to control the flavour intensity, the selected strain was first tested at different levels in a defined strain starter culture as well as in combination with a mixed strain starter culture. The results of population dynamics, sensory evaluation and analysis of volatile compounds pointed to the possibility of controlling both the cell numbers of strain B851 as well as the cheese flavour intensity resulting from this strain. Thus, B851 was used to enhance the flavour development of a Proosdij-type cheese made with a new thermophilic culture S1138, developed to prevent crack formation. The addition of culture B851 led to an increase in the overall flavour intensity, indicating that it is possible to tailor the flavour of cheese by using specifically selected cultures, even in combination with complex starter cultures.

Effect of Single Strain and Traditional Mixed Strain Starter Cultures on Rheological Properties of Wheat Dough and on Bread Quality

ABSTRACTInvestigations were made to test the effect of two different sourdough starter culture types on wheat dough and bread quality. Two single‐strain starter cultures consisting of well‐defined strains of lactic acid bacteria (Lactobacillus plantarum, L. brevis) and a traditional mixed‐strain sourdough culture (containing L. crispatus, L. pontis, and Saccharomyces cerevisiae) were evaluated for their effects on the rheological characteristics of wheat dough using both fundamental rheological and standard baking tests. Two other doughs were also evaluated, one which was chemically acidified to a comparable pH value by the addition of lactic acid, and a control which was not acidified. Dynamic oscillation tests were performed using a controlled stress rheometer. The phase angle and the absolute value of the complex dynamic modulus were measured for all doughs at frequencies of 0.1–10 Hz. The addition of sourdough prepared using single‐strain or mixed‐strain cultures significantly increased the phase angle and reduced the complex modulus of the doughs at all frequencies (P &lt; 0.05). Significant differences were found between the dough which was chemically acidified and those doughs which were biologically acidified. The addition of sourdough effected an increase in loaf specific volume relative to both the chemically acidified and the nonacidified doughs.

Characteristics of Manchego Cheese Manufactured from Raw and Pasteurized Ovine Milk and with Defined-Strain or Commercial Mixed-Strain Starter Cultures

Microbiological, chemical, rheological, and sensory characteristics of Manchego cheese manufactured with raw or pasteurized milk and with a defined-strain starter culture made up of four Lactococcus lactis ssp. lactis isolates from high quality artisanal Manchego cheese have been compared with those of cheese made with a commercial mixed-strain starter culture. Higher total viable counts and lower pH values after 24 h were detected in both raw and pasteurized milk cheeses made with the defined-strain starter culture, which survived better than did the commercial mixed-strain starter culture during ripening of pasteurized milk cheese. Differences in proteolysis of raw milk cheeses were reduced, whereas lower concentrations of residual caseins and higher concentrations of pH 4.6-soluble N and TCA-soluble N were observed in pasteurized milk cheeses made with the commercial mixed-strain starter culture. Elasticity, fracturability, and hardness were higher in raw and pasteurized milk cheeses made with the defined-strain starter culture. Flavor quality and intensity were not apparently affected by the starter culture used, whereas cheese bitterness was lower in raw and in pasteurized milk cheeses made with the defined-strain starter culture.

Conductimetric Analyses of Bacteriophage Infection of Two Groups of Bacteria in DL-Lactococcal Starter Cultures

Two conductimetric methods were used for simple and rapid detection of bacteriophages against the two major groups of bacteria in DL-lactococcal, mixed-strain starter cultures. Bacteriophages against the acid-producing bacteria Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris in a starter were analyzed in a Malthus® instrument using reconstituted skim milk as the medium. The presence of phages against Lactococcus lactis ssp. lactis biovar. diacetylactis strains in a starter could be analyzed by measurement of CO2 production with an indirect conductance method in the Rabit® instrument. For both methods, the presence of phages against bacteria in a starter increased the detection time. At 30°C and .1% inoculum, a very sensitive assay was obtained that detected phages against acid-producing strains in a starter within 3 to 4h. The presence of 40 to 1000 infectious phages was sufficient for detection with a mixed-strain starter culture. Bacteriophages against CO2- producing bacteria were detected after 7 to 9h with a .1% inoculum at 30°C. Bacteriophage infection of single strains of bacteria reduced total change in conductance. For pure strains of an acid- or a CO2-producing strain, the detection limits were 1 and 100 pfu, respectively.

Some observations on the use of the ‘API STREP’ bacterial identification system to classify strains of lactococci (lactic streptococci) from commercial starter cultures

Strains of Lactococcus sp. isolated from a commercial mixed strain starter culture were classified according to their cultural characteristics, as determined using the API STREP bacterial identification system. A correlation between the API STREP profile and plasmid profile of strains was observed. However, certain key tests for lactococci gave frequent anomalous or variable results, notably for failure to hydrolyse esculin, although most strains were able to acidify a medium containing esculin as sole carbohydrate source. This led to some unsatisfactory classifications. Correct storage of API STREP test reagents was important and the inclusion of controls is recommended. There is tentative evidence that the ability to ferment ribose rapidly is a plasmid encoded function in these bacteria.

The use of mesophilic cultures in the dairy industry.

The use of mesophilic starter cultures, containing group N Streptococcus and Leuconostoc species, in the dairy industry is examined. Bacteriophage attack is identified as the main cause of culture inhibition and criteria used to select stable mixed-strain starter cultures and phage-insensitive defined strains are established. The key aspects of culture propagation and storage are highlighted, as well as bulk-culture protection based on physical exclusion of phage and the use of phage-inhibitory media. Developments in the application of starter culture systems in different countries are examined and show that significant process control and elimination of phage problems can be achieved.

Acid-Producing Activity of Lyophilized Lactic Streptococcal Cheese Starter Concentrates

Freeze-dried concentrates of lactic streptococcal starter bacteria (single and mixed strains) were prepared in different lyophilization media and stored under vacuum and several gases. The concentrates were tested for survival and acid-producing activity after storage for up to a year by rehydration with water to the original prelyophilization volume. Acid production was measured during incubation at 30C for up to 6h and 22C for up to 18h. Lyophilization media included 11% nonfat milk, nonfat milk containing 15% sodium glycerophosphate, nonfat milk containing 10% maltose, and sucrose-peptone (10%-5%) solution. Storage conditions included vacuum-sealing and storage under atmospheres of air, argon, carbon dioxide, and nitrogen. Best results were with commercial mixed strain starter cultures of unknown strain composition which were purchased as direct vat set Cheddar cheese starters. Freeze dried concentrates of these cultures were active and stable at ambient temperatures (25C) for several months when vacuum-sealed and stored in amber containers filled with argon and when lyophilized in 11% nonfat containing 15% sodium glycerophosphate. For example, freeze dried concentrates of 10 such cultures when stored for 5 to 8 months at 25C produced an average percent titratable acidity of .97 (range .86 to 1.03) at 22C in 18h when inoculum was made into steamed (95C for 40min) nonfatmilk; when incubated at 30C for 6h, the average was .60% (range .35 to .80%) for freeze dried concentrates stored 3 to 4 mo. The apparent enhanced acid production by rehydrated concentrates incubated at 22C as compared to 30C suggests that such preparations may have greater application for direct inoculation of bulk starter milk than for cheese vat milk.

Inhibition of Enteropathogenic Escherichia coli by Homofermentative Lactic Acid Bacteria in Skimmilk

Inhibition of enteropathogenic Escherichia coli in skimmilk at 21 and 32 C by 0.25 and 2.0% of added Streptococcus lactis, Streptococcus cremoris, or a mixed strain starter culture was studied. After 15 h of fermentation, fermented milks were refrigerated at 7 C and then were tested periodically for survival of E. coli. Three methods for enumeration of E. coli during these fermentations were compared. They included trypticase soy agar (TSA) pour plates, violet red bile agar (VRB) pour plates, and TSA surface plating with a VRB overlay. Lactic cultures had similar inhibitory properties at 32 C, but there were differences at 21 C, with S. lactis being least inhibitory and the mixed strain culture most inhibitory. The VRB pour plate method gave poorest recovery of E. coli when fermentation was at 32 C and when fermented milks were refrigerated. The TSA surface plating method apparently allowed for recovery of injured E. coli cells and gave results similar to the TSA pour plate method.

THE ISOLATION OF A LYSOGENIC STRAIN FROM A MULTIPLE STRAIN STARTER CULTURE*

A commercial mixed strain starter culture which had been used for over 10 years without producing ‘slow cheeses’ in a factory produced a clear zone of inhibition surrounded by small plaques. Isolates from this culture proved to be lysogenic strains which released temperate phage after UV irradiation and induction. The phage susceptible indicator strain was successfully lysogenized by the temperate phages and then shown to produce phage after UV irradiation.

CULTURAL STUDIES ON STREPTOCOCCUS DIACETILACTIS AND OTHER MEMBERS OF THE LACTIC STREPTOCOCCUS GROUP

Cultural information about Streptococcus diacetilactis strains is tabulated along with similar data on Streptococcus lactis, Streptococcus cremoris, and certain Leuconostoc species. These data facilitate comparisons of bacteria commonly found in mixed-strain lactic starter cultures. Characteristic reactions which indicate simple means of differentiating between these organisms may be observed. The data are used to justify the recognition of S. diacetilactis as a third species of the lactic streptococcus group. Leuconostoc organisms are separated into two groups on the basis of dextran production on a sucrose medium.Details of a manometric method for measuring CO2production by single and mixed-strain starter cultures are given. The Warburg Respirometer was employed for this purpose and possible application of the method in the design of mixed-strain starter cultures for use in the dairy industry is discussed.

Bacteriophage-Lysis of Streptococcus Diacetilactis and its Effect on Biacetyl Production in Mixed-Strain Starter Cultures

Summary Bacteriophages specific for Streptococcus diacetilactis were isolated from cottage cheese whey sampled in several commercial dairy plants. Electron microscopy of one of the bacteriophages revealed that it was tadpole-shaped with an over-all length of about 180 mµ; growth studies indicated the virus had a latent period of 30 to 33min. and an average burst size of 44. Assays revealed that S. diacetilactis produced an average of five times more biacetyl in single-strain milk culture than other bacteria commonly used in controlled dairy fermentations. This aroma-producing organism was combined with one or more strains of Streptococcus lactis, Streptococcus cremoris, and Leuconostoc organisms in the presence and absence of homologous phages for S. diacetilactis . Selective phage-lysis resulted, with consequent marked reduction in biacetyl synthesis, but no effect on the rate of acid production. It is suggested that selective bacteriophage-lysis of S. diacetilactis organisms may occur under commercial conditions. This might account for some instances of variation in flavor and aroma of cultured dairy products.

Taxonomic Study of High Carbon Dioxide-Producing Lactic Acid Streptococci Isolated from Mixed-Strain Starter Cultures

Summary A taxonomic and serological investigation was carried out on strains of high CO 2 -producing lactic acid streptococci isolated from commercial mixed-strain starter cultures causing the floating curd defect in cottage cheese. Non-CO 2 -producing lactic acid streptococci found in mixed-strain starter cultures also were studied. Known cultures designated as Streptococcus lactis, Streptococcus cremoris, Streptococcus diacetilactis, Leuconostoc citrovorum , and Leuonostoc dextranicum were included for comparative purposes. The high CO 2 -producing lactic acid streptococci were identified in this study as S. diacetilactis. S. lactis and S. cremoris species easily were identified by salt tolerance, growth temperature and hydrolysis of arginine. S. diacetilactis gave characteristic cultural and serological reactions and, along with S. lactis and S. cremoris organisms, was distinguishable from Leuconostoc species by litmus milk reaction at 30° C. The name Streptococcus cremoris var. aromaticus was suggested to identify two cultures which seemed intermediate between S. cremoris and S. diacetilactis . Test methods employed did not distinguish between organisms designated as L. citrovorum and L. dextranicum , but the one culture of Leuconostoc mesenteroides used in the study was easily identified by its ability to liberate ammonia from arginine which none of the associative organisms could do.

A Test for the Detection of Lactic Bacteriophage Using 2,3,5-Triphenyltetrazolium Chloride

Summary In this study, 2,3,5-triphenyltetrazolium chloride (TTC) was used to measure inhibition of lactic cultures by bacteriophage. The procedure follows: Place 8.7ml. of 10% sterile reconstituted nonfat dry milk (NDM) in each of two sterile test tubes. To each tube add 0.2ml. of a lactic culture diluted 1:1 with sterile 10% NDM. Add 1ml. of serially diluted phage nitrate to one tube. To the other tube, remaining as the control, add 1ml. of sterile distilled water. Place sterile rubber stoppers in the tubes and invert twice to mix the contents. Incubate the tests for 4 hr. at 32° C. in a water bath. Add 0.1ml. of 1:25 aqueous solution of TTC per tube. Invert the tubes twice and incubate again for 30min. at 32° C. Compare the color in the tube with added filtrate to the control tube. If less color is developed than in the control, inhibition has occurred. Results of this test are available in 4.5 hr. Its sensitivity for bacteriophage is comparable, and in some cases better, than a litmus milk test (8) requiring from 12 to 16 hr. With mixed-strain lactic starter cultures, the TTC test indicates some cases of phage inhibition in 4.5 hr. that are not evident after extended incubation in the litmus milk test. By using unheated filtrate and filtrate heated to 80° C. for 10min., this test can be used to distinguish between inhibition of lactic cultures caused by heat-stable inhibitors and bacteriophage.