Chinese Journal of Animal Nutrition >

2026 , Vol. 38 >Issue 9: 6913 - 6924

DOI: https://doi.org/10.12418/CJAN2026.552

RESEARCH PAPER

Effects of Dietary Supplementation with Different Forms of Aurantiochytrium limacinum on Muscle Quality of Litopenaeus vannamei

  • LI Hongye , 1, 2 ,
  • DOU Shilong 1, 2 ,
  • TANG Zeming 1, 2 ,
  • WANG Qiuzhen 1, 2 ,
  • GUO Ran , 1, 2, *
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  • 1 Ocean College, Hebei Agricultural University, Qinhuangdao 066000, China
  • 2 Key Laboratory of Nutritional Regulation and Disease Control in Aquaculture of Hebei Province, Qinhuangdao 066000, China
* professor, E-mail:

Received date: 2026-01-15

  Online published: 2026-09-12

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Abstract

This experiment was conducted to investigate the effects of Aurantiochytrium limacinum on muscle quality of Litopenaeus vannamei. A total of 360 healthy juvenile Litopenaeus vannamei with an initial body weight of (0.26±0.01) g were selected and randomly divided into 3 groups with 3 replicates per group and 40 shrimp per replicate. Shrimp in the 3 groups were fed a basal diet (CON group), the basal diet supplemented with 3% Aurantiochytrium limacinum with intact cell wall (SL1 group) or 3% Aurantiochytrium limacinum with disrupted cell wall (SL2 group), respectively, with equal nitrogen and energy levels. The pre-test period lasted for 7 days, and the formal trial lasted for 56 days. The results showed as follows: 1) the muscle crude protein and collagen contents of the SL1 and SL2 groups were significantly higher than those of the CON group (P<0.05), and the muscle collagen content of the SL2 group was significantly higher than that of the SL1 group (P<0.05). 2) The muscle cooking loss rates of the SL1 and SL2 groups were significantly lower than that of the CON group (P<0.05). 3) The muscle cohesiveness of the SL1 group was significantly higher than that of the CON group (P<0.05), and the muscle hardness, gumminess, chewiness and cohesiveness of the SL2 group were significantly higher than those of the CON group (P<0.05). 4) The mRNA relative expression levels of serine/threonine-protein kinase (AKT), mechanistic target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), eukaryotic translation initiation factor 4E2 (EIF4E2), transforming growth factor-β (TGF-β), troponin C (TNNC) and myosin light chain (MYLC) in muscle of the SL1 and SL2 groups were significantly higher than those of the CON group (P<0.05). 5) The muscle malondialdehyde (MDA) content of the SL1 group was significantly lower than that of the other groups (P<0.05), while the muscle total antioxidant capacity (T-AOC) and MDA content of the SL2 group were significantly higher than those of the other groups (P<0.05); the mRNA relative expression levels of catalase (CAT) and superoxide dismutase (SOD) in muscle of the SL1 and SL2 groups were significantly higher than those of the CON group (P<0.05). 6) The myofiber diameter of the SL2 group was significantly lower than that of the other groups (P<0.05). In conclusion, dietary supplementation with Aurantiochytrium limacinum can activate the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signaling pathway and the expression of myogenic regulatory genes in muscle of Litopenaeus vannamei, and increase muscle crude protein and collagen contents. Aurantiochytrium limacinum with intact cell wall can improve muscle antioxidant capacity; Aurantiochytrium limacinum with disrupted cell wall can increase muscle collagen content, decrease myofiber diameter, and improve muscle textural properties.

Key words： Litopenaeus vannamei; Aurantiochytrium limacinum; cell disruption; muscle quality

Cite this article

LI Hongye , DOU Shilong , TANG Zeming , WANG Qiuzhen , GUO Ran . Effects of Dietary Supplementation with Different Forms of Aurantiochytrium limacinum on Muscle Quality of Litopenaeus vannamei[J]. Chinese Journal of Animal Nutrition, 2026 , 38(9) : 6913 -6924 . DOI: 10.12418/CJAN2026.552

2024年我国凡纳滨对虾(Litopenaeus vannamei)的养殖总产量达237.4万t,同比增长6.08%[1],是我国重要的水产经济动物,也是人类饮食中优质蛋白质的重要来源之一,可为人体提供多种必需营养素,包括优质蛋白质、必需矿物质及不饱和脂肪酸[2]。在健康饮食理念日益普及的背景下,消费者对凡纳滨对虾产品的品质偏好日趋精细,不仅关注其外观规格,更对其肉质的紧实度、弹性等质构特性及鲜美风味等内在品质给予了高度重视[3]。因此,凡纳滨对虾的肌肉品质与消费者接受程度密切相关。
鱼油是对虾配合饲料中不饱和脂肪酸的主要来源,但随着捕捞压力的增大以及饲料成本上涨的影响,水产养殖业面临严峻挑战[4],寻求非海洋鱼类来源的二十二碳六烯酸(DHA)和二十碳五烯酸(EPA)是破解此困境的路径之一。裂殖壶菌(Aurantiochytrium limacinum)是一种海洋微藻,其细胞内含有大量油脂,且富含DHA、类胡萝卜素及角鲨烯等活性物质[5],为鱼油的可持续替代提供了有效方案。此外,裂殖壶菌作为一种新型的饲料添加剂能够改善肌肉品质,有关研究发现,裂壶藻粉可通过减轻凡纳滨对虾肌肉中氧化损伤,改善肌肉质量[6]。肌肉品质是一个综合指标体系,涵盖营养特性、质构特性、微观结构及功能性成分等维度。相关研究表明,饲料添加裂殖壶藻能提高凡纳滨对虾肌肉质构特性,促进肌纤维生长和发育,改善凡纳滨对虾的肌肉品质[7]。
然而,微藻的细胞壁由几丁质和纤维素构成,完整的细胞壁可能会阻碍细胞内营养物质的释放与吸收[8]。目前关于饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉品质的研究较少。因此,本试验以凡纳滨对虾为研究对象,旨在评估饲料添加未破壁与破壁裂殖壶菌对凡纳滨对虾肌肉品质的影响,从而为优质对虾饲料的开发提供参考。

1 材料与方法

1.1 试验饲料

试验所用裂殖壶菌为本实验室保存的裂殖壶菌S3D菌株。参考朱婧瑶等[9]的液氮研磨法对裂殖壶菌进行破壁处理,取裂殖壶菌发酵液,冷冻干燥后,在预冷研钵中加入适量液氮研磨至细粉。未破壁与破壁裂殖壶菌营养水平见表1。
表1 不同形式裂殖壶菌的营养水平(干物质基础)

Table 1 Nutrient levels of different forms of Aurantiochytrium limacinum (DM basis)

项目
Items		 未破壁裂殖壶菌
Aurantiochytrium limacinum with intact cell wall		 破壁裂殖壶菌
Aurantiochytrium limacinum with disrupted cell wall
粗蛋白质CP 15.24 15.04
粗脂肪EE 5.24 27.00
粗灰分Ash 8.54 8.83
二十二碳六烯酸DHA 3.50 19.17

营养水平为实测值。

Nutrient levels were measured values.

1.2 试验设计与饲料

选取初始体重为(0.26±0.01) g的健康凡纳滨对虾幼虾360尾,随机分为3组,每组3个重复[养殖缸(40 cm×50 cm×60 cm,100 L)],每个重复40尾虾。3个组分别饲喂基础饲料(CON组)及在基础饲料中添加3%未破壁裂殖壶菌(SL1组)和添加3%破壁裂殖壶菌(SL2组)的等氮等能饲料。预试期7 d,正试期56 d。
试验饲料均是以鱼粉、豆粕及鸡肉粉为蛋白质源,玉米油为脂肪源配制的等氮(粗蛋白质水平约为41%)等能(粗脂肪水平约为8%),其组成及营养水平见表2。所有原料经粉碎后过80目筛,按既定配方比例初步混合,随后加入玉米油并在搅拌机中充分混匀,混合均匀的物料通过双螺杆挤条机挤压成型,制成粒径为1.2 mm的颗粒饲料,经自然风干后置于-20 ℃冰箱中保存以备用。
表2 试验饲料组成及营养水平(干物质基础)

Table 2 Composition and nutrient levels of experimental diets (DM basis)

项目
Items		 组别Groups
CON SL1 SL2
原料Ingredients
未破壁裂殖壶菌
Aurantiochytrium limacinum with intact cell wall		 3.00
破壁裂殖壶菌
Aurantiochytrium limacinum with disrupted cell wall		 3.00
鱼粉Fish meal 18.00 17.29 17.29
豆粕Soybean meal 25.00 25.00 25.00
鸡肉粉Chicken meal 17.00 17.00 17.00
面粉Flour 20.00 20.00 20.00
玉米油Corn oil 2.70 2.61 1.95
微晶纤维素Microcrystalline cellulose 10.29 8.09 8.75
卵磷脂Lecithin 0.50 0.50 0.50
氯化胆碱Choline chloride 0.50 0.50 0.50
黏合剂Adhesive 2.00 2.00 2.00
磷酸二氢钙Ca(H2PO4)2 2.00 2.00 2.00
预混料Premix1) 2.00 2.00 2.00
三氧化二钇Y2O3 0.01 0.01 0.01
合计Total 100.00 100.00 100.00
营养水平Nutrient levels2)
粗蛋白质CP 41.35 41.60 41.84
粗脂肪EE 8.07 8.09 8.05

1)预混料为每千克饲料提供 The premix provided the following per kg of diets:VA 10 mg,VB1 6 mg,VB2 5 mg,VB6 7.5 mg,VB12 4 mg,烟酰胺 niacinamide 50 mg,抗坏血酸 ascorbic acid 500 mg,泛酸钙 calcium pantothenate 20 mg,生物素 biotin 2.5 mg,叶酸 folic acid 5 mg,VE 200 mg,VK3 10 mg,VD3 5 mg,肌醇 inositol 100 mg,玉米蛋白粉 corn gluten meal 75 mg,CuSO4·5H2O 10 mg,FeSO4·H2O 300 mg,ZnSO4·H2O 200 mg,MnSO4·H2O 100 mg,KIO3 80 mg,Na2SeO3 67 mg,CoCl2·6H2O 5 mg,NaCl 100 mg,沸石 zeolite 638 mg。

2)营养水平为实测值。Nutrient levels were measured values.

1.3 饲养管理

养殖期间每天投喂4次,投喂量控制在虾体重的8%~12%,投喂时间为07:00、11:00、15:00、19:00,每天清晨换水1次,更换1/2的新鲜海水。试验期间24 h充气,水中溶解氧含量>5.0 mg/L,海水盐度为(31±1)‰(质量分数),水温为(26±2) ℃。每周测定pH及氨氮和亚硝酸盐含量,养殖期间pH为7.5~8.5,氨氮含量<0.2 mg/L,亚硝酸盐含量<0.2 mg/L。

1.4 样品采集

试验结束后,将凡纳滨对虾禁食24 h以排空消化道,然后使用丁香酚(50 mg/L)进行麻醉,从每重复随机挑选12尾虾进行解剖,以获取肌肉组织样本。其中,先取3尾虾用于肌肉营养物质含量测定;然后取3尾虾,将第1腹节用于肌肉质构分析,第2腹节用于肌肉组织学测定;再取3尾虾进行持水力测定;剩余3尾虾取肌肉组织,保存于-80 ℃,以备后续总RNA提取和测定肌肉胶原蛋白含量及生化指标。

1.5 指标测定

1.5.1 裂殖壶菌、饲料和肌肉常规营养物质含量及肌肉胶原蛋白含量测定

参照AOAC(2016)[10]的标准方法测定裂殖壶菌、饲料与肌肉的水分、粗蛋白质和粗脂肪含量,以及裂殖壶菌和肌肉的粗灰分含量;采用气相色谱法测定裂殖壶菌的DHA含量。肌肉中胶原蛋白含量通过羟脯氨酸含量计算,羟脯氨酸含量采用南京建成生物工程研究所的试剂盒测定,操作过程严格按照说明书进行。

1.5.2 肌肉质构特性分析

使用质构检测分析仪(TMS-Pilot,Food Technology Corporation,美国)质地剖面分析(TPA)模式检测肌肉样品的质构参数。选用P/36R型圆柱平面探头开展测定,取对虾第1腹节肌肉组织样品,样品厚度控制在4.0~5.0 mm。测试参数设定如下:检测速率30 mm/min,触发力0.75 N,形变率50%,单次循环位移量2.23 mm,断裂力阈值20.45 N。

1.5.3 肌肉持水力测定

肌肉滴水损失率和冷冻渗出率测定参考李雪婷[11]的方法,取2份约3.00 g虾肉分别称重(W1和W2),分别置于4 ℃和-20 ℃冰箱中24 h,拭去表面水分,分别称量末重(W3和W4),计算滴水损失率和冷冻渗出率。计算公式如下:
滴水损失率(%)=[(W1-W3)/W1]×100;
冷冻渗出率(%)=[(W2-W4)/W2]×100。
肌肉蒸煮损失率测定:取3.00 g虾肉至5 mL离心管中,称重(W5),将离心管盖剪掉,用封口膜将离心管管口封好,并在封口膜上扎个通气孔,后放入75 ℃水浴锅中水浴15 min,吸干肌肉表面水分后称重(W6)。计算公式如下:
蒸煮损失率(%)=[(W5-W6)/W5]×100。

1.5.4 肌肉抗氧化指标测定

取肌肉组织匀浆上清液测定抗氧化指标,包括丙二醛(MDA)含量及总抗氧化能力(T-AOC)。上述指标采用南京建成生物工程研究所试剂盒进行测定,测定方法按照试剂盒说明书进行。

1.5.5 肌肉组织学测定

取肌肉组织样品,用固定液进行固定,然后进行肌肉组织包埋、切片,再通过苏木精-伊红(HE)染色,处理完毕后用DM2500 LED光学显微镜(Leica,德国)进行观测,然后使用Image J软件测定肌纤维直径。

1.5.6 肌肉基因表达测定

提取肌肉组织的总RNA,并检测RNA完整性,然后测定RNA纯度和浓度,用荧光定量PCR(qPCR)检测和定量RNA特定序列,采用geNorm算法筛选内参基因,以甘油醛-3-磷酸脱氢酶(GAPDH)和TATA结合蛋白(TBP)作为最优内参基因组合,目的基因相对表达量以2个内参基因的几何平均值进行标准化。引物序列由生工生物工程(上海)股份有限公司合成,引物序列信息见表3。PCR反应体系配制如下:取1 μL逆转录产物,加入3.6 μL无菌水,随后依次加入0.2 μL正向引物、0.2 μL反向引物以及5 μL 2×ChamQ Universal SYBR qPCR Master Mix(购自南京诺唯赞生物科技股份有限公司),充分混匀后,使用CFX96 Touch荧光定量PCR仪(Bio-Rad,美国)进行检测。扩增程序设置为:首先在95 ℃预变性10 min;随后进行40个循环的扩增,每个循环包括95 ℃变性5 s和60 ℃退火延伸15 s这2个步骤,并在每个循环结束时采集荧光信号。采用2-ΔΔCt法计算目的基因mRNA相对表达量。
表3 引物序列

Table 3 Primer sequences

基因
Genes		 引物序列
Primer sequences (5'—3')		 GenBank登录号
GenBank accession No.
内参基因Reference genes
甘油醛-3-磷酸脱氢酶
GAPDH		 F:GCAACAGTTTTTCGCTGCAC
R: ACGAACACAAGGACATCTGC		 MG787341.1
TATA结合蛋白
TBP		 F:GACAACAAGGAGTGGTTTGGAG
R:AGGAGCACACCACATTGTTG		 XM_027352221.1
抗氧化相关基因Antioxidant related genes
过氧化氢酶
CAT		 F:TTGCAAGGTCGCCTCTTTTC
R:TTGCCATCAACACACATGGG		 AY518322.1
超氧化物歧化酶
SOD		 F:TTGGCTCCGAAGTTCAATGG
R:TTGCCGGGTTTAAGGTGAAC		 XM_027377625.1
肌肉品质性状相关基因Meat quality traits related genes
丝氨酸/苏氨酸蛋白激酶
AKT		 F:AAACCGAGTGTTGCAAAGGG
R:ACAGGCGGTCATTTGTTTGG		 KF163129.1
磷脂酰肌醇-3-激酶
PI3K		 F:ACCGTTTATTGCGTCGTGAG
R:CGAACTGCACAAATCCATGC		 XM_027359988.1
雷帕霉素机械性靶蛋白
mTOR		 F:TTGAACATGCGCTCGAACAC
R:AGTTTGTACGGCGGTCAAAC		 XM_027372359.1
核糖体蛋白S6激酶1
S6K1		 F:TGTGAACCGCCTTTCAAACC
R:AACAACTGGTGAGCATTGGC		 XM_027359033.1
真核翻译起始因子4E2
EIF4E2		 F:ACAAACCACTGCCACAAGTG
R:TGTGTTGTTGCCTGTTTGCC		 XM_027354394.1
转化生长因子-β
TGF-β		 F:TGCCCAGGTGCAAGAAAATG
R:GGCCTCTTTCTCTGCTTCAAAC		 XM_027353723.1
肌钙蛋白C
TNNC		 F:TGGAATTCAGGAGTTCGTCGAG
R:ATAACGAAAAGCCTCCCCTGAG		 XM_027381668.1
肌球蛋白轻链
MYLC		 F:TCCAAGAAGGCCAAGAAGACG
R:ATCTGGAAGCCCTCCTTGAAC		 XM_027376218.1

1.6 数据统计分析

采用SPSS 26.0软件对数据进行单因素方差分析,并采用Duncan氏法进行多重比较,结果用“平均值±标准差(mean±SD)”表示,P<0.05表示差异显著。

2 结果

2.1 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉常规营养物质和胶原蛋白含量及持水力的影响

由表4可知,各组间肌肉水分、粗脂肪、粗灰分含量无显著差异(P>0.05);SL1组、SL2组肌肉粗蛋白质和胶原蛋白含量显著高于CON组(P<0.05),且SL2组肌肉胶原蛋白含量显著高于SL1组(P<0.05)。与CON组相比,SL1组、SL2组肌肉蒸煮损失率均显著降低(P<0.05);各组间肌肉滴水损失率和冷冻渗出率无显著差异(P>0.05)。
表4 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉常规营养物质和胶原蛋白含量及持水力的影响

Table 4 Effects of dietary supplementation with different forms of Aurantiochytrium limacinum on contents of conventional nutrient and collagen, and water-holding capacity in muscle of Litopenaeus vannamei

项目
Items		 组别Groups
CON SL1 SL2
水分Moisture/% 79.12±0.55 77.65±0.85 78.19±0.21
粗脂肪EE/% DM 2.94±0.36 3.26±0.27 3.26±0.33
粗蛋白质CP/% DM 92.25±1.42a 95.30±0.41b 95.85±0.50b
粗灰分Ash/% DM 0.28±0.00 0.29±0.01 0.28±0.00
胶原蛋白Collagen/(μg/mg FW) 0.92±0.02a 1.17±0.04b 1.51±0.01c
滴水损失率Drip loss rate/% 2.46±0.61 2.23±0.44 1.82±0.62
冷冻渗出率Freeze exudation rate/% 1.87±0.33 1.65±0.14 1.54±0.50
蒸煮损失率Cooking loss rate/% 16.95±0.37b 13.46±1.11a 13.65±0.87a

同行数据肩标不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05)。下表同。

In the same row, values with different small letter superscripts mean significant difference (P<0.05), while with the same or no letter superscripts mean no significant difference (P>0.05). The same as below.

2.2 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉质构特性的影响

由表5可知,与CON组相比,SL1组肌肉内聚性显著升高(P<0.05),硬度、弹性、胶黏性、咀嚼性、黏附性均无显著差异(P>0.05);SL2组肌肉硬度、胶黏性、咀嚼性、内聚性均显著升高(P<0.05),弹性、黏附性无显著差异(P>0.05)。
表5 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉质构特性的影响

Table 5 Effects of dietary supplementation with different forms of Aurantiochytrium limacinum on muscle textural properties of Litopenaeus vannamei

项目
Items		 组别Groups
CON SL1 SL2
硬度Hardness/N 9.19±0.82a 10.71±0.61a 15.87±1.30b
弹性Springiness/mm 0.50±0.08 0.67±0.12 0.73±0.14
胶黏性Gumminess/% 1.69±0.38a 2.69±0.32a 5.57±2.02b
咀嚼性Chewiness/mJ 1.19±0.19a 2.00±0.60a 4.53±1.84b
黏附性Adhesiveness/N·mm 0.06±0.02 0.07±0.06 0.03±0.01
内聚性Cohesiveness/% 0.17±0.03a 0.25±0.02b 0.27±0.04b

2.3 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉品质性状相关基因表达的影响

由图1可知,与CON组相比,SL1组肌肉丝氨酸/苏氨酸蛋白激酶(AKT)、雷帕霉素机械性靶蛋白(mTOR)、核糖体蛋白S6激酶1(S6K1)、真核翻译起始因子4E2(EIF4E2)的mRNA相对表达量均显著升高(P<0.05),肌肉磷脂酰肌醇-3-激酶(PI3K)的mRNA相对表达量无显著差异(P>0.05);SL2组肌肉AKT、PI3K、mTOR、S6K1、EIF4E2的mRNA相对表达量均显著升高(P<0.05)。与CON组相比,SL1组和SL2组肌肉转化生长因子-β(TGF-β)、肌钙蛋白C(TNNC)、肌球蛋白轻链(MYLC)的mRNA相对表达量均显著升高(P<0.05)。
图1 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉品质性状相关基因表达的影响

数据柱标相同小写字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05)。图2同。

Fig.1 Effects of dietary supplementation with different forms of Aurantiochytrium limacinum on meat quality traits related gene expression of Litopenaeus vannamei

Value columns with the same small letter mean no significant difference (P>0.05), while with different small letters mean significant difference (P<0.05). The same as Fig.2.

2.4 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉抗氧化指标及抗氧化相关基因表达的影响

由表6可知,与CON组相比,SL1组肌肉MDA含量显著降低(P<0.05),肌肉T-AOC无显著差异(P>0.05);SL2组肌肉T-AOC和MDA含量均显著升高(P<0.05)。由图2可知,与CON组相比,SL1组和SL2组肌肉过氧化氢酶(CAT)、超氧化物歧化酶(SOD)的mRNA相对表达量均显著升高(P<0.05)。
表6 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉抗氧化指标的影响

Table 6 Effects of dietary supplementation with different forms of Aurantiochytrium limacinum on muscle antioxidant indices of Litopenaeus vannamei

项目
Items		 组别Groups
CON SL1 SL2
总抗氧化能力T-AOC/(U/mg prot) 4.33±0.09a 4.30±1.20a 7.95±0.39b
丙二醛MDA/(nmol/mg prot) 0.68±0.10b 0.25±0.08a 1.86±0.25c
图2 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉抗氧化相关基因表达的影响

Fig.2 Effects of dietary supplementation with different forms of Aurantiochytrium limacinum on antioxidant related gene expression in muscle of Litopenaeus vannamei

2.5 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉组织学的影响

由图3-A至图3-F可知,与CON组相比,SL1、SL2组肌纤维排列更紧密;与SL1组相比,SL2组肌肉组织结构的细化程度更高。由图3-G可知,SL1组、SL2组肌纤维直径显著低于CON组(P<0.05),且SL2组肌纤维直径显著低于SL1组(P<0.05)。
图3 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉组织学的影响

A:CON组肌纤维横切图 transverse section of muscle fibers in CON group (200×);B:SL1组肌纤维横切图 transverse section of muscle fibers in SL1 group (200×);C:SL2组肌纤维横切图 transverse section of muscle fibers in SL2 group (200×);D:CON组肌纤维纵切图 longitudinal section of muscle fibers in CON group (400×);E:SL1组肌纤维纵切图 longitudinal section of muscle fibers in SL1 group (400×);F:SL2组肌纤维纵切图 longitudinal section of muscle fibers in SL2 group (400×);G:肌纤维直径 myofiber diameter;“****”:P<0.000 1。

Fig.3 Effects of dietary supplementation with different forms of Aurantiochytrium limacinum on muscle histology of Litopenaeus vannamei

3 讨论

3.1 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉品质与肌纤维特性的影响

肌肉常规营养物质含量是评估肌肉品质的基本指标[12]。本研究发现,饲料添加裂殖壶菌可以显著提升凡纳滨对虾肌肉粗蛋白质含量。肖芬芬[13]研究发现,适量的裂殖壶藻渣可促进框鲤(Cyprinus carpio var. specularis)肝脏和全鱼中的蛋白质沉积并减少肝脏脂质积累;而在花鲈[14](Lateolabrax maculatus)和多鳞白甲鱼[15](Onychostomamacrolepis)的研究中表明,饲料中添加不同水平裂壶藻粕均显著降低肌肉的粗脂肪含量,而对肌肉粗蛋白质含量无显著影响;饲料添加裂壶藻可显著提高建鲤(Cyprinus carpio var. Jian)全鱼和肌肉的粗脂肪含量[16];而Neylan等[17]和Liao等[18]分别在裸盖鱼(Anoplopoma fimbria)和大口黑鲈(Micropterus salmoides)的研究发现,与鱼粉组相比,饲料添加裂殖壶菌对全鱼体成分无显著影响。以上研究结果与本试验不尽相同,这可能与所用裂殖壶菌成分不同以及物种特异性有关。
MYLC、TNNC是肌原纤维收缩的调节蛋白,其表达差异与肌肉发育状态密切相关,且能够通过改变肌原纤维特性影响肌肉品质[19-21]。本研究显示,饲料添加裂殖壶菌显著上调肌肉中MYLC、TNNC基因的表达。值得注意的是,研究表明,在三倍体鲫鱼和瓦氏黄颡鱼♀×长吻鮠♂(Pelteobagrus vachelli♀×Leiocassis longirostris♂)肌肉中,MYLC、TNNC基因表达上调可能与mTOR信号通路激活有关[22-23]。在本研究中,mTOR信号通路相关基因(PI3K、AKT、mTOR)的表达改变与肌肉粗蛋白质含量及肌肉生成调节基因的表达趋势一致,表明裂殖壶菌可能通过PI3K/AKT/mTOR信号通路促进凡纳滨对虾肌肉生长和肉品质提升。这可能与裂殖壶菌富含丰富的不饱和脂肪酸有关,相关研究结果表明,EPA和DHA可以通过激活雷帕霉素靶蛋白(TOR)信号通路来刺激细胞增殖,促进蛋白质合成,并抑制蛋白质降解[24]。在草鱼(Ctenopharyngodon idellus)中的研究表明[25],DHA诱导的肌肉蛋白质沉积可能与mTOR/S6K1信号通路的激活相关,该调控可能有助于脂质发挥“蛋白质节约作用”。
肌肉质构特性包括硬度、弹性、咀嚼性、胶黏性、黏附性和内聚性[26],而持水力能够反映可溶性物质在肌肉中的流失情况[27]。试验结果表明,与CON组相比,饲料添加未破壁和破壁裂殖壶菌均能够显著降低肌肉蒸煮损失率,而添加破壁裂殖壶菌可提高肌肉硬度、胶黏性、咀嚼性和内聚性,这一结果提示肌肉的持水力和质构特性得到明显改善。研究表明,肌肉胶原蛋白含量与肌肉持水力和质构特性呈正相关[28-31]。在本试验中,饲料添加破壁裂殖壶菌的对虾肌肉中胶原蛋白含量的显著高于其他组,表明破壁处理有利于裂殖壶菌中活性成分的释放与吸收。TGF-β信号通路在胶原蛋白合成的过程中起重要作用[32-34],在本研究中,饲料添加裂殖壶菌的对虾肌肉TGF-β基因表达显著上调,与肌肉胶原蛋白含量变化趋势相同,说明饲料添加裂殖壶菌通过激活TGF-β信号通路,促进了肌肉胶原蛋白的合成,并影响肌肉持水力和质构特性。

3.2 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉抗氧化能力的影响

肌肉的抗氧化能力是维持肌肉品质的关键,氧化反应会导致肌肉中脂质、蛋白质等营养物质发生变化,致使肌肉发生损伤、颜色褐变等不良反应[35]。T-AOC和MDA含量是重要的抗氧化指标[36-37]。在本试验中,与CON组相比,饲料添加未破壁裂殖壶菌对肌肉T-AOC无显著影响,但显著降低肌肉MDA含量,显著上调肌肉SOD、CAT基因表达。上述结果表明,未破壁裂殖壶菌可通过增强肌肉抗氧化相关基因的表达、减少脂质过氧化产物积累,从而抑制肌肉营养物质降解,改善肌肉品质。这可能与裂殖壶菌富含DHA等不饱和脂肪酸、蛋白质、矿物质、多糖、活性多肽等营养物质有关[38]。研究发现,补充DHA可缓解草鱼肌肉氧化应激[25];在低鱼粉饲料中添加裂殖壶菌能够提高凡纳滨对虾肌肉抗氧化能力[7],上述结论与本试验结果相符,类似结果在尼罗罗非鱼[39](Oreochromis niloticus)研究中也有报道。本试验中,饲料添加破壁裂殖壶菌后,对虾肌肉T-AOC显著提高,同时肌肉MDA含量显著升高。T-AOC升高表明对虾机体抗氧化系统被激活,抗氧化防御能力上调;而MDA含量升高反映脂质过氧化程度加剧,这可能与破壁处理释放的部分活性物质诱导体内自由基生成、超出抗氧化系统即时清除能力有关。

3.3 饲料添加不同形式裂殖壶菌对凡纳滨对虾肌肉组织学的影响

肌纤维是肌肉组织的基本构成单位,因此肌纤维的特性在一定程度上决定了肌肉品质[19]。而肌纤维直径和密度是评估肌肉品质的重要指标[40]。在本试验中,饲料添加裂殖壶菌显著减小肌纤维直径,而饲料添加破壁裂殖壶菌可使肌肉纤维排列更加紧密。已有研究表明,肌纤维细化及致密排列与肌肉弹性提升、质构优化有关[41]。结合本试验结果,推测上述肌纤维形态学改变可能有助于提高肌肉弹性,改善肌肉质构特性。这种现象的出现可能与裂殖壶菌富含丰富的DHA有关。已有研究证实,饲料添加DHA可降低团头鲂(Megalobrama amblycephala)的肌纤维直径,进而改善其肌纤维相关特性[42];同时,DHA可显著上调草鱼肌肉肌源性调控因子的mRNA表达,包括肌分化因子(MyoD)、生肌因子5(Myf5)、肌原调节因子4(Mrf4)及肌细胞生成素(Myog),从而改善肌纤维结构[43]。由此推测,裂殖壶菌可能是通过影响凡纳滨对虾肌纤维发育,降低肌纤维直径,从而改善对虾肌肉品质,提升口感。

4 结论

① 饲料添加裂殖壶菌可激活凡纳滨对虾肌肉中PI3K/AKT/mTOR信号通路及肌肉生成调节基因的表达,提高肌肉粗蛋白质和胶原蛋白含量。
② 与破壁裂殖壶菌相比,未破壁裂殖壶菌可以提高凡纳滨对虾肌肉抗氧化能力,减少氧化应激。
③ 与未破壁裂殖壶菌相比,破壁裂殖壶菌能够提高凡纳滨对虾肌肉胶原蛋白含量,减小肌纤维直径,改善肌肉质构特性。

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