Single-strain Starter Research Articles

Enzyme activities and volatile flavor compound compositions of tiger nut paste prepared using different fungal starter strains

Abstract In order to clarify effects of different fungal strains on fermented tiger nut paste enzyme activities and volatile flavor compounds composition, koji enzyme activities and volatile flavor substances of six tiger nut paste preparations (S1–S6) were compared. The results revealed that koji enzyme activities of S4, S5 and S6 were significantly higher than corresponding activities of S1, S2 and S3. Among them, S4 exhibited the highest enzyme activity levels for cellulase, xylanase, acid protease, glucosidase, and amylase. GC-MS analysis of the six paste preparations detected 43 volatile components, including 17 aldehydes, 8 alkenes, 4 heterocyclics, 2 ketones, 2 phenols and 7 other types of compounds. Notably, S4 had the most volatile compounds (28), including 5 volatile compounds which were not detected in the other tiger nut pastes. Results of principal component analysis and partial least squares discriminant analysis (PLS-DA) revealed differences in levels of 17 volatile flavor substances among the six tiger nut paste preparations. Notably, PLS-DA results achieved more significant separation and stronger clustering trends when used to discriminate among volatile flavor substances of paste preparations fermented using single-strain starters (S1, S2, S3) versus double-strain starters (S4, S5, S6) consisting of Aspergillus oryzae, Aspergillus niger and/or Mucor. The tiger nut paste (S4) prepared by mixed fermentation of A. oryzae and A. niger had a unique flavor, producing the highest enzyme activity and a wider variety of volatile flavor compounds. Therefore, the production of tiger nut paste through compound strain koji fermentation was a feasible strategy.

Starter culture-related changes in free amino acids, biogenic amines profile, and antioxidant properties of fermented red beetroot grown in Poland

Fermentation of two red beet cultivars (Wodan and Alto) with single-strain starter cultures consisting of selected strains of lactic acid bacteria (LAB) of plant origin (Weissella cibaria KKP2058, Levilactobacillus brevis ZF165) and a multi-strain culture (containing W. cibaria KKP2058, L. brevis ZF165, Lactiplantibacillus plantarum KKP1822, Limosilactobacillus fermentum KKP1820, and Leuconostoc mesenteroides JEIIF) was performed to evaluate their impact on betalains, free amino acids, formation of biogenic amines, and antioxidative properties of the juice formed. Next-generation sequencing data analysis used to identify the microbial community revealed that the starter cultures promoted the dominance of the genus Lactobacillus, and decreased the proportion of spoilage bacteria compared to spontaneously fermented juices. Generally, the fermentation process significantly influenced the amount of the analyzed compounds, leading in most cases to their reduction. The observed changes in the studied parameters depended on the starter culture used, indicating different metabolic activities of the LAB strains towards bioactive compounds. The use of multi-strain starter cultures allowed to largely prevent the reduction of betacyanins and histamine formation.

Effect of proteolytic starter culture isolated from Chinese Dong fermented pork (Nanx Wudl) on microbiological, biochemical and organoleptic attributes in dry fermented sausages

The effect of a proteolytic starter culture isolated from Nanx Wudl, on microbiological, biochemical and organoleptic attributes of dry fermented sausages was investigated during processing. Based on preliminary screening, the combination of Staphylococcus xylosus SX16 and Lactobacillus plantarum CMRC6, showing excellent proteolytic activity in vitro, was selected as the multi-strain starter (starter LS). For comparison, the single-strain starter culture of L. plantarum CMRC6 (starter LB) and non-inoculated control were also tested. During fermentation, lactic acid bacteria and staphylococci dominated the microbiota and suppressed the Enterobacteriaceae growth in LS-inoculated sausages. The addition of LS starter accelerated acidification and proteolysis during ripening, improving the contents of total free amino acids and several essential free amino acids (Phe, Ile and Leu). Volatile compounds analysis revealed that LS-fermented sausage showed the highest abundance of 3-methyl-1-butanol, probably due to the inoculated S. xylosus. The inoculation of LS starter improved the sensory properties of sausages, especially the flavor attribute. Therefore, S. xylosus SX16 and L. plantarum CMRC6 are promising candidates for inclusion as multi-strain starters in the manufacture of gourmet fermented dry sausage.

Impact of Saccharomyces cerevisiae and Metschnikowia fructicola autochthonous mixed starter on Aglianico wine volatile compounds.

Non-Saccharomyces yeasts are metabolically active during grape must fermentations and can contribute with enzymes and metabolites to enhance the complexity and to define the final wine aroma. Nowadays, the use of non-Saccharomyces yeasts in combination with Saccharomyces cerevisiae is a state-of-the art strategy to improve wine composition and/or wine sensory properties. The present paper deals with the new yeast strains of Metschnikowia fructicola and S. cerevisiae, that were selected as representatives of the yeast microbiota isolated from grapes and grape juice of Aglianico cultivar. S. cerevisiae was utilized both as single strain starter and in combination with M. fructicola in experimental fermentations of Aglianico must. The dynamic of yeast populations was evaluated during the fermentation process analyzing the wine volatile compounds profile. The volatile compounds were identified by SPME-GC/MS. The results, showed that the multiple indigenous yeast starter was able to modulate the volatile compounds profiles and improve the aromatic complexity of wine, with a higher content of esters and terpenes.

Sequential inoculum of Hanseniaspora guilliermondii and Saccharomyces cerevisiae for winemaking Campanino on an industrial scale.

In this study, the effect of sequential inoculation with non-Saccharomyces (Hanseniaspora guilliermondii) and Saccharomyces cerevisiae yeast on the distinctive characteristics of the Campanino white wine was investigated. For this purpose, three independent winemaking experiments were carried out on an industrial scale (batches A, B and C). In detail, the first one was carried out using the sequential inoculation technique while the other two, using a S. cerevisiae single-strain starter or no inoculation representing the control batches. Microbiological and chemical parameters and sensorial profiles of the wines were defined. Interestingly, the results showed that when sequential cultures (H. guilliermondii in a sequential mixture with S. cerevisiae) were used, a better wine aroma and quality was observed. More specifically, the wine obtained by sequential inoculation showed lower acetic acid values and enhanced volatile profiles than the wine from the control batches. Finally, sensorial analysis confirmed that the sequential cultures led to an improvement in wine flavour. Therefore, results suggest that the sequential inoculation using non-Saccharomyces and Saccharomyces yeast represents a biotechnological practice that can improve the quality features of traditional white wine. It has been shown for the first time that on an industrial scale H. guilliermondii could be used in sequential inoculum with S. cerevisiae in making white Campanino wine.

Utilization of indigenously isolated single strain starter cultures for the production of sourdough bread

<p>Sourdoughs were prepared with <em>Saccharomyces cerevisiae</em> (T<sub>0</sub>) and indigenously isolated starter cultures i.e<em> Lactobacillus brevis (</em>T<sub>1</sub>)<em>, Lactobacillus fermentum</em> (T<sub>2</sub>) and<em> Lactobacillus plantarum </em>(T<sub>3</sub>). Breads were prepared from all sourdoughs samples in triplicate and analyzed for pH, Total Titratable Acidity (TTA), loaf volume, microbial characteristics (total plate count and fungal count) and sensory profile (internal and external) in triplicate. The breads prepared from <em>Saccharomyces cerevisiae</em> (T<sub>0</sub>) exhibited the highest pH with the lowest TTA while T<sub>1 </sub>showed the lowest pH with the highest TTA. The T<sub>0</sub> breads got the highest values for loaf volume followed by T<sub>1</sub>. The breads produced with the addition of hetero-fermentative starter cultures (T<sub>1 </sub>and T<sub>2</sub>) showed resistance against the growth of the contaminating microorganisms. In the sensory evaluation, the breads produced with<em> </em>T<sub>1 </sub>ranked the best for color (crust and crumb), taste, aroma, texture and overall acceptability by the panelists. </p>

Two efficient nitrite-reducing Lactobacillus strains isolated from traditional fermented pork (Nanx Wudl) as competitive starter cultures for Chinese fermented dry sausage

Lactic acid bacteria isolated from traditional Dong pork product (Nanx Wudl) were investigated for their potential as starter cultures for Chinese fermented dry sausages. Based on preliminary screening, Lactobacillus plantarum CMRC6 and Lactobacillus sakei CMRC15, both showing excellent nitrite-reducing capacity, were used as single-strain starter cultures. For comparison, a commercial composite starter was also tested. In CMRC6 and CMRC15-inoculated sausages, lactic acid bacteria dominated the microflora and improved the microbiological safety by suppression of Enterobacteriaceae growth. Nitrite content of all inoculated sausages declined rapidly during ripening compared to non-inoculated. Texture profiles analysis showed inoculated sausages had more pronounced textural development during ripening. Sensory evaluation indicated CMRC6 and CMRC15-fermented sausages had comparable or more desirable organoleptic characteristics than sausage made with commercial starters. Therefore, CMRC6 and CMRC15 are promising candidates as multi-functional starter cultures for microbiological safety and residual nitrite control in gourmet Chinese dry sausage production.

Industrial application of selected lactic acid bacteria isolated from local semolinas for typical sourdough bread production

Four obligate heterofermentative lactic acid bacteria (LAB) strains (Weissella cibaria PON10030 and PON10032 and Leuconostoc citreum PON 10079 and PON10080) were tested as single strain starters, mono-species dual strain starters, and multiple strain starter for the preparation and propagation of sourdoughs for the production of a typical bread at industrial level. The kinetics of pH and TTA during the daily sourdough refreshments indicated a correct acidification process for all trials. The concentration of lactic and acetic acid increased consistently during fermentation. The resulting molar ratios between these two organic acids in the experimental trials were lower than those observed in the control trial. The microbiological investigation showed levels of approximately 109 CFU/mL in almost all sourdoughs and the comparison of the genetic polymorphisms of the dominating LAB with those of the pure cultures evidenced the persistence of the added strains over time. The resulting breads were evaluated for several quality parameters. The breads with the greatest height were obtained with the quadruple combination of leuconostocs and weissellas. The highest softness was registered for the breads obtained from fermentations performed by W. cibaria PON10032 alone and in combination. The different inocula influenced also the color, the void fraction, the cell density and the mean cell area of the breads. Different levels of acids, alcohols, aldehydes, esters, hydrocarbons, ketones, terpenes, furans and phenol were emitted by the breads. The sensory tests indicated the breads from the sourdoughs fermented with the seven LAB inocula as sweeter and less acidic than control breads and the breads from the trials with the highest complexity of LAB inoculums were those more appreciated by tasters. A multivariate approach found strong differences among the trials. In particular, control breads and the breads obtained with different starter LAB were quite distant and a more strict relation was found among the productions carried out by W. cibaria strains. This study proved the suitability of the selected strains of L. citreum and W. cibaria for industrial-scale level applications in sourdough bread production.

Evaluation of a single strain starter culture, a selected inoculum enrichment, and natural microflora in the processing of Tonda di Cagliari natural table olives: Impact on chemical, microbiological, sensory and texture quality

The processing of natural table olives is still empirical and far to be controlled. Taking natural fermentation (NF) as control, the performances of a single strain LAB starter culture (SSL) and a selected inoculum enrichment (SIE) were compared. The results showed that the SIE was more efficient in controlling spoiling microflora (Enterobacteriaceae spp.) compared to SSL and NF. Indeed, Enterobacteriaceae in SIE samples were not detectable starting from 10 days, while in SSL and NF samples starting from 30 days. Both starters rapidly acidified the brine, lowering the pH to safety levels (<4.0) after 12 days, while NF samples reached pH 4.3 after 45 days, and these values were kept constant up to 150 days. Moreover, both starters showed debittering activity, as stated through sensory analyses and HPLC analysis of phenols. Compared to NF samples, SIE and SSL extracts retained more hydroxytyrosol at the end of the process, and this could partially account for their higher scavenging activity. Texture profile analysis (TPA) showed that olives processed with SIE inoculum were firmer and more elastic, compared to SSL inoculated olives, thus resulting more similar to NF samples.

Microbiology of Cheddar cheese made with different fat contents using a Lactococcus lactis single-strain starter

Flavor development in low-fat Cheddar cheese is typified by delayed or muted evolution of desirable flavor and aroma, and a propensity to acquire undesirable meaty-brothy or burnt-brothy off-flavor notes early in ripening. The biochemical basis for these flavor deficiencies is unclear, but flavor production in bacterial-ripened cheese is known to rely on microorganisms and enzymes present in the cheese matrix. Lipid removal fundamentally alters cheese composition, which can modify the cheese microenvironment in ways that may affect growth and enzymatic activity of starter or nonstarter lactic acid bacteria (NSLAB). Additionally, manufacture of low-fat cheeses often involves changes to processing protocols that may substantially alter cheese redox potential, salt-in-moisture content, acid content, water activity, or pH. However, the consequences of these changes on microbial ecology and metabolism remain obscure. The objective of this study was to investigate the influence of fat content on population dynamics of starter bacteria and NSLAB over 9 mo of aging. Duplicate vats of full fat, 50% reduced-fat, and low-fat (containing <6% fat) Cheddar cheeses were manufactured at 3 different locations with a single-strain Lactococcus lactis starter culture using standardized procedures. Cheeses were ripened at 8°C and sampled periodically for microbiological attributes. Microbiological counts indicated that initial populations of nonstarter bacteria were much lower in full-fat compared with low-fat cheeses made at all 3 sites, and starter viability also declined at a more rapid rate during ripening in full-fat compared with 50% reduced-fat and low-fat cheeses. Denaturing gradient gel electrophoresis of cheese bacteria showed that the NSLAB fraction of all cheeses was dominated by Lactobacillus curvatus, but a few other species of bacteria were sporadically detected. Thus, changes in fat level were correlated with populations of different bacteria, but did not appear to alter the predominant types of bacteria in the cheese.

PRODUCTION OF PROBIOTIC YOGHURT (BIFILACT) BY USING SOME SPECIES OF LACTIC ACID BACTERIA

Probiotic lactic acid bacteria was used in manufacturing yoghurt from fortified whole milk. The product was stored under refrigeration for one week. The results showed that yoghurt manufacturing by using single or mixed strain bacteria was acceptable concerning acidity percent as it was 0.52 , 0.92 and 1.20 % using L.acidophilus , B.lactis and mixed culture , respectively for the fresh yoghurt. Total solid percentages of the fresh yoghurt were 16.47 , 16.48 and 15.93% for the three mentioned culture , respectively. Counts of Bifidobacteria in single strain starter and in mixed starter were higher followed by L.acidophilus in mixed starter, while counts were lowest in L.acidophilusin in single strain starter one week after storage counting were 81× 107 , 81× 107 , 36× 107 and 11× 107 c.f.u/ml. respectively, and these counts were within the acceptable ones of WHO and FAO values. Sensory evaluation showed that the product was acceptable for at least one week storage without any microbial or sensory changes which indicated that it can be used as a therapeutic product. Physical properties of the freeze dryed product were also studied and the results showed the ability for reconstruction and using it in capsule formulation.

Prevalence and impact of single-strain starter cultures of lactic acid bacteria on metabolite formation in sourdough

Flavour of type II sourdoughs is influenced by the ingredients, processing conditions, and starter culture composition. It is, however, not fully clear to what extent different sourdough lactic acid bacteria (LAB) contribute to flavour. Therefore, two types of flour (rye and wheat) and different LAB starter culture strains were used to prepare sourdoughs, thereby leaving the yeast microbiota uncontrolled. All LAB starter culture strains tested were shown to be prevalent and to acidify the flour/water mixture to pH values between 3.1 and 3.9 after 24h of fermentation. Multiple aldehydes, alcohols, ketones, and carboxylic acids were produced by the sourdough-associated microbiota throughout the fermentation period. Based on the organoleptic evaluation of breads produced with these sourdoughs, five LAB strains were selected to perform prolonged wheat and rye fermentations as to their capacity to result in an acidic (Lactobacillus fermentum IMDO 130101, Lactobacillus plantarum IMDO 130201, and Lactobacillus crustorum LMG 23699), buttermilk-like (Lactobacillus amylovorus DCE 471), or fruity flavour (Lactobacillus sakei CG1). Upon prolonged fermentation, higher metabolite concentrations were produced. For instance, L. sakei CG1 produced the highest amounts of 3-methyl-1-butanol, which was further converted into 3-methylbutyl acetate. The latter compound resulted in a fruity banana flavour after 48h of fermentation, probably due to yeast interference. Rye fermentations resulted in sourdoughs richer in volatiles than wheat, including 3-methyl-1-butanol, 2-phenylethanol, and ethyl acetate.

The influence of manufacture on the free D-amino acid content of Cheddar cheese

The changes in the concentration and those of composition of alanine, aspartic acid and glutamic acid enantiomers were investigated during manufacture of Cheddar cheese. The amount of D-alanine increased continuously during ripening following the liberation of L-alanine originated from the proteolysis of milk proteins. There was slightly more D-aspartic and D-glutamic acid in the dry matter of curd after pressing than before pressurization. The D-amino acid content and the ratio of the D-enantiomers related to the total amount of free amino acids differed significantly among cheeses produced with different single-strain starters. The D-amino acid composition changed during manufacture, but the influence of the strain selection was not significant on the D-amino acid pattern.

The origin of single strain starter culture usage for commercial Cheddar cheesemaking

The technology of using single strain starter cultures for commercial Cheddar cheesemaking is commonly believed to have originated in New Zealand in about 1934. However, W. S. Sutton of the New South Wales Department of Agriculture isolated pure strains of Streptococcus cremoris and, along with A. B. Shelton, conducted cheesemaking trials at the Hawkesbury Agricultural College, now the University of Western Sydney, from at least September 1930. Pure strains were being used in factories by October 1930 and, by July 1932, at least six factories were using pure single cultures. The technology was taken to New Zealand from New South Wales by Dr Moir.

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.

Multivariate statistical analysis of peptide profiles and free amino acids to evaluate effects of single-strain starters on proteolysis in miniature Cheddar-type cheeses

Abstract Multivariate statistical analysis of reverse phase-high-performance liquid chromatography (RP-HPLC) chromatograms of ethanol (70%)-soluble and -insoluble fractions and free amino acids from miniature Cheddar-type model cheeses was performed to evaluate effects of different single-strain starters on proteolysis. The statistical analysis was done by using principal component analysis (PCA) and hierarchical cluster analysis (HCA). Based on results from the statistical analysis, we were able to group strains according to their effects on chromatographic profiles and free amino acids. PCA, using variables standardised to zero mean, and the covariance matrix made it possible to identify peaks which were strongly influenced by the different starters. Multivariate statistical analysis of chromatographic profiles was a more objective and powerful approach for evaluating proteolysis in cheese, than visual assessment of the chromatograms.

Design of a phage-insensitive lactococcal dairy starter via sequential transfer of naturally occurring conjugative plasmids

The plasmid-free Lactococcus lactis subsp. cremoris MG1614 is highly phage sensitive and lacks lactose fermenting ability (Lac) and primary casein degrading ability (Prt). Food grade gene transfer systems were used to sequentially superimpose different phage defense systems on this background, resulting in a gradual increase in resistance to bacteriophage in the derivatives. pLP712, encoding Lac and Prt, was then transferred to one of these hosts, into which plasmids encoding adsorption inhibition, restriction modification, and abortive infection had already been introduced. This resulted in a phage-resistant strain which was successfully used as a single-strain starter for cheddar cheese manufacture under industrial conditions.

Use of a broad-host-range bacteriocin-producing Lactococcus lactis transconjugant as an alternative starter for salami manufacture

Lactococcus lactis DPC4268 is widely used for Cheddar cheese manufacture in Ireland where it is recognised for its reliable fast acid producing ability in dairy environments. A transconjugant of this strain, L. lactis DPC4275, was generated which produces the broad spectrum, two-component bacteriocin lacticin 3147, which is inhibitory to a variety of undesirable Gram-positive bacteria including Clostridium, Bacillus, Enterococcus, Listeria, Staphylococcus and Streptococcus. Both DPC4268 and DPC4275 were used as single strain starters for manufacture of salamis. These products were compared with a salami manufactured with a conventional starter ( Lactobacillus sake and Staphylococcus carnosus) in terms of pH development, a w-value, weight loss, colour development and sensory characteristics. Salamis produced with either lactococcal culture exhibited pH values below 5.1 and a w-values below 0.90 which is favourable for preservation and hygienic stability. In addition, these salamis had good sensory and colorimetric qualities. A minimum lacticin 3147 concentration of 640 AU/g was detected in the salamis which were produced with L. lactis DPC4275.

Inhibitory activity of a nisin-producing starter culture on Listeria innocua in raw ewes milk Manchego cheese

The inhibitory activity of nisin-producing Lactococcus lactis subsp. lactis ESI 515 on the survival of Listeria innocua during ripening of raw ewes milk Manchego cheese was investigated. After 60 days of ripening, counts of L. innocua in cheese were 4.08 log units lower than the control when Lc . lactis subsp. lactis ESI 515 was used as a single-strain starter. Nisin activity was detected in cheeses manufactured with Lc . lactis subsp. lactis ESI 515 throughout the ripening period.

Growth reduction of Listeria spp. caused by undefined industrial red smear cheese cultures and bacteriocin-producing Brevibacterium lines as evaluated in situ on soft cheese.

The undefined microbial floras derived from the surface of ripe cheese which are used for the ripening of commercial red smear cheeses have a strong impact on the growth of Listeria spp. In some cases, these microbial consortia inhibit Listeria almost completely. From such undefined industrial cheese-ripening floras, linocin M18-producing (lin+) (N. Valdés-Stauber and S. Scherer, Appl. Environ. Microbiol. 60:3809-3814, 1994) and -nonproducing Brevibacterium linens strains were isolated and used as single-strain starter cultures on model red smear cheeses to evaluate their potential inhibitory effects on Listeria strains in situ. On cheeses ripened with lin+ strains, a growth reduction of L. ivanovii and L. monocytogenes of 1 to 2 log units was observed compared to cheeses ripened with lin strains. Linocin M18 activity was detected in cheeses ripened with lin+ strains but was not found in those ripened with lin strains. We suggest that production of linocin M18 contributes to the growth reduction of Listeria observed on model red smear cheeses but is unsufficient to explain the almost complete inhibition of Listeria caused by some undefined microbial floras derived from the surface of ripe cheeses.