| Red sausage is a kind of cured meat products.Nitrite is often added in the production,but it is easy to combine with amine substances in the product to form strong carcinogen N-nitrosamine under acidic conditions.In order to reduce the content of N-nitrosamines in sausage products and improve their safety quality,in this study,a rapid and simultaneous determination method of nitrate and nitrite was developed by high performance capillary electrophoresis(HPCE).On the basis of this method,lactobacillus fermentation technology was applied to fermentation-free sausage products.The fermentation time and types of Lactobacillus fermentants were screened,and then in vitro model was established.A microbial nitrification inhibitor which can inhibit the formation of N-nitrosamine was explored in the proposed system and applied to the sausage to verify its effect.It can reduce the formation of N-nitrosamines in sausage without affecting the original flavor of the product.The main contents of this paper are as follows.1.HPCE was used to detect nitrate and nitrite,focusing on the suitable detection conditions and how to eliminate the influence of interfering ions..Under the condition of 214 nm,using capillary zone electrophoresis-direct UV method with a capillary column which has an inner diameter of 50 μm,a total length of 40 cm and an effective length of 30 cm,the separation buffer of p H value,concentration,additive amount of electroosmotic flow modifier(CTAB,sixteen alkyl three methyl bromide),excluding the impact of Cl-interference and voltage on separation effect.and method of inspection,to determine the recovery rate,linearity,detection limits and precision.Results: 400 mmol/L boric acid(adjusting p H=9 with 2 mol/L KOH solution)+100 mmol/L Na2SO4+0.5 mmol/L CTAB was used as separation buffer.The separation voltage was-5 k V,the operating temperature was 25℃,0.5 psi(injection pressure of 3 447.4 Pa)for 5 seconds,for samples under 500 mmol/L of salt,the NO3-and NO2-can be separated well in 6 min.2.In order to determine the effect of different fermentation time on the formation of N-nitrosamines in sausage.Four commercial starter strains were selected: SHI-59(Staphylococcus xylosus + Staphylococcus pentosus + Lactobacillus plantarum),WBX-43(Staphylococcus bovis + Staphylococcus xylose),PRO-MIX5(Staphylococcus xylosus + Lactobacillus sake + Lactobacillus plantarum),VBM-60(Staphylococcus xylosus + Staphylococcus pentosus + Lactobacillus),and three single strains: Lactobacillus curvatus(LC),Lactobacillus pentosus(LP),Lactobacillus sake(LS)were inoculated into salted sausage fillings with 107 CFU/g respectively,fermentation at 35℃ for 12,16,20,24 h after stuffing and filling,then sampling to determine the number of lactic acid bacteria in each group.The fermented sausage products are prepared by cooking(drying,cooking,smoking,baking)the samples at each fermentation time point,and the sensory indexes,p H,nitrite,nitrate,8 kinds of biogenic amines and 9 kinds of N-nitrosamines were determined.The results showed that the longer the fermentation time was,the lower the content of N-nitrosamine in sausage was.At this fermentation time point,PRO-MIX5 is particularly outstanding.After 12 hours of fermentation,the nitrite residue in the product can be significantly reduced(7.58 mg/kg),the total amount of nine N-nitrosamines is as low as 8.68 μg/kg,the N-dimethylnitrosamines(NDMA)content(2.91 μg/kg)was in line with the national limit standard.Secondly,LC bacteria can also significantly reduce the total amount of nine N-nitrosamines to 9.46 μg/kg,but the effect of reducing nitrite residues(47.73 mg/kg)is poor.3.At the fixed fermentation time(12h),different Lactobacillus starters were screened.Seven commercial Lactobacillus starters commonly added to fermented meat products: Staphylococcus xylose + Staphylococcus pentosus(THM-17),PRO-MIX5,Staphylococcus xylose + Staphylococcus meat + Lactobacillus sake(WBL-45),VHI-41,SHI-59,WBX-43,VBM-60,three strains(LC,LP,LS)were screened to inhibit the formation of N-nitrosamines,Five strains,Pediococcus Pentosans(PP),Staphylococcus xylose(SX),Staphylococcus bovis(SB),Lactobacillus sake-1(LS-1),Lactobacillus plantarum(LPT)isolated from commercial lactic acid bacteria starter were inoculated with 107 CFU/g in the cured minced meat.Fermentation for 12 hours after filling enema.After fermentation,the number of lactic acid bacteria and the total number of bacteria were measured.The finished products were prepared by cooking.The sensory,p H,color difference,texture,nitrite,nitrate,biogenic amines,N-nitrosamines and other indicators were determined.The results showed that LPT and SX were better than CK in application,which could significantly reduce the nitrite residue to 10.84 and 10.13 mg/kg,and the nitrate content was 38.27 and 52.09 mg/kg,respectively.LPT could significantly reduce the total amount of eight biogenic amines in the product.To 431.08 mg/kg,the two starters could reduce NDMA content to 1.29 and 2.51 μg/kg,respectively,and inhibit the formation of 9 N-nitrosamines to 19.68 and 17.96 μg/kg,respectively,which significantly improved the safety quality of sausage products.4.The lactic acid bacteria starter PRO-MIX5,LC,LPT and SX which can effectively inhibit the formation of N-nitrosamines in sausage were screened by preliminary experiments,and explore the mechanism of inhibiting N-nitrosamine.First,PRO-MIX5 was cultured in minced meat medium and MRS medium,and then screened step by step in nitrite degradation system and NDMA formation system,eliminating the effects of metabolites,intracellular enzymes and bacteria respectively.The fragments of lactic acid bacteria were obtained.The effect of NDMA inhibition was determined.Four kinds of lactic acid bacteria starter,kinds of inducers added to MRS medium and the amount of bacterial debris added were screened.Finally,the dominant starter and the best culture mode were determined.The results showed that lactic acid bacteria inhibited the formation of N-nitrosamines mainly by the function of bacterial debris.PRO-MIX5 had the best inhibiting effect on the formation of N-nitrosamines by using MRS broth medium containing 9 kinds of N-nitrosamines at the concentration of 0.04 μg/m L,in the range of 0.2%-1.5%,the inhibition rate of NDMA can reach about 60%.5.PRO-MIX5 fragments known as microbial nitrification inhibitor(MNI)were screened by preliminary exploration.MNI can inhibit NDMA formation in vitro simulation system.In order to verify the blocking effect of MNI on N-nitrosamines in sausage and its related quality,the sausage was prepared by adding MNI to the cured filling at the ratio of 0.05%,0.25% and 0.50% of meat quality.Meanwhile,the blank control check group was set up.The sausage was prepared by filling,enema,cooking.By measuring the sensory,p H,redness,elasticity,nitrite content,biogenic amine content and N-nitrosamine content of sausage,the effects of MNI on the sensory and safety quality of sausage products were explored.The results showed that compared with CK group,the nitrite residue in sausage added 0.05% MNI was 21.44 mg/kg,the nitrite degradation rate was 18.92%,the total inhibition rate of nine N-nitrosamines was 41.04%,and the inhibition rate of NDMA was 52.87%.At this time,the sensory evaluation score of sausage was the highest(26 points).There was no significant difference in the p H value,redness value,elasticity of sausage between CK group,but there were 8 kinds of total.The content of biogenic amines in CK group was 19.64% higher than CK group,but it didn’t exceed the limits prescribed by relevant regulations.The application effect was good. |
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