CN105779338A - 一种利用嗜热链球菌发酵乳清制备的冰淇淋乳脂肪替代物及方法 - Google Patents
一种利用嗜热链球菌发酵乳清制备的冰淇淋乳脂肪替代物及方法 Download PDFInfo
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- CN105779338A CN105779338A CN201610170086.1A CN201610170086A CN105779338A CN 105779338 A CN105779338 A CN 105779338A CN 201610170086 A CN201610170086 A CN 201610170086A CN 105779338 A CN105779338 A CN 105779338A
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- Prior art keywords
- streptococcus thermophilus
- whey
- ice cream
- lactalbumin
- butter oil
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
- A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
- A23G9/38—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing peptides or proteins
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- A—HUMAN NECESSITIES
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- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
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- A23G9/40—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds characterised by the dairy products used
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- A—HUMAN NECESSITIES
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Dairy Products (AREA)
- Confectionery (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种利用嗜热链球菌发酵乳清制备的冰淇淋乳脂肪替代物及方法。本发明提供了一株嗜热链球菌(Streptococcus thermophilus)GST‑6,其保藏号CGMCC NO.11864。本发明还提供了一种制备嗜热链球菌发酵产物或其冻干物的方法,包括如下步骤:在含有乳清蛋白的培养基中发酵嗜热链球菌(Streptococcus thermophilus),得到悬浊状发酵产物。本发明的实验证明,本发明发现了一株新的嗜热链球菌,利用其乳清发酵产物,制备具有溶解性好,持水力高,粘弹性大的乳脂肪替代物,用于制作冰激凌。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种利用嗜热链球菌发酵乳清制备的冰淇淋乳脂肪替代物及方法。
背景技术
冰淇淋是深受消费者喜爱的嗜食性乳制品,具有细腻的口感和独特风味,但传统的冰淇淋中脂肪含量较高,容易使消费者摄入过多的热量,增加罹患肥胖,糖尿病,高血压等多种代谢性疾病的风险。因而研究乳脂肪替代物,降低冰淇淋中的脂肪含量,开发低脂冰淇淋,降低冰淇淋食品的健康不利因素,对于冰淇淋的产品开发和消费者健康具有重要意义。按照我国的冰淇淋品类划分标准(商务部-贸易行业标准-SB/T10013-2008),全脂冰淇淋的脂肪含量应≥8%,半脂和低脂冰淇淋的脂肪含量不应超过6%,一般与普通冰淇淋相比脂肪含量减少50%以上。低脂冰淇淋开发的主要难题在于,脂肪是构成冰淇淋细腻口感和脂溶性风味物质的重要因素和来源,降低脂肪含量往往会影响冰淇淋风味、粘稠度、口感、融化性、乳化性、膨胀率和外观等产品指标,因此多年来国内外的专家学者在不断地研究能为广大消费者接受的低脂冰淇淋和无脂冰淇淋,目前国外已经有了相关的商业化产品,但国内该领域的产品较为匮乏。
乳清是牛乳中的酪蛋白凝固后排出的上清液,是干酪加工的副产物,全球每年产生的乳清量约4×107吨。乳清主要由水(93.5%),乳糖(4.8%),脂肪和蛋白质(0.5%)及矿物质(1.0%)等组成。乳清中的蛋白质,具有营养价值高、易消化吸收等特点,还具有较好的溶解、持水、成胶、粘合和乳化等加工特性,适合于作为脂肪的替代物,但过去的研究表明,未经处理的乳清蛋白对脂肪的替代作用有限。乳清中的乳糖是引起富营养化、过量氧消耗等环境危害的主要物质。生物消化、转化是实现有价废弃资源回收利用最有效的方法之一。
发明内容
本发明的一个目的是提供一株嗜热链球菌(Streptococcus thermophilus)。
本发明提供的一株嗜热链球菌(Streptococcus thermophilus)GST-6,其保藏号CGMCC NO.11864。
上述的嗜热链球菌(StreptococcuS thermophilus)GST-6或其发酵产物或其菌悬液或其培养液在制备胞外多糖或乳脂肪替代物或冰激凌中的应用也是本发明保护的范围。
本发明另一个目的是提供一种制备胞外多糖的方法。
本发明提供的方法,包括如下步骤:在含有乳清蛋白和乳清反应体系中发酵上述的嗜热链球菌(Streptococcus thermophilus),得到胞外多糖。
本发明第三个目的是提供一种制备乳脂肪替代物的方法。
本发明提供的方法,包括如下步骤:
1)在含有乳清蛋白和乳清反应体系中发酵上述的嗜热链球菌(Streptococcusthermophilus),得到发酵产物;
2)冻干所述发酵产物,得到乳脂肪替代物。
上述方法中,所述含有乳清蛋白和乳清反应体系中乳清蛋白和乳清的质量比为5:8。
上述方法中,所述含有乳清蛋白和乳清反应体系中乳清蛋白的质量百分含量为5%;
所述含有乳清蛋白和乳清反应体系中的乳清的质量百分含量为8%。
上述方法中,所述发酵的温度为42℃,所述发酵时间为16-24h。
由上述方法制备的胞外多糖也是本发明保护的范围。
由上述方法制备的乳脂肪替代物是本发明保护的范围。
本发明第三个目的是提供一种制备冰激凌的方法。
本发明提供的冰激凌的方法,包括如下步骤:用上述的乳脂肪替代物替代冰激凌原料中的脂肪,制备冰激凌。
GST-6于2015年12月11日,保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号),保藏编号为CGMCC NO.11864,分类命名为嗜热链球菌(Streptococcus thermophilus)。
本发明的实验证明,本发明以乳清和乳清蛋白为主要原料,调配乳清蛋白比例,通过新嗜热链球菌发酵,制备得到含有胞外多糖的发酵产物,将发酵产物冻干得到乳乳脂肪替代物,乳乳脂肪替代物可以替代冰激凌中的乳脂肪,使乳清可以再次利用,能够增加乳清的附加值,降低乳清带来的环境污染,节省干酪生产企业的生产成本。
附图说明
图1为嗜热链球菌乳清蛋白发酵液粘度随时间变化曲线。
图2为冰淇淋加工流程图。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、嗜热链球菌GST-6的获得
一、嗜热链球菌GST-6的分离、纯化
取5g待分离藏灵姑样品加入45mL无菌生理盐水中,震荡混匀后逐级稀释到适宜浓度,取50μL稀释后的菌液涂布于M17琼脂平板,于42℃培养72h。挑取平板上生长的乳酸菌典型单菌落(圆形,中等大小,凸起,微白色边缘整齐)于MRS琼脂培养基上反复划线,直至获得纯培养物为止,得到纯化单菌落命名GST-6。
二、嗜热链球菌GST-6
1、表型鉴定
挑取上述一分离得到的GST-6单菌落,在M17培养基中培养,取菌液,涂布于M17琼脂平板,于42℃厌氧培养24-48h,形成乳酸菌典型单菌落(圆形,中等大小,凸起,微白色边缘整齐)。
2、生理生化鉴定
挑取上述分离得到的GST-6单菌落进行革兰氏染色和过氧化氢酶试验,其中革兰氏染色阳性,细胞形态为链球状,同时过氧化氢酶试验为阴性的菌株,初步鉴定为链球菌属。
3、16S rDNA鉴定
提取菌株GST-6的基因组DNA进行片段扩增。采用乳酸菌16S rDNA基因的通用引物,正向引物:A27F 5′-AGCGGATCACTTCACACAGGACTACGGCTACCTTGTTACGA-3′,反向引物:A1495R 3′-GCAGAGTTCTCGGAGTCACGAAGAGTTTGATCCTGGCTCAG-5′,1.5%琼脂糖凝胶电泳。PCR产物委托北京鼎国生物有限公司进行测序,将测序结果(请提供测序结果为序列1如下所示)与NCBI数据库中的核酸序列进行比对。结果显示GST-6菌株与NCBI数据库中嗜热链球菌的16S rDNA序列相似度超过99%。
经过上述鉴定,GST-6为嗜热链球菌(Streptococcus thermophilus)。
GST-6于2015年12月11日,保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号),保藏编号为CGMCC NO.11864,分类命名为嗜热链球菌(Streptococcus thermophilus)。
实施例2、嗜热链球菌GST-6的应用
一、发酵产物的制备
1、嗜热链球菌GST-6发酵的条件摸索
将实施例1获得的嗜热链球菌GST-6CGMCC NO.11864按照接种量1%(质量百分含量)接种于发酵培养基中,42℃,静置培养32h,得到悬浊状发酵产物。
上述每1L发酵培养基:称取WPI90乳清蛋白50g(新西兰恒天然)、甜乳清粉(波兰Mlekovita,Production code:054)80g用水定容至1L。
2、检测发酵产物凝胶强度(黏度)
取10mL悬浊状发酵产物,用美国Brookfield技术公司的DV-III型(配有温度控制系统)旋转流变仪,选用SC4-18型号转子,检测发酵产物0-36h间的凝胶强度变化,结果如图1所示,可以看出,在42℃,静置培养16-24h条件下获得了最强的凝胶强度,超过36h发酵物的凝胶强度显著下降。
因此最佳发酵时间为16-24h。
3、发酵产物中胞外多糖的检测
1)粗多糖的制备
向上述1培养24h收集的发酵产物中加入80%(质量/体积)的三氯乙酸(TCA)水溶液至终浓度为4%(质量/体积),室温下搅拌2h,10000×g 4℃离心45min去除蛋白,收集上清液加入两倍体积的低温无水乙醇,于4℃冷藏静置12h,10000×g 4℃离心45min,收集沉淀透析,用蒸馏水溶解,装入分子截流量为8000~14000Da(上海易佰聚,产品型号:T25-14-005)的透析袋中透析48h,每8h换一次蒸馏水。再经浓缩、冷冻干燥制得粗多糖。
2)多糖含量检测
用苯酚硫酸法(赵宁,问亚琴,&潘秋红(2011).苯酚-硫酸法测定干红葡萄酒中的多糖含量.中外葡萄与葡萄酒(05),9-12.)测定胞外多糖含量,约为4.84mg/g(发酵产物)。
3)胞外多糖纯化
DEAE-Sepharose Fast Flow离子交换柱层析纯化,上述1)制备的粗多糖用蒸馏水溶解后(20mg/mL),经DEAE-Sepharose Fast Flow离子交换柱层析(2.6×40cm,GE Healthcare,产品目录号:17-0709-01),上样量100mg,依次用蒸馏水(1-30管)、0.2mol/L NaCl(31-70管)、0.5mol/L NaCl(71-120管)线性梯度洗脱,洗脱速度为1mL/min,每管收集5mL,采用苯酚硫酸法(赵宁,问亚琴&潘秋红,2011)逐管检测多糖含量,按多糖含量检测值分别合并收集单一峰组分,去离子水透析,冷冻干燥,得到离子交换层析纯化后多糖。
再将离子交换层析纯化后多糖用0.9%(w/v)的NaCl水溶液溶解后(2mg/mL),经Sepharose CL-6B凝胶柱层析(2.5×50cm),上样量5mg,用0.9%NaCl水溶液脱,洗脱速度为0.5mL/min,洗脱时间240min。每管收集5mL,逐管检测多糖含量,按多糖含量检测值分别合并收集单一峰组分,去离子水透析,冷冻干燥,得到纯化的胞外多糖。
4)胞外多糖GC-MS分析
衍生化后经GC-MS分析:
a、单糖水解,称取5mg上述3)获得的纯化的胞外多糖EPS样品至安瓿瓶中,再加入2mL的2mol/L三氟乙酸(TFA),用酒精喷灯封口,于烘箱中120℃水解2h,水解液减压蒸干后,再加入少量甲醇减压蒸干(重复5次)以除去残留的TFA,然后加少量水溶解,冷冻干燥,即得完全酸水解单糖样品;
b、糖腈乙酸酯衍生物的制备,精密称取各单糖标准品(鼠李糖、阿拉伯糖、木糖、果糖、甘露糖、葡萄糖、半乳糖,美国Sigma公司)5mg、NaBH4 30mg和5mg肌醇至安瓿瓶中,慢慢滴入2mL蒸馏水,边加边震荡,室温下使还原反应成糖醇(反应2h),慢慢滴入几滴冰乙酸以中和过量的NaBH4,边加边震荡,直至不再产生气泡。
将旋转蒸发仪调至60℃真空旋蒸至样品完全干燥,之后每次用0.1%(v/v)盐酸甲醇溶液2mL复溶后再次蒸干,重复4-5次以除去硼酸根。处理完毕将产物置于105℃烘箱脱水15min,拿出加入吡啶和乙酸酐各0.5mL,用酒精喷灯封口,沸水浴反应1h,所得产物经微孔膜(0.22μm)除去杂质,进行气相色谱分析。完全酸水解后EPS样品衍生物的制备同样按照此方法制备。
C、气相色谱条件,色谱仪:Agilent 7890A气相色谱仪;色谱柱:DB225毛细管柱(30m×0.25mm);检测器:氧火焰离子检测器(FID);流速:1mL/min;分流比:1:50;柱温:220℃;检测器温度:250℃;进样口温度:250℃;进样量:1μL。结果表明,上述纯化后胞外多糖由葡萄糖和半乳糖按2:1比例构成。
一维核磁分析:用重水D2O(sigma公司)配置5mg/ml和40mg/ml的样品分别用于1H NMR和13C NMR检测,检测采用Bruker AVANCE 600MHz核磁(Bruker Group,瑞士)在25℃,用5mm倒置探头检测.)结果显示,多糖由6个糖单元的重复单糖结构组成。
二、嗜热链球菌GST-6发酵产物的应用
1、乳脂肪替代物PWP的制备
将实施例1获得的嗜热链球菌GST-6CGMCC NO.11864按照接种量1%(质量百分含量)接种于发酵培养基中,42℃,静置培养24h,得到悬浊状发酵产物。
将上述悬浊状发酵产物进行预冻,得到预冻产物;再将预冻产物冻干,得到冻干粉即为乳脂肪替代物PWP。
预冻条件:-40~-70℃预冻6-12h,冻结厚度不应超过5cm;较佳的预冻条件为-70℃,12h,冻结厚度4cm;
冻干条件为-50℃,0.2Mpa真空,冻干72h。
2、乳脂肪替代物PWP在制备冰淇淋中的应用
1)制备冰淇淋
将上述1获得的乳脂肪替代物PWP和乳脂肪替代物Simplesse(Nutra-Sweet公司)分别进行冰淇淋制备,配方按如下表1所示:
表1冰淇淋浆料配方表(%质量百分含量)
冰淇淋加工流程如图2所示。
2)、冰淇淋检测
(1)冰淇淋的质构参数
冰淇淋样品、器具及质构仪在冷藏室平衡至-15℃,用手术刀切取1.5×1.5×1.5cm正方体样品,采用压缩模式,探头TA10,夹具TA-RT-KI,负载单元:4500g,下压速度5mm/s,下压距离10mm,触发负载0.1g,每组样品做3平行实验。
结果如表2所示。
表2冰淇淋及浆料的质构参数
(2)微生物指标
检测采用国标GB/T 4789.18-2003中记述的方法。
结果如表3所示。
表3冰淇淋微生物指标
由表2可知,乳脂肪替代物PWP与商品蛋白乳脂肪替代物Simplesse相当,在硬度,浆料粘度,浆料冰点,膨胀率和冰淇淋融化速度等产品指标上二者较为接近。冰淇淋pH值本发明乳脂肪替代物低于商品Simplesse,感观品尝有酸及水果香气。另外,产品微生物指标符合国家要求(见表3)。
Claims (10)
1.一株嗜热链球菌(Streptococcus thermophilus)GST-6,其保藏号CGMCC NO.11864。
2.权利要求1所述的嗜热链球菌(Streptococcus thermophilus)GST-6或其发酵产物或其菌悬液或其培养液在制备胞外多糖或乳脂肪替代物或冰激凌中的应用。
3.一种制备胞外多糖的方法,包括如下步骤:在含有乳清蛋白和乳清反应体系中发酵权利要求1所述的嗜热链球菌(Streptococcus thermophilus),得到胞外多糖。
4.一种制备乳脂肪替代物的方法,包括如下步骤:
1)在含有乳清蛋白和乳清反应体系中发酵权利要求1所述的嗜热链球菌(Streptococcus thermophilus),得到发酵产物;
2)冻干所述发酵产物,得到乳脂肪替代物。
5.根据权利要求3或4所述的方法,其特征在于:
所述含有乳清蛋白和乳清反应体系中乳清蛋白和乳清的质量比为5:8。
6.根据权利要求3-5中任一所述的方法,其特征在于:
所述含有乳清蛋白和乳清反应体系中乳清蛋白的质量百分含量为5%;
所述含有乳清蛋白和乳清反应体系中的乳清的质量百分含量为8%。
7.根据权利要求3-6中任一所述的方法,其特征在于:
所述发酵的温度为42℃,所述发酵时间为16-24h。
8.由权利要求2或3所述方法制备的胞外多糖。
9.由权利要求4-6中任一所述方法制备的乳脂肪替代物。
10.一种制备冰激凌的方法,包括如下步骤:用权利要求9所述的乳脂肪替代物替代冰激凌原料中的脂肪,制备冰激凌。
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