Antimicrobial, Growth, Acid and Aroma Properties of the Dominant Microflora Isolated at Different Stages of Gouda Cheese Production

Abstract

The microflora of Gouda cheese is dominated by starter culture lactic acid bacteria (SLAB) and non-starter organisms, which are not deliberately added. Both of these groups of microorganisms are responsible for development of the cheese flavor and organoleptic properties during ripening. This study generally aimed to characterize the dominant microbial species at different stages of Gouda cheese production in order to identify non-starter bacterial strains with positive effects for possible application as starter culture adjuncts in fermented dairy matrices. Ten (10) independent samples from the Gouda cheese production line at Sanatos Dairies (U) Limited were collected at each of the following stages of production: raw milk, pasteurized milk, non-spiced Gouda cheese of one week, non-spiced Gouda cheese of one month, and spiced Gouda cheese of one month. The samples were enumerated within 8 h, for total viable counts (TVC), yeast and molds, total Lactococci, Staphylococcus spp., total coliforms/E. coli and total LAB using standard protocols. After enumeration of plates, colonies with different morphologies were purified (isolated) and examined for cell morphologies, Gram reaction and biochemical characteristics for presumptive identification and subsequently speciated the bacterial isolates using 16S rDNA sequence analysis. The dominant species were examined for genetic diversity using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering. Selected isolates from each of the major clusters were evaluated for antimicrobial activity, growth, acid and aroma production capacity in Ultra High Temperature (UHT) milk in order to prospect their possible application as starter culture adjuncts in fermented milk. LAB (2.34 - 7.77 log CFU/g) dominated the microflora of raw milk and cheese samples. TVC in respective samples corresponded with LAB counts implying that LAB were the major organisms in different samples examined. Of the 125 microbial isolates, 121 (96.8%) were found to be bacteria and were presumptively identified as Staphylococcus spp. (26.5%), Streptococcus, or Enterococcus (23.9%), Klebsiella, Enterobacter, Eschericia coli, Salmonella, Proteus, Serratia, or Yersinia (19%), Bacillus, Streptomyces, Listeria or Corynebacterium (9.9%) and Lactobacillus or Clostridium were 14.1%. 16S rDNA sequence analysis delineated 28 species with Lactococcus lactis (12.5%), Streptococcus lutetiensis (8.8%) and Lactobacillus plantarum (5%) being the major LAB species detected in all cheese samples; the rest of the isolates were either spoilage or pathogenic bacteria that could have found their way into the cheese post production. UPGMA clustering of these organisms associated them according to their different species and source of isolation; L. lactis from non-spiced Gouda cheese matured for one week were clustered together with L. lactis from spiced Gouda cheese matured for one month implying similar genetic relatedness. Each of the S. lutetiensis and L. plantarum clusters were only obtained from spiced Gouda cheese matured for one month. Thus, Lactococcus lactis and Lactobacillus plantarum were evaluated further for growth, antimicrobial activity against selected pathogenic bacterial strains, acid and aroma production properties. Single and mixed cultures of these organisms had similar growth patterns. All isolates inhibited E. coli, Salmonella and S. aureus on solid media assays but only one isolate of Lc. lactis (Gw-05) inhibited E. coli and Salmonella in the cell-free supernatant assay. In single culture, Lc. lactis had better acid production properties than Lb. plantarum. Mixed of these organisms enhanced acid production. During short term ripening, L. plantarum synthesized more organic acids, ketones, esters and aldehydes (13.7%, 2.3%, 9.3%, and 0.8%, respectively) than L. lactis which mostly produced organic acids and esters (38.7%, 6.6%, respectively). Co-culture of these organisms inhibited aroma production; only three organic acids and one ester were produced. During long term ripening, each of L. lactis and L. plantarum only produced organic acids and esters. However, L. plantarum produced more esters than L. lactis. Co-culture of the organisms enhanced aroma production resulting from synthesis of organic acids, esters and one ketone. It was therefore concluded that LAB dominate the microflora of Gouda cheese production. L. lactis has better prospects for application as primary starter culture in dairy products because they promote rapid acidification of milk, which is desirable for curd formation. Short-term fermentation of milk by single culture of L. lactis and L. plantarum enhance aroma production, while long term ripening enhances aroma production in mixed culture. Single cultures of these organisms could therefore enhance aroma formation in yoghurt, which requires short-term fermentation, whereas mixed culture could be applied in cheese, which requires long-term ripening.