发酵剂筛选及双蛋白干酪成熟中的理化性质研究
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摘要
干酪是一类营养丰富的乳制品,由于风味等原因在我国发展缓慢。干酪的生产过程中,原料、发酵剂、加工工艺和成熟条件等决定干酪的质构和风味。本课题主要研究自选发酵剂和添加大豆蛋白对双蛋白干酪理化、质构及风味变化的影响,为双蛋白干酪的生产、质量评定和开发提供理论依据。
第一,课题通过对六株乳酸菌在发酵过程中发酵液的酸度、pH值、胞外多糖、粘度、游离氨基氮、双乙酰等指标进行分析,筛选出在豆乳和牛乳中都能优势生长的两株乳酸菌L. Lactis subsp. Cremoris QH27-1和L. Lactis subsp. Lactis XZ3303作为制作双蛋白干酪的发酵剂。优化了发酵剂的增菌培养基和冻干保护剂配方,结果表明,培养基配方中添加15%脱脂乳碱性蛋白酶酶解液,2%葡萄糖和1%Vc可以促进两种乳球菌的联合生长；在15%脱脂乳中添加5%葡萄糖、3%甘油、1%谷氨酸钠和1%VC能够为QH27-1和XZ3303菌株提供最好的保护效果,在冷冻干燥工艺中维持较高的活菌数,达到3.25×1010cfu/mL。
第二,通过对双蛋白干酪凝乳工艺条件优化,确定双蛋白干酪生产较佳工艺为豆乳添加比例为5.98%,CaCl2浓度为0.44‰,凝乳酶添加量为7.0×10-3g·mL-1,此时双蛋白凝乳的质地参数硬度达到最大值为63.926g,对照组的凝乳硬度为67.777g,按此凝乳条件制作双蛋白干酪收率为9.92%,与对照组干酪为9.79%相近。
第三,双蛋白干酪成熟过程中,乳酸菌数量是随着干酪成熟时间的延长呈现先增加后减少的趋势；利用自选发酵剂制作的纯牛乳干酪(NSE)和双蛋白干酪(ASE)的成熟度均较高,成熟六个月后两者的成熟度都可达到23%左右；并且WSN、12%TCA-SN、5%PTA-SN的质量含量随着干酪成熟时间的延长逐渐升高,SDS-PAGE电泳分析结果表明,发酵剂对大豆蛋白及酪蛋白有较强的降解作用；微生物中的脂肪酶分解脂肪产生游离脂肪酸,提供了风味物质基础。
第四,利用GC-MS联用分析成熟2个月和6个月干酪的风味物质变化并鉴定。结果表明：双蛋白和对照组干酪随着成熟时间的延长,风味物质的种类和含量相应增加。通过风味物质的主成分分析(PCA),证明成熟干酪风味中起主要作用的是醇、醛、酸和酯类。风味物质的聚类分析表明,NSC和NSE的干酪间距离最近,而两者都与ASE和ASC干酪的距离较远,说明发酵剂对干酪风味的影响小于豆乳添加的影响。
第五,利用质构仪对双蛋白干酪在成熟过程中的质构参数分析表明,随着成熟时间的延长,干酪的硬度逐渐下降,黏聚性和弹性有所增加,咀嚼性呈先增加后下降的趋势；扫描电镜(SEM)观察干酪的微观结构,添加豆乳造成双蛋白干酪质地松散,表面及内抛面粗糙。利用自选发酵剂制作的双蛋白干酪,其质地较商品发酵剂有明显改善。感官评价分析结果表明,豆乳的添加对干酪的质地和感官特性均无不良影响,自选发酵剂生产的双蛋白干酪,随着成熟时间的延长,风味和组织状态最优,说明应用自选发酵剂生产双蛋白干酪可以在一定程度上改善双蛋白干酪的质构特征和感官品质,为其能够得到广泛应用提供了可行性。
第六、双蛋白干酪含有16种氨基酸,含量较对照组有一定的提高,其所含的氨基酸与牛乳的氨基酸取长补短,提高了营养价值。干酪多肽样品中,除ASE干酪对大肠杆菌有一定的抑制效果,其他组多肽对大肠杆菌和金黄色葡萄球菌均无抑制作用。四种干酪多肽样品的总抗氧化能力得到提高,DPPH清除能力和羟基自由基消除能力基本没有变化。
本研究的双蛋白干酪,风味物质丰富,适合中国人的口味,为双蛋白干酪的推广提供了可行性。
Cheeses are rich nutritional dairy products, which were development slowly in China for taste and other reasons. Flavor of cheese is an important indicator to the quality of cheese. Raw materials, starters, processing and maturation conditions are all influence the flavor of cheese. It was researched that adding soy protein and experimental starters influence physical and chemical, rheological, microstructural and flavor changes. It was provided the theoretical basis to the production, quality assessment and development of double-protein cheeses.
This study includes the following eight aspects:
First, Selected and blended of starters used in double protein cheeses:Acidity, pH, extracellular polysaccharides, viscosity, free amino nitrogen, diacetyl and others were tested of six strains of lactic acid bacteria. XZ3303 and QH27-1 were growth well in soy milk and milk, and were selected as starters used in production of double protein cheeses. And optimization starter enrichment medium and freeze-drying protective agents:Skimmed milk hydrolyzed with alkaline protease 15%+glucose 2%+VC 1% can promote the growth of the two joint lactis, is the best growth medium. Skimmed milk 15%+glucose 5%+glycerol 3% +MSG 1%+VC 1% can protect L. Lactis subsp. Cremoris QH27-1 and L. Lactis subsp. Lactis XZ3303 in the freeze-drying process, and maintain a high number of viable cells to achieve good protection.
Second, Optimization conditions of double protein cheese curd, the better conditions of double protein cheese curd were obtained by analyzed texture and status of double protein curd. The conditions were adding 5.98% soymilk, CaCl2 concentration was 0.44‰, rennet concentration was 7.0×10-4g·mL-1.
Third, Compositions changing of double protein milk cheeses and milk cheeses during ripening were compared. The amount of lactic acid bacteria is increased firstly and then reduced gradually, through the tests in ripening time of double protein cheeses. Milk cheese made with experimental starters (NSE) and double protein cheese added soy milk made with experimental starters (ASE) have higher ripening degree. The amount of WSN,12%TCA-SN, 5%PTA-SN were increased with cheese ripening. Experimental starters have strong degradation to soy protein and casein whicn was analysis with electrophoretic. Lipases of microbe play an important role to produce short chain and long chain fatty acids in ripening time. Lipases can hydrolysis soy milk which made the species and the amount of unsaturated free fatty acids were increased in the cheeses added soy milk.
Fourth, GC-MS was used on analysis flavor compounds in cheeses matured 2 months and 6 months. The species and the amount of flavor substances were increase of double protein cheeses and control cheeses in ripening time. Some kinds of alcohols, aldehydes, acids and esters have large contributions to flavor in cheese were obtained by principal component analysis (PCA). With the maturation time, double protein cheese with experimental starters received the highest scores in flavor and organizations of the sensory evaluation results.
Fifth, It is no adversely effect on texture, flavor and sensory characteristics as added soy milk. L. lactis subsp. Cremoris QH27-1 and L. lactis subsp. Lactis XZ3303 can also improve the quality of double protein cheddar cheese. Double protein cheeses produced with experimental starters can improve texture characteristics and sensory quality, and are more suitable to Chinese tastes than others. It is feasibility to be widely used. Added soy milk was significantly influence the microstructure of cheeses, which made the cheeses softness, rough of surface and inner surface. The cheeses were producted with experimental starters have clesrly improved compared to the cheeses producted with commercial starters.
Sixth, By amino acid composition analysis, total amino acid of double protein cheeses was significantly higher than control cheeses. From the analysis result of 16 kinds of amino acids, every kinds of amino acid in double-protein cheeses are higher than the control. The amino acids of soy protein are supplementary with milk protein, so made the double protein cheeses have more nutrition.
With the antimicrobial activity trial of peptide in cheeses, four cheeses can not inhibit Staphylococcus aureus, and only double protein cheese producted with experimental starters inhibit E. coli. The substances of four cheeses all have antioxidant capacity, by the tests of the total antioxidant capacity, DPPH radical scavenging ability and hydroxyl radical scavenging ability.
This article studied systematicly on cheese production process, and changes of composition, protein and fat hydrolysis, microstructure, nutrition, texture and flavor characteristics on double protein cheese using selecting starters or commercial starters, compare with pure milk cheese. It can be drawn double protein cheese making of selecting starters which is rich flavor and taste for the Chinese people had nutritious, antioxidant activity and antibacterial activity against E. coli. It was feasibility to promoting development of double protein provides.
第一,课题通过对六株乳酸菌在发酵过程中发酵液的酸度、pH值、胞外多糖、粘度、游离氨基氮、双乙酰等指标进行分析,筛选出在豆乳和牛乳中都能优势生长的两株乳酸菌L. Lactis subsp. Cremoris QH27-1和L. Lactis subsp. Lactis XZ3303作为制作双蛋白干酪的发酵剂。优化了发酵剂的增菌培养基和冻干保护剂配方,结果表明,培养基配方中添加15%脱脂乳碱性蛋白酶酶解液,2%葡萄糖和1%Vc可以促进两种乳球菌的联合生长；在15%脱脂乳中添加5%葡萄糖、3%甘油、1%谷氨酸钠和1%VC能够为QH27-1和XZ3303菌株提供最好的保护效果,在冷冻干燥工艺中维持较高的活菌数,达到3.25×1010cfu/mL。
第二,通过对双蛋白干酪凝乳工艺条件优化,确定双蛋白干酪生产较佳工艺为豆乳添加比例为5.98%,CaCl2浓度为0.44‰,凝乳酶添加量为7.0×10-3g·mL-1,此时双蛋白凝乳的质地参数硬度达到最大值为63.926g,对照组的凝乳硬度为67.777g,按此凝乳条件制作双蛋白干酪收率为9.92%,与对照组干酪为9.79%相近。
第三,双蛋白干酪成熟过程中,乳酸菌数量是随着干酪成熟时间的延长呈现先增加后减少的趋势；利用自选发酵剂制作的纯牛乳干酪(NSE)和双蛋白干酪(ASE)的成熟度均较高,成熟六个月后两者的成熟度都可达到23%左右；并且WSN、12%TCA-SN、5%PTA-SN的质量含量随着干酪成熟时间的延长逐渐升高,SDS-PAGE电泳分析结果表明,发酵剂对大豆蛋白及酪蛋白有较强的降解作用；微生物中的脂肪酶分解脂肪产生游离脂肪酸,提供了风味物质基础。
第四,利用GC-MS联用分析成熟2个月和6个月干酪的风味物质变化并鉴定。结果表明：双蛋白和对照组干酪随着成熟时间的延长,风味物质的种类和含量相应增加。通过风味物质的主成分分析(PCA),证明成熟干酪风味中起主要作用的是醇、醛、酸和酯类。风味物质的聚类分析表明,NSC和NSE的干酪间距离最近,而两者都与ASE和ASC干酪的距离较远,说明发酵剂对干酪风味的影响小于豆乳添加的影响。
第五,利用质构仪对双蛋白干酪在成熟过程中的质构参数分析表明,随着成熟时间的延长,干酪的硬度逐渐下降,黏聚性和弹性有所增加,咀嚼性呈先增加后下降的趋势；扫描电镜(SEM)观察干酪的微观结构,添加豆乳造成双蛋白干酪质地松散,表面及内抛面粗糙。利用自选发酵剂制作的双蛋白干酪,其质地较商品发酵剂有明显改善。感官评价分析结果表明,豆乳的添加对干酪的质地和感官特性均无不良影响,自选发酵剂生产的双蛋白干酪,随着成熟时间的延长,风味和组织状态最优,说明应用自选发酵剂生产双蛋白干酪可以在一定程度上改善双蛋白干酪的质构特征和感官品质,为其能够得到广泛应用提供了可行性。
第六、双蛋白干酪含有16种氨基酸,含量较对照组有一定的提高,其所含的氨基酸与牛乳的氨基酸取长补短,提高了营养价值。干酪多肽样品中,除ASE干酪对大肠杆菌有一定的抑制效果,其他组多肽对大肠杆菌和金黄色葡萄球菌均无抑制作用。四种干酪多肽样品的总抗氧化能力得到提高,DPPH清除能力和羟基自由基消除能力基本没有变化。
本研究的双蛋白干酪,风味物质丰富,适合中国人的口味,为双蛋白干酪的推广提供了可行性。
Cheeses are rich nutritional dairy products, which were development slowly in China for taste and other reasons. Flavor of cheese is an important indicator to the quality of cheese. Raw materials, starters, processing and maturation conditions are all influence the flavor of cheese. It was researched that adding soy protein and experimental starters influence physical and chemical, rheological, microstructural and flavor changes. It was provided the theoretical basis to the production, quality assessment and development of double-protein cheeses.
This study includes the following eight aspects:
First, Selected and blended of starters used in double protein cheeses:Acidity, pH, extracellular polysaccharides, viscosity, free amino nitrogen, diacetyl and others were tested of six strains of lactic acid bacteria. XZ3303 and QH27-1 were growth well in soy milk and milk, and were selected as starters used in production of double protein cheeses. And optimization starter enrichment medium and freeze-drying protective agents:Skimmed milk hydrolyzed with alkaline protease 15%+glucose 2%+VC 1% can promote the growth of the two joint lactis, is the best growth medium. Skimmed milk 15%+glucose 5%+glycerol 3% +MSG 1%+VC 1% can protect L. Lactis subsp. Cremoris QH27-1 and L. Lactis subsp. Lactis XZ3303 in the freeze-drying process, and maintain a high number of viable cells to achieve good protection.
Second, Optimization conditions of double protein cheese curd, the better conditions of double protein cheese curd were obtained by analyzed texture and status of double protein curd. The conditions were adding 5.98% soymilk, CaCl2 concentration was 0.44‰, rennet concentration was 7.0×10-4g·mL-1.
Third, Compositions changing of double protein milk cheeses and milk cheeses during ripening were compared. The amount of lactic acid bacteria is increased firstly and then reduced gradually, through the tests in ripening time of double protein cheeses. Milk cheese made with experimental starters (NSE) and double protein cheese added soy milk made with experimental starters (ASE) have higher ripening degree. The amount of WSN,12%TCA-SN, 5%PTA-SN were increased with cheese ripening. Experimental starters have strong degradation to soy protein and casein whicn was analysis with electrophoretic. Lipases of microbe play an important role to produce short chain and long chain fatty acids in ripening time. Lipases can hydrolysis soy milk which made the species and the amount of unsaturated free fatty acids were increased in the cheeses added soy milk.
Fourth, GC-MS was used on analysis flavor compounds in cheeses matured 2 months and 6 months. The species and the amount of flavor substances were increase of double protein cheeses and control cheeses in ripening time. Some kinds of alcohols, aldehydes, acids and esters have large contributions to flavor in cheese were obtained by principal component analysis (PCA). With the maturation time, double protein cheese with experimental starters received the highest scores in flavor and organizations of the sensory evaluation results.
Fifth, It is no adversely effect on texture, flavor and sensory characteristics as added soy milk. L. lactis subsp. Cremoris QH27-1 and L. lactis subsp. Lactis XZ3303 can also improve the quality of double protein cheddar cheese. Double protein cheeses produced with experimental starters can improve texture characteristics and sensory quality, and are more suitable to Chinese tastes than others. It is feasibility to be widely used. Added soy milk was significantly influence the microstructure of cheeses, which made the cheeses softness, rough of surface and inner surface. The cheeses were producted with experimental starters have clesrly improved compared to the cheeses producted with commercial starters.
Sixth, By amino acid composition analysis, total amino acid of double protein cheeses was significantly higher than control cheeses. From the analysis result of 16 kinds of amino acids, every kinds of amino acid in double-protein cheeses are higher than the control. The amino acids of soy protein are supplementary with milk protein, so made the double protein cheeses have more nutrition.
With the antimicrobial activity trial of peptide in cheeses, four cheeses can not inhibit Staphylococcus aureus, and only double protein cheese producted with experimental starters inhibit E. coli. The substances of four cheeses all have antioxidant capacity, by the tests of the total antioxidant capacity, DPPH radical scavenging ability and hydroxyl radical scavenging ability.
This article studied systematicly on cheese production process, and changes of composition, protein and fat hydrolysis, microstructure, nutrition, texture and flavor characteristics on double protein cheese using selecting starters or commercial starters, compare with pure milk cheese. It can be drawn double protein cheese making of selecting starters which is rich flavor and taste for the Chinese people had nutritious, antioxidant activity and antibacterial activity against E. coli. It was feasibility to promoting development of double protein provides.
引文
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