| Molds are widely distributed in nature and found to be the common spoilage microorganism in bread and other baked goods.In view of the potential toxicity of chemical preservatives,biological preservatives with safe,efficient and other characteristics becomes the hotspot in recent years.As food-grade microorganisms,Propionibacterium freudenreichii and lactic acid bacteria have the potential to produce organic acids and other antifungal compounds.This thesis first isolates the main spoilage molds from bread and other bakery products,and then through strain screening,mixed culture of Propionibacterium freudenreichii and lactic acid bacteria,mixed fermentation and optimization of fermentation conditions,to improve the antifungal activity of the mixed bacteria and fermentation broth as well as application effect in the actual system.The main conclusions are as follows:(1)Isolation and identification of spoilage mold in bakery productsNine spoilage mold strains were separated and purified from 10 different bakery products.No.01-09 molds belong to Aspergillus niger,Penicillium commune,Aspergillus flavus,Cladosporium cladosporioides,Penicillium citrinum,Aspergillus chevalieri strain,Aspergillus versicolor,Aspergillus griseus and Alternaria angustiovoidea respectively.The detection rate and the earliest detection time of the 9spoilage molds showed that 01 Aspergillus niger,02 Penicillium commune and 03Aspergillus flavus were the main spoilage molds in bakery products.Therefore,the three molds were used in the subsequent antibacterial experiments.(2)Study on the antibacterial activity of the mixture of Propionibacterium freudenreichii and lactic acid bacteriaPropionibacterium freudenreichii D1,D6,and lactic acid bacteria L9,L11 were selected for their higher antifungal activity from 6 strains of Propionibacterium freudenreichii and 15 strains of lactic acid bacteria.The mixed culture of strains D6 and L9 showed strong synergistic effect and their lowest concentrations 1×10~7CFU/mL and1×10~5 CFU/mL,respectively,were needed for the highest antibacterial activity.The cell concentration of P.freudenreichii D6 and L.plantarumL9 reached 2.94 times and 2.31times,respectively,in the fermenter which were controlled p H value supplemented with carbon and nitrogen sources than that of non-fed control.It was found that spraying the mixed culture of D6 and L9 can completely inhibit the growth of penicillium on the bread slice during the 21 days storage when 100 mold spores were challenged;the durability test results showed that the sprayed mixed culture can extended the shelf life of bread from 4 to 11 days.Counting the number of viable cells of the two bacteria,it was found that Propionibacterium D6 maintained the initial number,while the lactic acid bacteria grew from the initial 5.9 log CFU/g to the highest value 7.5 log CFU/g.The concentration of lactic acid and acetic acid in bread sprayed with the mixed culture all increased and reached 0.215±0.002 and 0.155±0.003mg/g,respectively.In addition,when the mixed culture was incorporated into the dough,it was found that the fermentation and sensory characteristics of the bread were not affect,and the shelf life were extended from 3 to 6 days;the challenging test with 100 mold spores showed that the incorporated mixed bacterial can completely inhibit the growth of Aspergillus niger and Aspergillus flavus within 14 days,and delay the growth of Penicillium by 2 days.(3)Study on the antibacterial activity of the mixed fermentation broth of Propionibacterium freudenreichii and lactic acid bacteriaAmong the 15 strains of lactic acid bacteria and 6 strains of Propionibacterium freudenreichii,the lactic acid bacteria L5,L9 and Propionibacterium freudenreichii D2and D5 strains were selected for their fermentation broth with higher antifungal activity.L5 and D2 are the best combination as their mixed fermentation broth(8 days,no mold growth)has higher antibacterial activity than a single fermentation broth(4.5 days,mold growth).The maximum spore germination rate of Aspergillus flavus in the mixed fermentation broth group is(82.44±4.47%)and mycelial growth rate(8.49±0.17mm/d)were significantly lower than that of a single fermentation broth(96.03±1.13%,9.50±0.29mm/d).High temperature and protease treatment had no effect on the antifungal activity of the mixed fermentation broth.However,the p H can significantly affect its antifungal activity.Therefore,it is speculated that the main antifungal active substance in the mixed fermentation broth is organic acid.The highest content of the main organic acid acetic acid and propionic acid in the single fermentation broth is7.378±0.201 and 10.911±0.403mg/mL,while the value in the mixed fermentation increased to 12.575±0.701 and 19.293±0.839mg/mL,respectively.Propionic acid,followed by acetic acid,was the main antibacterial component in the mixed fermentation broth,and low-content azelaic acid,4-Hydroxybenzoic acid,citric acid,succinic acid,DL-p-hydroxyphenyllactic acid,L-3 phenyllactic acid,etc.,exert a bacteriostatic effect through a synergistic effect.(4)Optimization of mixed fermentation conditions of Propionibacterium freudenreichii D2 and Lactobacillus paracasei L5 and the application of the fermentation brothThe optimal fermentation conditions for Propionibacterium freudenreichii D2 and Lactobacillus paracasei L5 are as follow:fermentation time is 84h;the fermentation temperature is 30?;the fermentation p H value was controlled at 6.0;the optimal inoculation concentrations of D2 and L5 are 1×10~8 CFU/mL and 1×10~6 CFU/mL,respectively.The optimal initial glucose concentration is 4%,and the optimal nitrogen source and concentration are 1%soy peptone and 1%yeast extract FM802.Under these conditions,the production cost was reduced and the antifungal activity of the fermentation broth increased from the initial 9.96 AU/mL to 12.16 AU/mL.In-situ application experiment results showed that when 2AU/g fermentation liquid is added to the dough,the sensory evaluation and TPA value of the bread were good,and the shelf life is extended at least 22 days as compared to the control. |
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