A. Corsetti

ANTIMOULD ACTIVITY OF SOURDOUGH LACTIC ACID BACTERIA: IDENTIFICATION OF A MIXTURE OF ORGANIC ACIDS PRODUCED BY LACTOBACILLUS SANFRANCISCO CB1

Abstract Sourdough lactic acid bacteria, cultivated in wheat flour hydrolysate, produced antimould compounds. The antimould activity varied greatly among the strains and was mainly detected within obligately heterofermentative Lactobacillus spp. Among these, Lb. sanfrancisco CB1 had the largest spectrum. It inhibited moulds related to bread spoilage such as Fusarium, Penicillium, Aspergillus and Monilia. A mixture of acetic, caproic, formic, propionic, butyric and n-valeric acids, acting in a synergistic way, was responsible for the antimould activity. Caproic acid played a key role in inhibiting mould growth.

Latent Bioactive Peptides in Milk Proteins: Proteolytic Activation and Significance in Dairy Processing

Referee: Mr. Dick Fitzgerald, Ph.D., Life Science Department, University of Limerick, Limerick, Ireland After a brief description of the properties of bioactive peptides, the proteolytic activation of the bioactive sequences from milk protein precursors is discussed. The ability of proteolytic enzymes from various sources, especially from lactic acid bacteria, to release bioactive peptides and the physiological and biotechnological significance of these peptides in dairy products are reviewed.

Phenotypic and molecular identification and clustering of lactic acid bacteria and yeasts from wheat (species Triticum durum and Triticum aestivum) sourdoughs of Southern Italy.

The microflora of 25 wheat sourdoughs from the Apulia region, Southern Italy, was characterized. The sourdoughs were mainly produced from Triticum durum wheat. The number of lactic acid bacteria and yeasts ranged from ca. log 7.5 to log 9.3 colony forming units (cfu)/g and from log 5.5 to log 8.4 cfu/g, respectively. About 38% of the 317 isolates of lactic acid bacteria were identified by conventional physiological and biochemical tests. Phenotypic identification was confirmed by 16S rDNA and 16S/23S rRNA spacer region PCR. Overall, 30% of the isolates were identified as Lactobacillus sanfranciscensis, 20% as Lb. alimentarius, 14% as Lb. brevis, 12% as Leuconostoc citreum, 7% as Lb. plantarum, 6% as Lactococcus lactis subsp. lactis, 4% as Lb. fermentum and Lb. acidophilus, 2% as Weissella confusa and 1% as Lb. delbrueckii subsp. delbrueckii. Some of these species have not been previously isolated from sourdoughs. Since bakers yeast is widely used in sourdough production, Saccharomyces cerevisiae was largely found. The phenotypical relationships within the main lactic acid bacteria identified were established by using cluster analysis. A microbial map of the 25 sourdoughs was plotted showing characteristic associations among lactic acid bacteria and differences in the lactic acid bacteria species which were mainly due to the species of wheat flour, use of bakers yeast and type of bread.

Characterization of non-starter lactic acid bacteria from Italian ewe cheeses based on phenotypic, genotypic, and cell wall protein analyses.

ABSTRACT Non-starter lactic acid bacteria (NSLAB) were isolated from 12 Italian ewe cheeses representing six different types of cheese, which in several cases were produced by different manufacturers. A total of 400 presumptive Lactobacillus isolates were obtained, and 123 isolates and 10 type strains were subjected to phenotypic, genetic, and cell wall protein characterization analyses. Phenotypically, the cheese isolates included 32% Lactobacillus plantarumisolates, 15% L. brevis isolates, 12% L. paracasei subsp. paracasei isolates, 9% L. curvatus isolates, 6% L. fermentum isolates, 6%L. casei subsp. casei isolates, 5% L. pentosus isolates, 3% L. casei subsp.pseudoplantarum isolates, and 1% L. rhamnosusisolates. Eleven percent of the isolates were not phenotypically identified. Although a randomly amplified polymorphic DNA (RAPD) analysis based on three primers and clustering by the unweighted pair group method with arithmetic average (UPGMA) was useful for partially differentiating the 10 type strains, it did not provide a species-specific DNA band or a combination of bands which permitted complete separation of all the species considered. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis cell wall protein profiles clustered by UPGMA were species specific and resolved the NSLAB. The only exceptions were isolates phenotypically identified asL. plantarum and L. pentosus or as L. casei subsp. casei and L. paracaseisubsp. paracasei, which were grouped together. Based on protein profiles, Italian ewe cheeses frequently contained four different species and 3 to 16 strains. In general, the cheeses produced from raw ewe milk contained a larger number of more diverse strains than the cheeses produced from pasteurized milk. The same cheese produced in different factories contained different species, as well as strains that belonged to the same species but grouped in different RAPD clusters.

The sourdough microflora. Interactions between lactic acid bacteria and yeasts: metabolism of carbohydrates

Interactions betweenLactobacillus brevis subsp.lindneri CB1,L. plantarum DC400,Saccharomyces cerevisiae 141 andS.exiguus M14 from sourdoughs were studied in a co-culture model system using a synthetic medium. The lack of competition for maltose whenS.exiguus M14 was present in co-culture with each of the lactic acid bacteria (LAB) enhanced the bacterial cell yield and lactic and acetic acid production.L.brevis subsp.lindneri CB1 resting cells hydrolysed maltose and accumulated glucose in the medium, allowing the growth of maltose negative yeast.S.cerevisiae 141 competed greatly with each of the LAB for glucose and only withL.plantarum DC400 for fructose, causing a decrease in the bacterial cell number and in acid production. As a result of the glucose and fructose availability after the invertase activity of both yeasts,L.plantarum DC400 grew optimally in the presence of sucrose as a carbon source. All of the interactions indicated were confirmed by studying the behaviour of the co-cultures in wheat flour hydrolysate.

Interactions between yeasts and bacteria in the smear surface-ripened cheeses

In the initial phase of ripening, the microflora of bacterial smear surface-ripened cheeses such as Limburger, Taleggio, Brick, Münster and Saint-Paulin and that of surface mould-ripened cheeses such as Camembert and Brie may be similar, but at the end of the ripening, bacteria such as Brevibacterium spp., Arthrobacter spp., Micrococcus spp., Corynebacterium spp. and moulds such as Penicillium camemberti are, respectively, the dominant microorganisms. Yeasts such as Candida spp., Cryptococcus spp., Debaryomyces spp., Geotrichum candidum, Pichia spp., Rhodotorula spp., Saccharomyces spp. and Yarrowia lipolytica are often and variably isolated from the smear surface-ripened cheeses. Although not dominant within the microorganisms of the smear surface-ripened cheeses, yeasts establish significant interactions with moulds and especially bacteria, including surface bacteria and lactic acid bacteria. Some aspects of the interactions between yeasts and bacteria in such type of cheeses are considered in this paper.

Microbiological and biochemical characteristics of Canestrato Pugliese cheese made from raw milk, pasteurized milk or by heating the curd in hot whey.

Canestrato Pugliese cheeses were produced from raw ewes milk (R and R(II) cheeses), pasteurized ewes milk (P cheese) and by heating the curd in hot whey according to a traditional protocol (T cheese). R(II) differed from R cheese mainly by having been produced from raw milk with a higher number of somatic cells, 950.000 vs. 750.000 ml(-1), respectively. Compared to P and T cheeses, R and R(II) cheeses had a higher concentration (one or two orders of magnitude) of cheese-related bacteria such as adventitious mesophilic lactobacilli, enterococci and staphylococci. At the end of ripening, all cheeses contained less than 1.0 log cfu g(-1) of total and fecal coliforms, and Escherichia coli and Staphylococcus aureus were not detected. As shown by phenotypic identification and RAPD-PCR, R cheese contained the largest number of mesophilic lactobacilli species and the greatest diversity of strains within the Lactobacillus plantarum species. Primary proteolysis did not differ appreciably among the cheeses. On the contrary, both urea-PAGE and the RP-HPLC analyses of the water-soluble N fractions showed the more complex profiles in cheeses produced by raw milks. R and R(II) cheeses had the highest values of water-soluble N/total N (ca. 30%) and the highest concentration of total free amino acids (ca. 40 mg g(-1) which approached or exceeded those reported for Italian cheeses with very high level of proteolysis during ripening. The main differences between R-R(II) and P-T cheeses were the concentrations of aspartic acid, proline, alanine, isoleucine, histidine and lysine. The water-soluble extracts of R and R(II) cheeses contained levels of amino-, imino- and di-peptidase activities, which were about twice those found in P and T cheeses. Cheeses differed slightly in the concentration of total free fatty acids that ranged between 1673 and 1651 mg kg(-1) in R and R(II) cheeses, and 1397 and 1334 mg kg(-1) in P and T cheeses. Butyric, caproic, capric, palmitic, oleic and linoleic acids were found at the highest concentrations.

Characterization of bacteriocin-like inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity

Aims:u2002 To identify and characterize bacteriocion‐producing lactic acid bacteria (LAB) in sourdoughs and to compare in vitro and in situ bacteriocin activity of sourdough‐ and nonsourdough LAB.

Comparison of the microbiological, compositional, biochemical, volatile profile and sensory characteristics of three Italian PDO ewes’ milk cheeses

Abstract Three batches of three Italian ewes’ milk cheeses (Canestrato Pugliese, Fiore Sardo and Pecorino Romano), which were manufactured under the Protected Denomination of Origin (PDO), were compared for microbiological, compositional, biochemical, volatile profile and sensory characteristics. After ca. 1 year of ripening, the three cheeses had a complex microbial population, composed mainly of non-starter lactic acid bacteria (NSLAB); several species of mesophilic lactobacilli and enterococci were identified. Pecorino Romano had a very high level of NaCl, 8.7±0.1% w/w. The level of pH 4.6-soluble nitrogen increased in the order: Canestrato Pugliese>Fiore Sardo>Pecorino Romano. Urea-PAGE electrophoresis showed that the hydrolysis of α s1 -casein was more consistent than β -casein. RP-HPLC profiles of the ethanol-soluble and ethanol-insoluble fractions of the pH 4.6-soluble nitrogen showed differences between the cheeses which agreed with the content of free amino acids. Glutamic acid, histidine, valine, isoleucine, leucine and phenylalanine were the free amino acids present at the highest levels in all the cheeses. Sixty-two volatile components were identified by Gas Chromatography-Mass Spectrometry analysis of steam distillates of the cheeses. The volatile profile of the three cheeses differed significantly. Esters were the main volatiles in the Canestrato Pugliese cheese but were the lowest in Fiore Sardo cheese. Ketones and alcohols were the principal class of volatile components in Fiore Sardo and Pecorino Romano cheeses, respectively. Lactones and products of the breakdown of the sulphur-containing amino acids were found at considerable levels in all cheeses, but aldehydes were present at low levels. Only 30 carboxylic acids were identified; Canestrato Pugliese had the highest content of total acids. Butanoic, hexanoic, octanoic and decanoic acids represented the 30–33% of the total carboxylic acids in the cheeses. The cheeses were subjected to descriptive sensory analysis; 19 discriminating and 2 descriptive attributes were analysed by Principal Component Analysis (PCA). The sensory characteristics of the three ewes’ milk cheeses were distinct.

Volatile compound and organic acid productions by mixed wheat sour dough starters: influence of fermentation parameters and dynamics during baking

Lactobacillus brevis subsp. lindneri CB1, Lactobacillus plantarum DC400 and Saccharomyces cerevisiae 141 or Saccharomyces exiguus M14 were used as starters to produce wheat sour dough breads. Sour doughs with higher relative percentage of yeast fermentation products (1-propanol, 2-methyl-1-propanol, 3-methyl-1-butanol and ethanol) and with higher total peak area of volatile compounds, or with a more complete profile (higher amounts of ethylacetate and lactic and acetic acids, and the presence of carbonyl compounds) were produced by the associations between lactic acid bacteria (LAB) and S. cerevisiae 141 or S. exiguus M14, respectively. Low temperature (25°C) and sour dough firmness (dough yield 135) were appropriate for LAB souring activities but limited yeast metabolism. Raising the temperature to 30°C and semi-fluid sour doughs gave more complete volatile profiles. Flour ash content from 0·55–1% positively influenced the total amount of volatiles and lactic and acetic acid productions. While at 3 h the sour dough was mainly characterized by iso-alcohols, an increase of leavening time up to 9 h gave a total amount of volatiles about three times higher than that at 5 h and strengthened the LAB contribution. The additions of fructose and citrate to the dough enhanced the acetic acid and volatile synthesis by LAB, respectively. After baking, the ethanol disappeared, 2-methyl-1-propanal was synthetized, lactic and acetic acids remained constant, the total amount of volatiles was reduced to a level