羊肉发酵干香肠品质特性及挥发性风味变化及其形成机理研究
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摘要
本研究以内蒙古羊肉和羊尾脂肪为原料,接种由产香肉葡萄球菌(St.carnosus)、戊糖乳杆菌(L.pentosus)和戊糖片球菌(P.pentosaceus)复合而成的发酵剂以及添加天然香辛料(黑胡椒和孜然),采用相同的工艺配方和工艺条件生产四组羊肉发酵香肠,分别为对照组(CO)、发酵剂组(SC)、发酵剂+黑胡椒组(SC+BP)和发酵剂+黑胡椒+孜然组(SC+BP+C)。同时采用先进的HPLC、GC、DHS和GC-MS等技术,重点研究复合发酵剂、香辛料和工艺参数对羊肉发酵香肠品质特性和挥发性风味成分的影响,探索羊肉发酵香肠挥发性风味成分的变化及其形成机理,旨在为下一步羊肉发酵香肠的工业化生产和优良发酵剂及新产品的开发提供理论依据。具体研究内容和结果如下:
分析了羊肉发酵香肠在加工和贮藏过程中理化特性的变化规律。结果发现:试验各组的pH值在发酵阶段(第1.5天)迅速下降到5.0以下,显著低于对照组(p<0.05),在干燥成熟和贮藏过程中pH值始终维持在5.3以下;试验组Aw值在成熟后(第7天)均下降到0.88左右(p<0.05),在贮藏过程中继续下降到0.85;水分含量在干燥阶段显著下降(p<0.05);脂肪含量和蛋白质含量在整个加工和贮藏过程中变化不明显,差异不显著(p>0.05)。
游离氨基酸和游离脂肪酸随加工和贮藏时间的变化规律表明:试验组蛋白质发生了很强的水解变化,贮藏后期试验组的蛋白水解指数(PI)在14%～16%之间,非蛋白氮(NPN)和总游离氨基酸(TFAA)的含量在贮藏过程中升高速度最快,表现出蛋白质随温度升高以及发酵剂的使用而快速水解的态势,尤其是发酵剂+黑胡椒+孜然组的非蛋白氮(NPN)和总游离氨基酸(TFAA)变化均显著高于对照组(p<0.05);大部分游离氨基酸(FAA)在干燥成熟过程中含量显著升高(p<0.05),均增加了5～10倍以上,其中谷氨酸(Glu)、亮氨酸(Leu)、赖氨酸(Lys)、丙氨酸(Ala)、缬氨酸(Val)和精氨酸(Arg)含量较高。试验组和对照组的硫代巴比妥酸(TBARS)值变化范围在0～2mg/kg之间,没有出现脂肪的过度氧化酸败,试验组的TBARS值在成熟后低于对照组(自然发酵)。棕榈酸(C16:0)、硬脂酸(C18:0)和油酸(C18:1)是发酵香肠生产过程中的主要游离脂肪酸,其中单不饱和脂肪酸中油酸(C18:1)含量呈显著升高趋势(p<0.05),饱和脂肪酸(SFA)中的硬脂酸(C18:0)和羊蜡酸(C10:0)占总游离脂肪酸(TFFA)的百分含量在发酵香肠贮藏过程中呈下降趋势,而且试验组C18:0的百分含量低于对照组。SC+BP组和SC+BP+C组的单不饱和脂肪酸(MUFA)和多不饱和脂肪酸(PUFA)占总游离脂肪酸(TFFA)的百分含量在贮藏过程中明显下降;总游离脂肪酸(TFFA)在贮藏过程中显著上升(p<0.05),说明温度提高有利于TFFA的释放。
试验各组乳酸菌在发酵香肠成熟和贮藏过程中始终处于优势菌状态并保持着较高的水平(108cfu/g),显著高于对照组(p<0.05),细菌总数(106cfu/g)在成熟后和贮藏过程中显著低于对照组(108cfu/g)(p<0.05),而葡萄球菌/微球菌在贮藏后期显著高于对照组(p<0.05),本试验添加发酵剂和香辛料有利于保证产品的安全性和贮藏性。
对成熟后(7天)及贮藏过程(14天、21天和35天)各组发酵香肠进行了感官评价,结果建立了色泽、风味、组织状态和滋味与整体可接受性之间的线性模型公式为Y=0.08X1-0.147X2+0.222X3+0.836X4 (R2=0.999),感官特性权重依次为滋味>组织状态>风味>色泽。试验组的组织状态、多汁性、滋味和整体可接受性在贮藏中期(21天)感官评分明显增加,其膻味下降也较对照组明显,说明添加发酵剂和香辛料可以降低羊肉发酵香肠中的膻味,并提高其整体可接受性。蛋白水解指数(PI)和TBARS值与发酵香肠的整体可接受性呈正相关,蛋白水解指数(PI)的权重系数远远大于TBARS值,说明发酵香肠的整体可接受性主要决定于非蛋白氮、小肽和游离氨基酸等蛋白水解产物。
利用动态顶空制样(DHS)/吹扫捕集(P & T)技术结合气-质联机(GC-MS)对羊肉发酵香肠在灌肠后(0天)、发酵后(1.5天)、成熟后(7天)、贮藏中期(21天)和贮藏后期(35天)的挥发性风味成分进行了分析鉴定。在对照组、发酵剂组、发酵剂+黑胡椒组和发酵剂+黑胡椒+孜然组分别检出93种、83种、85种和90种挥发性风味成分,这些成分可归类为醛类、萜类、烷烃类、醇类、酮类、酸类、芳烃类、酯类、含氮化合物、含硫化合物和呋喃类化合物,其中酯类和烷烃类种类最多,分别占总种类数的16%～22%和15%～20%。
在发酵结束(1.5天),试验组检出的挥发性风味种类较少,但却检出大量的S-乳酸异丙酯/乳酸异戊酯,同时检出大量碳水化合物的水解产物2,3-丁二酮(奶油香)和3-羟基-2-丁酮(黄油味)。发酵剂+黑胡椒组检出了4-甲基庚烷、3-甲基壬烷和4-甲基癸烷等支链烷烃。本试验各组检出了3-甲基丁醛(黑巧克力)、己醛(青草香)、辛醛(草香,甜香)、三甲基吡嗪(坚果烤香)、甲氧基乙酸乙酯(甜香)等关键芳香化合物,它们主要形成于成熟过程中的氨基酸Strecker降解和脂肪水解氧化以及美拉德反应等,对发酵香肠的风味贡献较大。来源于黑胡椒的石竹烯(辛辣气味)、β-水芹烯(胡椒气味)、3-蒈烯(辛辣味)是添加香辛料羊肉发酵香肠的主要风味。
In this study, these sausages were manufactured with mutton and sheep tail fat obtained from sheep fed in Inner Mongolia and inoculated the strain of Staphylococcus carnosus in combination with acidifying strain of Lactobacillus pentosus and Pediococcus pentosaceus as starter culture and spices for the production. 4 groups fermented sausages manufacture at the same technology and formula were named control group (CO), starter culture group (SC), starter culture and black pepper group ( SC+BP ), starter culture and black pepper and cumin (SC+BP+C), respectively. The aims of this work were to investigate the effects of quality characteristics and formation mechanism of volatile flavor compounds of mutton fermented dry sausages with starter cultures combination and spices during ripening and storage by HPLC, GC, DHS and GC-MS. A further aim was to provide a theory basis in realizing the industrial production of mutton fermented sausages and developing idea commercial starter cultures and new products in the future. The contents and results were as follows.
The changes of physico-chemical properties of mutton dry fermented sausages during ripening and storage were analyzed. The results showed that: the pH values of fermented sausages inoculated the starter culture decreased ( p<0.05 ) , dropped to below 5.0 at the end of the fermentation ( 1.5 days ) and were significantly lower than the control; the pH values of all sausages increased slowly and the spice groups increased ( p<0.05 ) during the drying and ripening and kept below 5.3;the Moisture, Aw values of all sausages decreased gradually, and Aw values decreased from 0.88 to 0.85 after ripening; the changes of protein and fat contents were slight(p>0.05).
The changes of the FFA (free fat acid) and the FAA (free amino acid) of mutton fermented sausages were studied during ripening and storage. The results showed that: the proteolysis indexes (PI) of the all sausages were percent 14 to 16 and the NPN and TFAA contents increased gradually during processing and enhance sharply during the middle storage. The changes of NPN and TFAA of batch SC+BP+C were significantly higher than the control(p<0.05). The contents of most FAA increased quickly above tenfold at the drying and ripening stage(p<0.05)and Arg, Glu, Leu, Lys, Ala and Val were dominant in concentration. Since the concentrations of most FAA in all sausages were 5-10 times of their corresponding sensory thresholds, the FAA might have make an important contribution to the formation of mutton fermented sausages flavor. The changes of TBARS values in all sausages were 0 mg/kg to 2 mg/kg and were not fat excessive oxidation. In general, the FFA of all the samples showed a significant increase during ripening and storage. Large amount of free fatty acids were accumulated during the storage and oleic acid,palmitic acid and stearic acid were the most abundant free fatty acids in treatment.
The oleic acid contents of MUFA were improved dramatically (p<0.05), while the proportion of capric acid and stearic acid decreased during the storage , especially the sausages inoculated starter culture combination and spices were lower than the control (uninoculated). The proportion of MUFA and PUFA in the batch SC+BP and SC+BP+C decreased obviously during storage. These indicated that the MUFA and PUFA were the key fatty acids in lipid oxidation. Temperature had great effect on lipase and microbial enzyme, in this study, the TFFA increased significantly during the storage(p<0.05).
A few significant differences were found in counts of microorganism between the three types of sausages and the control. Lactic acid bacteria (LAB) in sausages made with starter cultures combination and spices increased to 108cfu/g ( p<0.05 ) and kept this level in storage period, while the total bacteria counts were significantly lower than in the control and Micrococci-staphylococci were higher than the control ( p<0.05 ) at the middle period of storage. The addition of combined starter cultures and spices improved the quality attributes and shelf-life of mutton fermented dry sausages.
The sensory index of all sausages at the end of ripening, earlier storage, middle storage and late storage were evaluated. The relativity of the color, flavor, texture, taste and the quality was evaluated by formula Y=0.08X1-0.147X2+0.222X3+0.836X4 (R2=0.999) and the sensory weight were successively taste > texture > flavor > color, which indicated that taste played an important role. The sensory scores of taste and the quality with combined starter cultures and spices quickly increased at the middle storage and the goaty flavor was lower than the control, which indicated that inoculating starter cultures and spices could decrease the goaty flavor and improve the quality. The correlation of the proteolysis index (B1), TBARS (B2) and the quality were evaluated by formula Y=42.354 B1+0.457 B2 (R2=0.981), the weight coefficient of PI was much larger than of the TBARS, which showed NPN, small peptide and FAA were determinant in the quality of mutton fermented sausages.
The volatile flavor compounds in all treated fermented sausages at the end of clyster, end of fermentation, end of ripening, middle stage and late stage were extracted and identified by dynamic headspace sampling (DHS) and purge-trap (P & T) methods and GC/MS system. There were 93, 83, 85 and 90 flavor compounds were detected with capillary column HP-5 from the batch CO, SC, SC+BP and SC+BP+C, respectively. These volatile flavor compounds in mutton fermented sausages could be clustered in the following chemical families: alkanes and alkenes, aromatic hydrocarbons, alcohols, aldehydes, ketones, carboxylic acids, esters, nitrogenous compounds, sulphur compounds, furan compounds and terpenoids, which showed different changing rules, especially esters and alkanes (alkenes) were more than the others whose proportions were 16%-22% and 15%-20% respectively.
The sorts of volatile flavor in all treated fermented sausages was small at the end of fermentation, but large amount of (S)-Isopropyl lactate / Isoamy lactate ,2,3-Butanedione (cream) and 3-hydroxy-2-butanone (butter) were detected, which were originated from the carbohydrate catabolism. The branched chain alkane, such as 4-methyl-decane, 3-methyl-nonane, 4-methyl-heptane et al. were detected in the SC+BP batch. The key aroma-active compounds were 3-methylbutanal (dark chocolate), hexanal (grass), octanal (green, sweet), methoxy-acetic ethyl ester (sweet), trimethyl-Pyrazine (nut, roast), which were originated from lipid degradation and oxidation, amino acid Strecker reaction and the odor-active compounds contributed to fermented sausages odor together. beta.- Phellandrene (pepper), 3-Carene (pungent) and Caryophyllene (pungent) originated from the black pepper were main flavor in the mutton fermented dry sausages added spices.
分析了羊肉发酵香肠在加工和贮藏过程中理化特性的变化规律。结果发现:试验各组的pH值在发酵阶段(第1.5天)迅速下降到5.0以下,显著低于对照组(p<0.05),在干燥成熟和贮藏过程中pH值始终维持在5.3以下;试验组Aw值在成熟后(第7天)均下降到0.88左右(p<0.05),在贮藏过程中继续下降到0.85;水分含量在干燥阶段显著下降(p<0.05);脂肪含量和蛋白质含量在整个加工和贮藏过程中变化不明显,差异不显著(p>0.05)。
游离氨基酸和游离脂肪酸随加工和贮藏时间的变化规律表明:试验组蛋白质发生了很强的水解变化,贮藏后期试验组的蛋白水解指数(PI)在14%～16%之间,非蛋白氮(NPN)和总游离氨基酸(TFAA)的含量在贮藏过程中升高速度最快,表现出蛋白质随温度升高以及发酵剂的使用而快速水解的态势,尤其是发酵剂+黑胡椒+孜然组的非蛋白氮(NPN)和总游离氨基酸(TFAA)变化均显著高于对照组(p<0.05);大部分游离氨基酸(FAA)在干燥成熟过程中含量显著升高(p<0.05),均增加了5～10倍以上,其中谷氨酸(Glu)、亮氨酸(Leu)、赖氨酸(Lys)、丙氨酸(Ala)、缬氨酸(Val)和精氨酸(Arg)含量较高。试验组和对照组的硫代巴比妥酸(TBARS)值变化范围在0～2mg/kg之间,没有出现脂肪的过度氧化酸败,试验组的TBARS值在成熟后低于对照组(自然发酵)。棕榈酸(C16:0)、硬脂酸(C18:0)和油酸(C18:1)是发酵香肠生产过程中的主要游离脂肪酸,其中单不饱和脂肪酸中油酸(C18:1)含量呈显著升高趋势(p<0.05),饱和脂肪酸(SFA)中的硬脂酸(C18:0)和羊蜡酸(C10:0)占总游离脂肪酸(TFFA)的百分含量在发酵香肠贮藏过程中呈下降趋势,而且试验组C18:0的百分含量低于对照组。SC+BP组和SC+BP+C组的单不饱和脂肪酸(MUFA)和多不饱和脂肪酸(PUFA)占总游离脂肪酸(TFFA)的百分含量在贮藏过程中明显下降;总游离脂肪酸(TFFA)在贮藏过程中显著上升(p<0.05),说明温度提高有利于TFFA的释放。
试验各组乳酸菌在发酵香肠成熟和贮藏过程中始终处于优势菌状态并保持着较高的水平(108cfu/g),显著高于对照组(p<0.05),细菌总数(106cfu/g)在成熟后和贮藏过程中显著低于对照组(108cfu/g)(p<0.05),而葡萄球菌/微球菌在贮藏后期显著高于对照组(p<0.05),本试验添加发酵剂和香辛料有利于保证产品的安全性和贮藏性。
对成熟后(7天)及贮藏过程(14天、21天和35天)各组发酵香肠进行了感官评价,结果建立了色泽、风味、组织状态和滋味与整体可接受性之间的线性模型公式为Y=0.08X1-0.147X2+0.222X3+0.836X4 (R2=0.999),感官特性权重依次为滋味>组织状态>风味>色泽。试验组的组织状态、多汁性、滋味和整体可接受性在贮藏中期(21天)感官评分明显增加,其膻味下降也较对照组明显,说明添加发酵剂和香辛料可以降低羊肉发酵香肠中的膻味,并提高其整体可接受性。蛋白水解指数(PI)和TBARS值与发酵香肠的整体可接受性呈正相关,蛋白水解指数(PI)的权重系数远远大于TBARS值,说明发酵香肠的整体可接受性主要决定于非蛋白氮、小肽和游离氨基酸等蛋白水解产物。
利用动态顶空制样(DHS)/吹扫捕集(P & T)技术结合气-质联机(GC-MS)对羊肉发酵香肠在灌肠后(0天)、发酵后(1.5天)、成熟后(7天)、贮藏中期(21天)和贮藏后期(35天)的挥发性风味成分进行了分析鉴定。在对照组、发酵剂组、发酵剂+黑胡椒组和发酵剂+黑胡椒+孜然组分别检出93种、83种、85种和90种挥发性风味成分,这些成分可归类为醛类、萜类、烷烃类、醇类、酮类、酸类、芳烃类、酯类、含氮化合物、含硫化合物和呋喃类化合物,其中酯类和烷烃类种类最多,分别占总种类数的16%～22%和15%～20%。
在发酵结束(1.5天),试验组检出的挥发性风味种类较少,但却检出大量的S-乳酸异丙酯/乳酸异戊酯,同时检出大量碳水化合物的水解产物2,3-丁二酮(奶油香)和3-羟基-2-丁酮(黄油味)。发酵剂+黑胡椒组检出了4-甲基庚烷、3-甲基壬烷和4-甲基癸烷等支链烷烃。本试验各组检出了3-甲基丁醛(黑巧克力)、己醛(青草香)、辛醛(草香,甜香)、三甲基吡嗪(坚果烤香)、甲氧基乙酸乙酯(甜香)等关键芳香化合物,它们主要形成于成熟过程中的氨基酸Strecker降解和脂肪水解氧化以及美拉德反应等,对发酵香肠的风味贡献较大。来源于黑胡椒的石竹烯(辛辣气味)、β-水芹烯(胡椒气味)、3-蒈烯(辛辣味)是添加香辛料羊肉发酵香肠的主要风味。
In this study, these sausages were manufactured with mutton and sheep tail fat obtained from sheep fed in Inner Mongolia and inoculated the strain of Staphylococcus carnosus in combination with acidifying strain of Lactobacillus pentosus and Pediococcus pentosaceus as starter culture and spices for the production. 4 groups fermented sausages manufacture at the same technology and formula were named control group (CO), starter culture group (SC), starter culture and black pepper group ( SC+BP ), starter culture and black pepper and cumin (SC+BP+C), respectively. The aims of this work were to investigate the effects of quality characteristics and formation mechanism of volatile flavor compounds of mutton fermented dry sausages with starter cultures combination and spices during ripening and storage by HPLC, GC, DHS and GC-MS. A further aim was to provide a theory basis in realizing the industrial production of mutton fermented sausages and developing idea commercial starter cultures and new products in the future. The contents and results were as follows.
The changes of physico-chemical properties of mutton dry fermented sausages during ripening and storage were analyzed. The results showed that: the pH values of fermented sausages inoculated the starter culture decreased ( p<0.05 ) , dropped to below 5.0 at the end of the fermentation ( 1.5 days ) and were significantly lower than the control; the pH values of all sausages increased slowly and the spice groups increased ( p<0.05 ) during the drying and ripening and kept below 5.3;the Moisture, Aw values of all sausages decreased gradually, and Aw values decreased from 0.88 to 0.85 after ripening; the changes of protein and fat contents were slight(p>0.05).
The changes of the FFA (free fat acid) and the FAA (free amino acid) of mutton fermented sausages were studied during ripening and storage. The results showed that: the proteolysis indexes (PI) of the all sausages were percent 14 to 16 and the NPN and TFAA contents increased gradually during processing and enhance sharply during the middle storage. The changes of NPN and TFAA of batch SC+BP+C were significantly higher than the control(p<0.05). The contents of most FAA increased quickly above tenfold at the drying and ripening stage(p<0.05)and Arg, Glu, Leu, Lys, Ala and Val were dominant in concentration. Since the concentrations of most FAA in all sausages were 5-10 times of their corresponding sensory thresholds, the FAA might have make an important contribution to the formation of mutton fermented sausages flavor. The changes of TBARS values in all sausages were 0 mg/kg to 2 mg/kg and were not fat excessive oxidation. In general, the FFA of all the samples showed a significant increase during ripening and storage. Large amount of free fatty acids were accumulated during the storage and oleic acid,palmitic acid and stearic acid were the most abundant free fatty acids in treatment.
The oleic acid contents of MUFA were improved dramatically (p<0.05), while the proportion of capric acid and stearic acid decreased during the storage , especially the sausages inoculated starter culture combination and spices were lower than the control (uninoculated). The proportion of MUFA and PUFA in the batch SC+BP and SC+BP+C decreased obviously during storage. These indicated that the MUFA and PUFA were the key fatty acids in lipid oxidation. Temperature had great effect on lipase and microbial enzyme, in this study, the TFFA increased significantly during the storage(p<0.05).
A few significant differences were found in counts of microorganism between the three types of sausages and the control. Lactic acid bacteria (LAB) in sausages made with starter cultures combination and spices increased to 108cfu/g ( p<0.05 ) and kept this level in storage period, while the total bacteria counts were significantly lower than in the control and Micrococci-staphylococci were higher than the control ( p<0.05 ) at the middle period of storage. The addition of combined starter cultures and spices improved the quality attributes and shelf-life of mutton fermented dry sausages.
The sensory index of all sausages at the end of ripening, earlier storage, middle storage and late storage were evaluated. The relativity of the color, flavor, texture, taste and the quality was evaluated by formula Y=0.08X1-0.147X2+0.222X3+0.836X4 (R2=0.999) and the sensory weight were successively taste > texture > flavor > color, which indicated that taste played an important role. The sensory scores of taste and the quality with combined starter cultures and spices quickly increased at the middle storage and the goaty flavor was lower than the control, which indicated that inoculating starter cultures and spices could decrease the goaty flavor and improve the quality. The correlation of the proteolysis index (B1), TBARS (B2) and the quality were evaluated by formula Y=42.354 B1+0.457 B2 (R2=0.981), the weight coefficient of PI was much larger than of the TBARS, which showed NPN, small peptide and FAA were determinant in the quality of mutton fermented sausages.
The volatile flavor compounds in all treated fermented sausages at the end of clyster, end of fermentation, end of ripening, middle stage and late stage were extracted and identified by dynamic headspace sampling (DHS) and purge-trap (P & T) methods and GC/MS system. There were 93, 83, 85 and 90 flavor compounds were detected with capillary column HP-5 from the batch CO, SC, SC+BP and SC+BP+C, respectively. These volatile flavor compounds in mutton fermented sausages could be clustered in the following chemical families: alkanes and alkenes, aromatic hydrocarbons, alcohols, aldehydes, ketones, carboxylic acids, esters, nitrogenous compounds, sulphur compounds, furan compounds and terpenoids, which showed different changing rules, especially esters and alkanes (alkenes) were more than the others whose proportions were 16%-22% and 15%-20% respectively.
The sorts of volatile flavor in all treated fermented sausages was small at the end of fermentation, but large amount of (S)-Isopropyl lactate / Isoamy lactate ,2,3-Butanedione (cream) and 3-hydroxy-2-butanone (butter) were detected, which were originated from the carbohydrate catabolism. The branched chain alkane, such as 4-methyl-decane, 3-methyl-nonane, 4-methyl-heptane et al. were detected in the SC+BP batch. The key aroma-active compounds were 3-methylbutanal (dark chocolate), hexanal (grass), octanal (green, sweet), methoxy-acetic ethyl ester (sweet), trimethyl-Pyrazine (nut, roast), which were originated from lipid degradation and oxidation, amino acid Strecker reaction and the odor-active compounds contributed to fermented sausages odor together. beta.- Phellandrene (pepper), 3-Carene (pungent) and Caryophyllene (pungent) originated from the black pepper were main flavor in the mutton fermented dry sausages added spices.
引文
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