孙佳静, 李貌, 孙志洪, 唐志如, 张相鑫, 陈进超. 胶红酵母产类胡萝卜素固态发酵工艺[J]. 中国农业科学, 2018, 51(10): 1982-1994 https://doi.org/10.3864/j.issn.0578-1752.2018.10.017
SUN JiaJing, LI Mao, SUN ZhiHong, TANG ZhiRu, ZHANG XiangXin, CHEN JinChao. The Solid-State Fermentation Process of Rhodotorula Mucilaginosa for Producing Carotenoids[J]. Scientia Acricultura Sinica, 2018, 51(10): 1982-1994 https://doi.org/10.3864/j.issn.0578-1752.2018.10.017

0 引言

【研究意义】类胡萝卜素是一类广泛存在于自然界中的有色多烯类化合物,主要包括β-类胡萝卜素、虾青素、叶黄素和番茄红素等。天然类胡萝卜素因其抗氧化、防癌、增强免疫力等保健作用受到越来越多人的青睐^[1]。因此,开展胶红酵母产胡萝卜素研究具有重要意义。【前人研究进展】类胡萝卜素混合物和β-胡萝卜素都没有基因毒性,类胡萝卜素可以通过调节细胞信号和基因表达抵抗癌症^[2],且可以抑制基因突变和抵抗环境诱导的基因毒性剂^[3]。类胡萝卜素已被FAO和WHO定为A类营养色素,在50多个国家和地区获准作为营养、着色双重功用的食品添加剂,被广泛应用于保健食品及医药和化妆品工业^[4]。目前,类胡萝卜素主要利用微生物和高等植物进行生产。从植物中提取天然类胡萝卜素不仅受地域条件、原料来源、提取效率等限制,而且生产成本较高。近年来,利用微生物生产类胡萝卜素受到越来越多的关注。生产类胡萝卜素的微生物主要包括三孢布拉霉（Blakesleatrispora）和红酵母类。三孢布拉霉虽然产率高,但发酵工艺较难调控,而且液态发酵普遍存在着生产工艺复杂、能耗高、环境污染大、成本高等缺点。胶红酵母是一种主要合成β-胡萝卜素的红酵母^[5],其通过固态发酵生产类胡萝卜素的工艺与液态发酵法相比,具有能耗低、培养基简单、生产过程节水、操作简便易行等优点^[6]。【本研究切入点】红酵母合成类胡萝卜素的效率受许多因素的影响,例如,培养基组成、发酵条件^[7]。目前,有关利用胶红酵母进行固态发酵生产类胡萝卜素的研究较少。本研究选用胶红酵母作为发酵菌种,主要利用农业副产品麸皮,并辅以其他发酵原料作为固态发酵底物原料,旨在探究发酵底物原料（麸皮、豆粕、玉米粉、玉米浆、米糠、硫酸铵、磷酸氢二钾和硫酸镁等）配比和发酵条件（接种量、发酵温度、底物含水量、发酵时间和底物pH）对类胡萝卜素产量、胶红酵母活菌数和发酵产物营养价值的影响。【拟解决的关键问题】确定胶红酵母最优的发酵底物配比和发酵条件;制定高效生产类胡萝卜素的胶红酵母发酵工艺。

1 材料与方法

试验于2015—2016年在西南大学生物饲料与分子营养实验室进行。

1.1 主要仪器

双目生物显微镜（B203LEDR,重庆奥特光学仪器有限公司）;高速冷冻离心机（ST 8,赛默飞世尔科技公司）;台式低速离心机（TDZ4-WS,湘仪公司）;涡旋仪（WH-3,上海沪西分析仪器有限公司）;恒温培养箱（HH?BII?500-BS,上海跃进医疗器械厂）;pH计（PHS-4C+,成都世纪方舟科技有限公司）;高压蒸汽灭菌器（MJ-54A,施都凯仪器设备有限公司）;数显恒温水浴锅（HH-8,常州国华电器有限公司）;超纯水系统（SDLD-R,重庆市澳凯龙医疗器械研究有限公司）;洁净工作台（SW-CJ-2D,苏净集团苏州安泰空气技术有限公司）。

1.2 试验材料

（1）菌种来源：重庆市北碚区柑桔研究所果园分离出的胶红酵母。该菌株已于2015年9月24日送至中国典型培养物保藏中心保藏,分类命名为胶红酵母TZR₂₀₁₄,保藏编号为CCTCC NO：M 2015574,保藏地址为武汉大学。菌落特征如下：菌落呈圆形、橙红色、隆起,胶质黏稠,边缘整齐,表面光滑,易于挑起。
（2）PDA培养基：称取马铃薯200 g、葡萄糖20 g、琼脂20 g,溶于1 000 mL水中,自然pH,115℃灭菌20 min。
（3）种子培养基：称取葡萄糖20 g、蛋白胨10 g、酵母提取物10 g,溶于1 000 mL自来水中,自然pH,115℃灭菌20 min。

1.3 胶红酵母固态发酵底物的优化

从满足胶红酵母生长必需的营养素以及经济性原则考虑,本研究选择麸皮、豆粕、玉米粉、玉米浆、米糠、硫酸铵、磷酸氢二钾和硫酸镁8种原料作为发酵底物,在借鉴前人研究基础上^[8,9],采用Design-Expert软件中的Mixture Design设计固态发酵底物配比,各原料的范围如下：麸皮50%—80%、豆粕6%—20%、玉米粉3%—15%、米糠2%—14%、玉米浆2%—10%、硫酸铵0.4%—2.5%、磷酸二氢钾0.05%—0.5%和硫酸镁0.03%—0.3%。具体设计如表1所示。
Table 1
表1
表1胶红酵母固态发酵底物优化设计
Table 1The optimized design of the substrates for the solid-state fermentation of Rhodotorula mucilaginosa (%)

序号 Order number	麸皮 Wheat bran	豆粕 Soybean meal	玉米 Maize	米糠 Rice bran	玉米浆 Maize syrup	硫酸铵 Ammonium sulphate	磷酸二氢钾 Monopotassium phosphate	硫酸镁 Magnesium sulphate
1	73.4	9.51	5.00	6.64	2.43	2.50	0.33	0.17
2	77.7	6.00	3.00	8.10	4.16	0.74	0.08	0.19
3	79.8	7.93	3.00	2.00	6.59	0.46	0.05	0.18
4	59.5	19.3	4.44	10.2	4.45	1.89	0.05	0.08
5	71.4	7.89	15.0	2.00	2.04	1.30	0.18	0.22
6	67.7	10.0	4.60	14.0	2.00	1.27	0.23	0.24
7	63.4	14.4	13.6	3.70	2.44	1.57	0.50	0.28
8	52.3	19.8	13.6	10.6	2.00	1.57	0.15	0.03
9	77.0	7.53	9.97	2.98	2.05	0.40	0.05	0.03
10	58.1	15.5	9.15	12.2	4.03	0.40	0.50	0.06
11	58.1	18.2	12.9	5.01	3.10	2.50	0.11	0.15
12	53.8	16.6	7.79	11.3	8.59	1.16	0.50	0.28
13	62.1	13.3	8.73	8.05	6.00	1.53	0.28	0.09
14	69.4	6.37	3.00	11.9	6.39	2.44	0.25	0.30
15	56.0	18.3	5.81	7.81	10.0	1.74	0.13	0.26
16	76.1	8.99	3.00	4.93	4.38	2.48	0.06	0.03
17	59.5	19.3	4.44	10.2	4.45	1.89	0.05	0.08
18	62.3	16.4	10.1	7.38	2.94	0.61	0.05	0.22
19	57.7	15.6	6.41	10.9	8.82	0.40	0.19	0.03
20	56.4	17.7	14.0	3.66	7.32	0.85	0.10	0.04
21	70.2	20.0	3.06	3.21	2.63	0.68	0.21	0.05
22	62.1	13.3	8.73	8.05	6.00	1.53	0.28	0.09
23	77.7	6.00	3.00	8.10	4.16	0.74	0.08	0.19
24	65.9	7.38	13.8	7.01	4.86	0.40	0.42	0.27
25	57.6	12.2	13.8	4.76	8.84	2.35	0.20	0.30
26	63.6	6.00	14.0	4.73	9.90	1.54	0.12	0.10
27	73.4	9.51	5.00	6.64	2.43	2.50	0.33	0.17
28	77.7	6.00	3.00	8.10	4.16	0.74	0.08	0.19
29	79.8	7.93	3.00	2.00	6.59	0.46	0.05	0.18
30	79.8	7.93	3.00	2.00	6.59	0.46	0.05	0.18
31	59.5	19.3	4.44	10.2	4.45	1.89	0.05	0.08
32	67.7	10.0	4.60	14.0	2.00	1.27	0.23	0.24
33	62.1	13.3	8.73	8.05	6.00	1.53	0.28	0.09
34	69.4	6.37	3.00	11.9	6.39	2.44	0.25	0.30
35	76.1	8.99	3.00	4.93	4.38	2.48	0.06	0.03
36	62.3	16.4	10.1	7.38	2.94	0.61	0.05	0.22

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取-80℃冻存的胶红酵母接种于PDA培养基上,28℃培养72 h,进行菌种的复苏。再取PDA培养基上的胶红酵母菌接种于种子培养基上,置于恒温振荡器中于200 r/min、28℃培养24 h,即得胶红酵母种子液。分别秤取按表1设计的36种发酵底物132 g,混匀,并将水分含量调节为65%,平均分成4份,其中1份做空白对照,另外3份作为一个处理的3次重复,用于排除培养基中原有的类胡萝卜素对试验的影响。置于250 mL三角瓶密封灭菌,待冷却后按发酵底物质量体积的5%接种。8层纱布密封后置于30℃培养箱培养72 h。

1.4 发酵条件优化

在获取最佳发酵底物配比的基础上,借鉴前人研究结果^[10,11]设定接种量、发酵时间、发酵温度、发酵pH和发酵底物含水量的梯度（表2）,并对以上5个参数进行L₁₆(4⁵)正交设计（表3）。
Table 2
表2
表2胶红酵母固态发酵条件优化梯度
Table 2The optimized gradients of the conditions for the solid-state fermentation of Rhodotorula mucilaginosa

梯度 Gradients	接种量 Inoculum (%)	发酵时间 Fermentation time (h)	发酵温度 Fermentation temperature (℃)	pH	含水量 Moisture content (%)
A	5.0	60.0	26.0	4.0	60.0
B	7.5	72.0	28.0	5.0	65.0
C	10.0	84.0	30.0	6.0	70.0
D	12.5	96.0	32.0	7.0	75.0

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Table 3
表3
表3胶红酵母固态发酵条件的优化设计
Table 3The optimized design of the conditions for the solid-state fermentation of Rhodotorula mucilaginosa

序号 Order number	接种量 Inoculum amount (%)	发酵时间 Fermentation time (h)	发酵温度 Fermentation temperature (℃)	pH	含水量 Moisture content (%)
1	A	A	A	A	A
2	A	B	B	B	B
3	A	C	C	C	C
4	A	D	D	D	D
5	B	A	B	C	D
6	B	B	A	D	C
7	B	C	D	A	B
8	B	D	C	B	A
9	C	A	C	D	B
10	C	B	D	C	A
11	C	C	A	B	D
12	C	D	B	A	C
13	D	A	D	B	C
14	D	B	C	A	D
15	D	C	B	D	A
16	D	D	A	C	B

A, B, C and D represents the relative gradients of inoculum amount, fermentation time, fermentation temperature, pH value, and moisture content in table 2, respectively表中A、B、C、D分别代表表2中相对应的发酵梯度
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胶红酵母复苏以及得到种子液的步骤同上。称取上述研究得到的最优发酵底物16组,每组132 g,混匀,并将水分含量调节为65%,平均分成4份,其中一份做空白对照,然后置于250 mL三角瓶密封灭菌,按表3设置的固态发酵条件进行发酵,其中pH用氢氧化钠和盐酸进行调节。

1.5 胶红酵母固态发酵工艺优化对底物养分的影响

发酵底物以及发酵产物灭菌、混匀后,在60℃烘干、粉碎,用于测定样品中的水分、粗蛋白质、粗脂肪、粗纤维、粗灰分、无氮浸出物、钙、磷和氨基酸含量。

1.6 测定方法

（1）胶红酵母活菌数：取5 g发酵底物或产物置于锥形瓶内,在锥形瓶内加入100 mL 0.85%的生理盐水,置于恒温振荡器中于200 r/min振荡20 min,用血球计数板进行计数。
固态发酵产物含胶红酵母细胞数（y,CFU/g）的计算方法如下：
y={[（80个小方格内细胞个数）/80×400×10⁴×稀释的倍数]×100}/5
（2）类胡萝卜素：测定过程主要参考杨文等^[12]和王岁楼等^[13]介绍的方法,并对部分操作步骤进行了改进。具体测定步骤如下：称取10 g样品并用50 mL 0.85%的生理盐水稀释,八层纱布过滤后菌液于4 800 r/min下离心10 min。收集菌体,置于50℃恒温箱中烘干至恒重。称取干酵母0.1 g,加入3 mol·L^-1的盐酸2.4 mL,静止1 h,转至沸水浴中加热4 min,迅速冷却,然后4 000 r/min离心10 min,弃上清液。水洗后再离心,重复2次,即得细胞碎片。在细胞碎片中加入4 mL丙酮溶液,室温漩涡振荡15 min后,4 000 r/min离心10 min,取上清液,重复浸提2次,所得上清液即为类胡萝卜素提取液。类胡萝卜素含量按下列公式计算：
色素的含量（μg·g^-1干菌体）=Aλ×D×V/0.16×W
式中：Aλ为475 nm波长处的吸光值,D为测定试样时的稀释倍数,V为丙酮的用量（mL）,W为酵母
菌重量（g）,0.16为类胡萝卜素的摩尔消光系数。
（3）水分：将样品（105±2）℃烘箱烘干至恒重。
（4）粗蛋白质：采用凯氏定氮法,用半自动凯氏定氮仪进行测定。
（5）粗脂肪：采用乙醚浸提法,用脂肪测定仪进行测定。
（6）粗灰分：将样品在（550±20）℃下灰化至恒重。
（7）粗纤维：采用滤袋技术进行测定。
（8）钙：采用高锰酸钾滴定法进行测定。
（9）磷：采用钼黄比色法进行测定。
（10）氨基酸：采用全自动氨基酸分析仪进行分析。

1.7 统计分析

试验数据使用SAS 9.0和Design-Expert进行统计分析。P<0.05表示差异显著。

2 结果

2.1 固态发酵底物优化

由表4所示,发酵底物18所产的类胡萝卜素含量最高,而发酵底物17经过发酵后产物中的活菌数量最高,考虑到本研究是以类胡萝卜素产量为主要目标,因此,将发酵底物18作为最优发酵底物,供后续的研究使用。
Table 4
表4
表4胶红酵母固态发酵底物优化对类胡萝卜素产量和菌体数量的影响
Table 4Effects of the optimization of the subtrates for the solid-state fermentation of Rhodotorula mucilaginosa on the carotenoid production and the number of bacteria

序号 Order number	类胡萝卜素 Carotenoid (μg·kg-1)	发酵料含菌数 Number of bacteria (109 CFU/g)
1	1 673±28.8	2.22±0.40
2	1 329±81.6	2.42±0.38
3	1 715±125	3.08±0.41
4	1 767±47.2	3.28±0.41
5	1 293±76.6	2.85±0.30
6	1 503±94.2	2.82±0.35
7	1 332±24.2	2.85±0.33
8	1 699±70.4	2.94±0.50
9	1 404±73.9	2.70±0.23
10	1 751±159	2.59±0.29
11	1 673±77.7	2.42±0.23
12	1 767±56.8	2.82±0.27
13	1 757±135	2.61±0.39
14	1 785±64.1	2.42±0.22
15	1 902±22.1	3.43±0.45
16	1 470±38.7	2.45±0.33
17	1 831±79.4	3.52±0.23
18	1 923±109	3.13±0.39
19	1 695±100	2.87±0.26
20	1 383±69.6	2.61±0.33
21	1 296±133	3.27±0.36
22	1 329±78.8	2.80±0.28
23	1 758±49.3	2.77±0.43
24	1 776±182	2.85±0.19
25	1 630±51.9	2.67±0.31
26	1 744±79.3	2.91±0.35
27	1 695±32.3	2.14±0.26
28	1 372±53.0	2.32±0.26
29	1 656±63.1	3.01±0.27
30	1 685±73.9	3.07±0.41
31	1 785±88.5	3.22±0.30
32	1 479±72.4	2.79±0.23
33	1 732±103	2.59±0.23
34	1 787±61.3	2.56±0.27
35	1 502±30.9	2.51±0.23
36	1 848±98.7	2.87±0.25

n=3; The value is mean±SD. The same as belown=3;数值为平均值±标准差。下同
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2.1.1 试验的数学模型分析 以表4数据为基础,用Design-Expert软件对类胡萝卜素含量和胶红酵母菌数进行分析,得到模型的性噪比（Adeq Precision）和R²预测值（Pred R-Squared）,如表5所示。当模型的信噪比大于4时表示模型是比较合适的。数据显示类胡萝卜素和胶红酵母菌数模型的性噪比均大于4,表明数据均是可取的,且具有较高的精密度值。当R²预测值为负值时意味着预测模型比实际模型更合适。该试验模型分析的结果均为负值,表明预测模型比实际模型具有更理想的效果。
Table 5
表5
表5模型的性噪比及R² 预测值
Table 5The Adeq Precision and Pred R-Squared of the model

项目 Items	类胡萝卜素 Carotenoids	胶红酵母菌数 Number of Rhodototula mucilaginosa
模型的信噪比 Adeq Precision	6.23	5.32
R2 预测值 Pred R-Squared	-0.095	-0.031

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Table 6
表6
表6发酵底物与胶红酵母类胡萝卜素产量及菌体数量的相关性
Table 6The correlation between the fermentation substrates of Rhodototula mucilaginosa and the carotenoid yield or number of bacteria

因素 Factor	类胡萝卜素 Carotenoids	P值 P-value	胶红酵母数 Number of Rhodototula mucilaginosa	P值 P-value
麦麸 Wheat bran	-0.336	0.045	-0.370	0.067
豆粕 Soybean meal	0.200	0.242	0.510	0.001
玉米粉 Maize	-0.049	0.777	0.005	0.978
米糠 Rice bran	0.329	0.050	-0.027	0.874
玉米浆 Maize syrup	0.344	0.040	0.183	0.284
硫酸铵 Ammonium sulphate	0.094	0.586	-0.296	0.080
磷酸二氢钾 Monopotassium phosphate	-0.009	0.960	-0.276	0.104
硫酸镁 Magnesium sulphate	0.196	0.253	-0.069	0.687

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由表6可知,胶红酵母发酵产物中类胡萝卜素含量与固态发酵底物中麦麸的添加量呈显著负相关（r=-0.336,P=0.045）;固态发酵底物中豆粕含量与发酵产物中胶红酵母活菌数呈显著正相关（r=0.510,P=0.001）;玉米浆的添加量与类胡萝卜素产量呈显著正相关（r=0.344,P=0.040）。此外,发酵底物中米糠添加量与发酵产物中类胡萝卜素含量正相关（r=0.329,P=0.050）。
2.1.2 试验结果验证 表7为通过Design-Expert软件对各指标进行分析后,以尽可能达到发酵产物中类胡萝卜素含量和胶红酵母菌数最大的一个最佳配方。
以表7配方为基础对两个指标进行模型预测,得出预测值如表8所示。再按照该配方进行发酵试验,将实测值与预测值进行显著性检验。由表8可知,类胡萝卜素的实测值略高于预测值,但差异不显著（P>0.05）。
Table 7
表7
表7胶红酵母固态发酵底物优化配方
Table 7The optimized formulation of the substrates for the solid-state fermentation of Rhodotorula mucilaginosa (%)

麦麸 Wheat bran	豆粕 Soybean meal	玉米粉 Maize	米糠 Rice bran	玉米浆 Maize syrup	硫酸铵 Ammonium sulphate	磷酸二氢钾 Monopotassium phosphate	硫酸镁 Magnesium sulphate
52.5	20.0	3.00	14.0	10.0	0.40	0.05	0.04

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Table 8
表8
表8胶红酵母类胡萝卜素产量的模型预测值与实测值
Table 8The model predictive values and measured data of the carotenoid production of Rhodotorula mucilaginosa

指标 Index	模型预测值 Model predictive value	实测值 Measured data	SEM	P值 P-value
类胡萝卜素 Carotenoids (μg·kg-1)	1 923	1 937	22.6	0.65
活菌数 Number of bacteria (109 CFU/g)	3.2	3.18	0.38	0.31

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2.2 固态发酵条件优化

用正交设计软件对类胡萝卜素含量和胶红酵母细胞数进行逐个分析处理,找出最佳的配方方案,得到的试验方案如表9所示。由表9可知,就发酵产物中活菌数来看,极差R_C>R_E>R_D>R_A>R_B,因此C因素（发酵温度）被确定为最重要的影响因素,并且发酵温度在第4水平时（32℃）发酵产物中活菌数最多;之后进行主次因素依次分析,得到获取最多活菌数的发酵条件为C₄E₁D₁A₁B₂。而从发酵产物中类胡萝卜素含量进行比较,极差R_C>R_D>R_B>R_E>R_A,因此,因素C是最重要的影响因素,并且发酵温度第2水平时（28℃）类胡萝卜素含量最高;然后进行主次因素依次分析,得到获取最高类胡萝卜素产量的发酵条件为C₂D₃B₂E₁A₂。针对不同发酵目的的优化发酵方案不完全相同,如表10所示。
Table 9
表9
表9胶红酵母固态发酵条件优化的正交试验结果
Table 9The orthogonal test results of the optimized conditions for the solid-state fermentation of Rhodotorula mucilaginosa

序号 Order number	A		B		C			D	E	发酵料含菌数 Number of bacteria in fermented materials (109 CFU/g)	类胡萝卜素含量 Carotenoid content (μg·kg-1)
1	1		1		1			1	1	4.03±0.29	1 377±30.5
2	1		2		2			2	2	3.60±0.31	3 149±50.8
3	1		3		3			3	3	2.78±0.40	2 150±93.5
4	1		4		4			4	4	3.53±0.41	2 126±75.6
5	2		1		2			3	4	2.57±0.25	3 150±77.8
6	2		2		1			4	3	3.29±0.23	1 914±65.5
7	2		3		4			1	2	3.64±0.43	1 902±46.2
8	2		4		3			2	1	2.82±0.35	2 378±87.9
9	3		1		3			4	2	2.96±0.26	1 651±55.4
10	3		2		4			3	1	3.49±0.28	3 068±32.5
11	3		3		1			2	4	3.02±0.34	2 012±69.8
12	3		4		2			1	3	3.41±0.39	2 447±103.5
13	4		1		4			2	3	3.27±0.38	2 350±45.6
14	4		2		3			1	4	2.73±0.37	1 752±29.6
15	4		3		2			4	1	3.31±0.26	2 686±56.4
16	4		4		1			3	2	3.29±0.21	2 192±33.8
k1	3.49		3.21		3.41			3.45	3.41	C4E1D1A1B2 RC>RE>RD>RA>RB
k2	3.08		3.28		3.22			3.18	3.37
k3	3.22		3.19		2.82			3.03	3.19
k4	3.15		3.26		3.48			3.27	2.96
R	0.41		0.09		0.66			0.42	0.45
k1	2201		2132		1874			1870	2377
k2	2336		2471		2858			2472	2224	C2D3B2E1A2 RC>RD>RB>RE>RA
k3	2294		2187		1983			2640	2215
k4	2245		2286		2362			2094	2260
R	135		338		984			770	161

A-E represents inoculum amount, fermentation time, fermentation temperature, pH, and moisture content, respectively. n=3; the value is mean±SD. The same as belowA—E分别代表接种量、发酵时间、发酵温度、pH、含水量;n=3;数值为平均值±标准差。下同
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Table 10
表10
表10胶红酵母固态发酵条件优化方案
Table 10e optimized scheme of the conditions for the solid-state fermentation of Rhodotorula mucilaginosa

项目Items	因素水平（由主到次）Factors level (from the main to the second)
细胞数 Cell number	C4（32℃）	E1（60%）	D1（4.0）	A1（5.0%）	B2（60 h）
类胡萝卜素Carotenoids	C2（28℃）	D3（6.0）	B2（72 h）	E1（60%）	A2（7.5%）

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为了更直观地反映各产物在不同因素水平下的变化,本研究还做了关于胶红酵母细胞数和类胡萝卜素含量的相关方差分析,分别如表11、表12所示。由表11可知,接种量、发酵温度、pH、含水量对发酵产物中胶红酵母活菌数均有极显著的影响（P<0.001）,其中发酵温度影响最大,其次是含水量,之后依次是接种量和pH。由表12得,发酵时间、发酵温度和pH对发酵产物中类胡萝卜素含量的影响均为极显著（P<0.01）,其中发酵温度的影响最大,其次为pH,最后是发酵时间。
Table 11
表11
表11发酵条件对胶红酵母菌体数量影响的方差分析
Table 11The variance analysis of the effects of fermentation conditions on the number of Rhodototula mucilaginosa

变异来源 Sources of variation	偏差平方和 Sum of squares of deviations	自由度 Freedom	方差 Variance	F值 F value	Fa Fa	显著水平 Significant level
接种量Inoculum amount	0.38	3	0.13	16.9	F0.01（3,6）=9.78	***
发酵时间Fermentation time	0.02	3	0.01		F0.05（3,6）=4.76
发酵温度Fermentation temperature	1.05	3	0.35	47.0	F0.1（3,6）=3.29	***
pH	0.37	3	0.12	16.7	F0.25（3,6）=1.78	***
含水量Moisture content	0.51	3	0.17	22.8		***
误差Error	0.02	3	0.01
修正误差Corrected error	0.05	6	0.01
总和Sum	2.35

*** represents P<0.001; ** represents P<0.01. The same as below***代表P<0.001;**代表P<0.01。下同
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Table 12
表12
表12发酵条件对发酵产物中类胡萝卜素含量影响的方差分析
Table 12The variance analysis of the effects of fermentation conditions on the carotenoid content

变异来源 Sources of variation	偏差平方 Sum of squares of deviations	自由度 Freedom	方差 Variance	F值 F value	Fa Fa	显著水平Significant level
接种量 Inoculum amount	41472	3	13 824		F0.01（3,9）=6.99
发酵时间 Fermentation time	265432	3	88 477	5.32	F0.05（3,9）=3.86	**
发酵温度 Fermentation temperature	2374448	3	791 483	47.5	F0.1（3,9）=2.81	***
pH	1476072	3	492 024	29.6	F0.25（3,9）=1.63	***
含水量Moisture content	66880	3	22 293
误差Error	41472	3	13 824
修正误差Corrected error	149824	9	16 647
总和Sum	4265776

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根据影响主次性以及显著性程度,含水量对发酵产物中胶红酵母活菌数影响显著,最佳含水量取E₂（60%）;发酵时间对发酵产物中类胡萝卜素含量影响显著,且最佳发酵时间为B₂（72 h）;接种量活菌细胞数影响极显著,最佳接种量取A₁（5.0%）;pH对活菌数和类胡萝卜素含量影响均显著,因pH选取应优先考虑类胡萝卜素含量,本试验最佳pH取D₃（6.0）;发酵温度对活菌数和类胡萝卜素含量影响均显著,因本试验以产类胡萝卜素为主要优化目标,最佳发酵温度取C₂（28℃）。最佳优化条件下发酵产物中类胡萝卜素含量与胶红酵母菌数如表13所示,对比表4可看出,发酵条件优化后,发酵产物中类胡萝卜素含量与活菌数得到明显增加。
Table 13
表13
表13最优发酵工艺下类胡萝卜素产量和胶红酵母数量
Table 13The carotenoid production and Rhodotorula mucilaginosa number under the optimal fermentation process

指标 Index	试验值 Test values
类胡萝卜素 Carotenoids (μg·kg-1)	4 535±32.5
发酵料含菌数 Number of bacteria of fermentation (109 CFU/g)	3.79±0.29

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2.3 胶红酵母固态发酵工艺优化对底物养分的影响

2.3.1 发酵前后底物常规营养成分对比分析 由表14可知,发酵后粗蛋白质、粗灰分和粗纤维含量显著高于发酵前（P<0.05）;发酵后含水量和粗脂肪含量显著低于发酵前;而发酵前后钙和磷的含量没有显著差异。
Table 14
表14
表14胶红酵母发酵底物常规营养成分在发酵前后的变化
Table 14The changes of conventional nutrient composition in substrate for Rhodotorula mucilaginosa after fermentation (%)

项目 Items	发酵底物Fermentation substrate	发酵产物Fermentation product	SEM	P 值 P-value
粗纤维 Crude fiber	10.4b	11.6a	0.10	<0.05
粗蛋白质 Crude protein	25.9 b	28.4a	0.13	<0.05
水分 Moisture	12.6a	11.0b	0.24	<0.05
粗脂肪 Crude fat	3.25a	2.73b	0.13	<0.05
粗灰分 Ash	6.87 b	7.83a	0.04	<0.05
磷 Phosphorus	0.51	0.51	0.01	0.116
钙 Calcium	0.55	0.56	0.03	0.712

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2.3.2 发酵前后底物氨基酸结果分析 由表15可知,发酵后苏氨酸、谷氨酸、脯氨酸含量显著高于发酵前（P<0.05）。
Table 15
表15
表15胶红酵母发酵底物氨基酸含量在发酵前后的变化
Table 15The changes of amino acid content in substrate in substrate for Rhodotorula mucilaginosa after fermentation (g /100 g)

项目 Items	发酵前 Before fermentation	发酵后 After fermentation	SEM	P 值 P-value
天门冬氨酸 Aspartic acid	1.65	1.70	0.03	0.29
苏氨酸 Threonine	0.72b	0.86a	0.02	<0.05
丝氨酸 Serine	0.90	0.92	0.01	0.40
谷氨酸Glutamate	3.47b	3.80a	0.04	<0.05
甘氨酸 Glycine	0.99	0.99	0.01	0.99
丙氨酸 Alanine	1.16	1.20	0.01	0.65
半胱氨酸 Cysteine	0.24	0.20	0.09	0.23
缬氨酸 Valine	0.94	0.98	0.04	0.56
蛋氨酸 Methionine	0.19	0.20	0.01	0.10
异亮氨酸 Isoleucine	0.76	0.82	0.05	0.12
亮氨酸 Leucine	1.52	1.60	0.06	0.38
酪氨酸 Tyrosine	0.43	0.50	0.04	0.21
苯丙氨酸 Phenylalanine	0.94	1.08	0.04	0.07
赖氨酸 Lysine	1.03	1.11	0.05	0.29
组氨酸 Histidine	0.60a	0.24b	0.06	<0.05
精氨酸 Arginine	1.30	1.21	0.06	0.33
脯氨酸 Proline	2.22b	2.98a	0.04	<0.05
总量 Total	19.5	20.9	0.51	0.16

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3 讨论

本试验结果表明,发酵产物中类胡萝卜素含量与固态发酵底物中麦麸的添加量呈显著负相关。这一结果同过去的报道并不完全一致。众所周知,适量添加麸皮可以增加培养基的蓬松程度,有助于氧气的流通,促进好氧菌的生长^[14]。但是,发酵底物中麸皮含量过高会影响酵母菌的发酵,其原因在于麸皮纤维含量高,会影响酵母菌的正常生长^[15]。为最大限度地利用廉价底物资源,本研究选择了52.5%的麸皮含量作为最适浓度。与麦麸结果相反,发酵底物中豆粕含量与发酵产物中胶红酵母活菌数呈极显著正相关;发酵底物中玉米浆的含量与发酵产物中类胡萝卜素的产量呈显著正相关,可能是这些物质含有酵母生长所必需的蛋白质、氨基酸、维生素、微量元素及某些生长因子。
除麦麸、豆粕、玉米浆、米糠外,其他底物（玉米、硫酸铵、磷酸二氢钾、硫酸镁）与发酵产物中类胡萝卜素含量及胶红酵母活菌数均未达到显著相关。王岁楼等^[16]也报道,K⁺和Mg²⁺并不能提高红酵母的类胡萝卜素产量。但有研究表明,KH₂PO₄、MgSO₄和(NH₄)₂SO₄对发酵产物中类胡萝卜素含量的影响均为正效应,因此,建议提高发酵底物中上述物质的实际水平^[17,18]。另有研究表明,当K₂HPO₄和MgSO₄的浓度分别为1 g·L^-1和10 g·L^-1时,海洋红酵母Y2细胞生物量最高;当K₂HPO₄和MgSO₄的浓度分别为2.5 g·L^-1和8 g·L^-1时,类胡萝卜素产量最高^[19]。因此,推测在一定剂量范围内,K⁺、PO₄^3-、Mg²⁺、NH₄⁺、SO₄²⁺与类胡萝卜素产量可能表现出剂量-效应关系,但超出一定范围,剂量-效应则不明显,甚至出现胶红酵母的生长或类胡萝卜素的合成受到抑制的情况^{[20,21,22,23]}。
在本研究中,温度对类胡萝卜素产量的影响最大,其次是pH,这一结果与赵紫华等^[24]报道的相反。有关资料显示,红酵母发酵产类胡萝卜素温度一般在30℃左右^[25],且固态发酵适宜的温度在25—35℃^[26,27],这些报道与本试验结果基本相符。关于发酵底物最适pH的报道也不尽相同,AKSU和EREN^[28]报道,胶红酵母产类胡萝卜素的最适pH为7.0;张闯^[29]研究得出红酵母发酵产类胡萝卜素的最适pH为6.0;还有报道指出,胶红酵母DBVPG 3853产类胡萝卜素的最适值pH为5.8^[16]。关于发酵时间,本试验结果与梁晓华等^[19]报道一致,而与王岁楼等^[16]、刘卉琳^[20]的报道不一致,原因可能是菌种、发酵底物或所采取的发酵工艺（固态与液态）不同。
本研究表明,胶红酵母产胡萝卜素的固态发酵工艺经优化后,发酵底物中的粗蛋白质提高9.67%,粗纤维提高11.5%,粗脂肪降低16%,钙和磷含量没有显著变化。此外,发酵后苏氨酸、谷氨酸、脯氨酸浓度显著高于发酵前,且总氨基酸含量提高了7.19%。通过发酵提高蛋白质或氨基酸含量在过去的研究中已得到广泛证实。ZHANG等^[30]研究表明,脱毒的麻籽饼粕经固态发酵后粗脂肪显著减少了5.53%,粗蛋白质含量增加15.1%。王建军^[31]研究表明,利用混菌固态发酵黄酒糟后,发酵产物中共检测出17种氨基酸,除组氨酸、苯丙氨酸和脯氨酸外,其他14种氨基酸的含量均有不同程度的提高。孙展英^[32]报道,利用曲霉X3发酵可使粗纤维含量降低51.3%,本研究发酵产物粗纤维升高可能与酵母菌基本不能利用纤维素类多糖有关。

4 结论

前人对胶红酵母的研究多采用液态发酵,本试验本着降低生产成本和环保的原则,采用固态发酵的方式。胶红酵母固态发酵产类胡萝卜素底物的最佳配比为麦麸52.5%、豆粕20.0%、玉米粉3.00%、米糠14.0%、玉米浆10.0%、硫酸铵0.40%、磷酸二氢钾0.05%和硫酸镁0.04%;最佳发酵条件为菌液接种量5.0%、发酵时间72.0 h、发酵温度28.0℃、pH 6.0、底物含水量60.0%。通过对胶红酵母固态发酵底物与发酵条件的优化,提高了发酵产物中类胡萝卜素产量,并改善了发酵底物的营养价值,使胶红酵母不仅可以生产类胡萝卜素,而且其发酵后的产物也可以直接用来饲喂动物。但胶红酵母固态产物对动物生产性能和在动物日粮中的最适添加量还需进一步探究。
The authors have declared that no competing interests exist.

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[1]	BANZATTO D, FREITA L A D,MUTTON M J R. Carotenoid production by Rhodotorula rubra cultivated in sugarcane juice, molasses, and syrup. Ciência e Tecnologia de Alimentos, 2013, 33(33): 14-18. [本文引用: 1]
[2]	TANG F Y.The silver bullet for cancer prevention: Chemopreventive effects of carotenoids .Biomedicine, 2012, 2(3): 117-121.https://doi.org/10.1016/j.biomed.2012.06.004URL [本文引用: 1]摘要 Cancer has been a leading cause of death in many countries. Chemoprevention of various types of human cancer using dietary nutrients has received a lot of attention and interest in the past decade. Recently, carotenoids have been shown to prevent tumor growth and progression. Carotenoids demonstrated chemopreventive capability by interrupting several stages of cancer including initiation, promotion, progression, and metastasis. The molecular mechanisms of actions are through the modulation of cell-signaling pathways and gene expression. The results of our study suggested that carotenoids could act as chemopreventive agents against the growth and progression of human cancer cells.
[3]	BHAGAVATHY S, SUMATHI P.Evaluation of antigenotoxic effects of carotenoids from green algae Chlorococcum humicola using human lymphocytes. Asian Pacific Journal of Tropical Biomedicine, 2012, 2(2):109-117.https://doi.org/10.1016/S2221-1691(11)60203-7URLPMID:3609264 [本文引用: 1]摘要 To identify the available phytochemicals and carotenoids in the selected green algae and evaluate the potential genotoxic/antigenotoxic effect using lymphocytes. Organic solvent extracts of Chlorococcum humicola (C. humicola) were used for the phytochemical analysis. The available carotenoids were assessed by HPLC, and LC-MS analysis. The genotoxicity was induced by the benzo(a)pyrene in the lymphocyte culture, the genotoxic and antigenotoxic effects of algal carotenoids with and without genotoxic inducer were evaluated by chromosomal aberration (CA), sister chromatid exchange (SCE) and micronucleus assay (MN). The results of the analysis showed that the algae were rich in carotenoids and fatty acids. In the total carotenoids lutein, 尾-carotene and 伪-carotene were found to be present in higher concentration. The frequency of CA and SCE increased by benzo(a)pyrene were significantly decreased by the carotenoids ( P<0.05 for CA, P<0.001 for SCE). The MN frequencies of the cells were significantly decreased by the treatment with carotenoids when compared with the positive controls ( P<0.05). The findings of the present study demonstrate that, the green algae C. humicola is a rich source of bioactive compounds especially carotenoids which effectively fight against environmental genotoxic agents, the carotenoids itself is not a genotoxic substance and should be further considered for its beneficial effects.
[4]	NELIS H J, DELEENHEER A P.Microbial sources of carotenoid- pigments used in foods and feeds . Journal of Applied Bacteriology, 1991, 70(3): 181-191.URL [本文引用: 1]
[5]	PERRIER V, DUBREUCQ E, GALZY P.Fatty-acid and carotenoid composition of rhodotorula strains. Archives of Microbiology, 1995, 164(3): 173-179. [本文引用: 1]
[6]	董娟, 郑晓吉, 孙静涛, 史学伟. 红酵母产类胡萝卜素影响因素及检测方法研究进展 . 粮食与油脂, 2013, 26(3): 49-51.URL [本文引用: 1]摘要 该文介绍类胡萝卜素主要组成和功能,对红酵母产类胡萝卜素发酵方法、发酵促进因子和破壁技术几个影响因素分别进行阐述,对类胡萝卜素分离与鉴定方法研究现状进行探讨,并展望未来发展方向。 DONG J, ZHENG X J, SUN J T, SHI X W.Research advance in influencing factors of carotenoid producted by red yeasts and detection methods .Cereals & Oils, 2013, 26(3): 49-51. (in Chinese)URL [本文引用: 1]摘要 该文介绍类胡萝卜素主要组成和功能,对红酵母产类胡萝卜素发酵方法、发酵促进因子和破壁技术几个影响因素分别进行阐述,对类胡萝卜素分离与鉴定方法研究现状进行探讨,并展望未来发展方向。
[7]	FERRAO M, GARG S.Studies on effect of media components on growth and α-carotene production by Rhodotorula graminis RC04. Journal of Cell Tissue Reseach, 2011, 11(1): 2551-2556. [本文引用: 1]
[8]	HERNANDEZ-ALMANZA A, CESAR MONTANEZ J, AGUILAR- GONZALEZ M A, MARTINEZ-AVILA C, RODRIGUEZ- HERRERA R, N.AGUILAR C.Rhodotorula glutinis as source of pigments and metabolites for food industry. Food Bioscience, 2014, 5:64-72.https://doi.org/10.1016/j.fbio.2013.11.007URL [本文引用: 1]摘要 Rhodotorula glutinis is a pigmented yeast, part of the Basidiomycota phylum, particularly important for food industries because its biotechnological potential and safety implications. Various strains of Rhodotorula present important features such as the production of large amounts of carotenoids, single-cell proteins from ethanol, acetic acid and acetaldehyde. This review describes and discusses the biology and biotechnology of Rhodotorula glutinis, focusing on development, microbial physiology and biochemical pathways, as well as fermentation systems for commercially viable industrial scale production of pigments.
[9]	KOT A M, BLAZEJAK S, KURCZ A, GIENTKA I, KIELISZEK M.Rhodotorula glutinis-potential source of lipids, carotenoids, and enzymes for use in industries. Applied Microbiology and Biotechnology, 2016, 100(14): 6103-6117.https://doi.org/10.1007/s00253-016-7611-8URLPMID:4916194 [本文引用: 1]摘要 Rhodotorula glutinisis capable of synthesizing numerous valuable compounds with a wide industrial usage. Biomass of this yeast constitutes sources of microbiological oils, and the whole pool of fatty acids is dominated by oleic, linoleic, and palmitic acid. Due to its composition, the lipids may be useful as a source for the production of the so-called third-generation biodiesel. These yeasts are also capable of synthesizing carotenoids such as 尾-carotene, torulene, and torularhodin. Due to their health-promoting characteristics, carotenoids are commonly used in the cosmetic, pharmaceutical, and food industries. They are also used as additives in fodders for livestock, fish, and crustaceans. A significant characteristic ofR. glutinisis its capability to produce numerous enzymes, in particular, phenylalanine ammonia lyase (PAL). This enzyme is used in the food industry in the production ofl-phenylalanine that constitutes the substrate for the synthesis of aspartame鈥攁 sweetener commonly used in the food industry.
[10]	ROADJANAKAMOLSON M, SUNTORNSUK W.Production of beta-carotene-enriched rice bran using solid-state fermentation of Rhodotorula glutinis. Journal of Microbiology and Biotechnology, 2010, 20(3): 525-531. [本文引用: 1]
[11]	HERNANDEZ-ALMANZA A, MONTANEZ-SAENZ J, MARTINEZ- AVILA C, RODRIGUEZ-HERRERA R, N.AGUILAR C. Carotenoid production byRhodotorula glutinis YB-252 in solid-state fermentation. Food Bioscience, 2014(7): 31-36.https://doi.org/10.1016/j.fbio.2014.04.001URL [本文引用: 1]摘要 Carotenoids represent a group of valuable molecules for the pharmaceutical, chemical, food and feed industries. In this study, a Plackett–Burman design was used to evaluate the effect of pH, moisture, carbon source, inoculum concentration, time and temperature of incubation and the concentration of inducer. Also, the influence of a metabolic inhibitor of the β-carotene accumulation was tested, demonstrating that a level of 125ppm permitted the accumulation of lycopene without concomitant production of β-carotene. Results showed that the highest obtained lycopene concentration was 340mgL611. Among the parameters evaluated, the inducer concentration, the fermentation time and the percentage of moisture in the system, are factors having a statistically significant effect on carotenoid production by Rhodotorula glutinis YB-252 in solid-state fermentation.
[12]	杨文, 吉春明. 一种简单的胞壁破碎方法 . 微生物学通报, 1995, 22(1): 58-59.URL [本文引用: 1]摘要 介绍一种简单，快速的胞壁破碎方法－酸－热处理法。破碎效果好且成本低廉。酸浓度大小和热处理时间长短对破碎效果有很大的影响。正交试验结果表明。破碎条件的最佳组合为ＨＣｌ浓度３ｍｏｌ／Ｌ，ＨＣｌ用量６ｍｌ／ｇ干细胞浸泡时间６０ｍｉｎ，热处理时间３ｍｉｎ。 YANG W, JI C M.A simple method of crushing cell wall .Microbiology, 1995, 22(1): 58-59. (in Chinese)URL [本文引用: 1]摘要 介绍一种简单，快速的胞壁破碎方法－酸－热处理法。破碎效果好且成本低廉。酸浓度大小和热处理时间长短对破碎效果有很大的影响。正交试验结果表明。破碎条件的最佳组合为ＨＣｌ浓度３ｍｏｌ／Ｌ，ＨＣｌ用量６ｍｌ／ｇ干细胞浸泡时间６０ｍｉｎ，热处理时间３ｍｉｎ。
[13]	王岁楼, 张鑫, 张平之. 红酵母类胡萝卜素提取方法研究 .食品与机械, 2000(6): 14-16.URL [本文引用: 1] WANG S L, ZHANG X, ZHANG P Z.The extraction of carotenoids fromRhodotorula . Food & Machinery, 2000(6): 14-16. (in Chinese)URL [本文引用: 1]
[14]	张玉诚, 薛白, 达勒措, 李秋瑾, 何宇. 混菌固态发酵白酒糟开发为蛋白质饲料的条件优化及营养价值评定 . 动物营养学报, 2016, 28(11): 3711-3720.https://doi.org/10.3969/j.issn.1006-267x.2016.11.042URL [本文引用: 1]摘要 本试验旨在优化白地霉、米曲霉、绿色木霉和枯草芽孢杆菌混菌固态发酵白酒糟开发为蛋白质饲料的条件,并评定其营养价值。将白地霉、米曲霉、绿色木霉和枯草芽孢杆菌按照1∶1∶1∶1混合后按10%接种到培养基中,采用L16(54)正交试验设计,共5个发酵条件,分别为基料、尿素、磷酸二氢钾、p H、水分,每个条件4个变量,共16组发酵条件。按条件配制好的混合物放置于(30±2)℃中培养72 h。对发酵前后真蛋白质、粗纤维含量进行极差分析确定最优条件,再比较最优条件发酵前后白酒糟营养水平和氨基酸组成的变化。结果显示:1)基料按照80%白酒糟、10%麸皮、5%玉米粉、5%菜籽粕配比,尿素添加量为1.5%,磷酸二氢钾添加量为0.7%,p H为5、水分为50%时发酵效果最好,为最优发酵条件。2)最优条件下发酵后白酒糟与发酵前相比,真蛋白质含量提高了57.85%(P0.01);粗纤维、酸性洗涤纤维、中性洗涤纤维、粗脂肪含量分别降低了42.39%、31.95%、27.73%、21.48%(P0.01);钙、磷含量分别提高了16.67%和68.18%(P0.01);总氨基酸含量提高了24.47%,其中赖氨酸、蛋氨酸、苏氨酸、缬氨酸、亮氨酸、异亮氨酸和脯氨酸含量分别提高了109.68%、38.09%、39.39%、71.43%、28.93%、10.87%和3.70%。综上可得,利用白地霉、米曲霉、绿色木霉和枯草芽孢杆菌混菌发酵白酒糟的最佳条件是:基料组成80%白酒糟、10%麸皮、5%玉米粉、5%菜籽粕,尿素1.5%,磷酸二氢钾0.7%,p H 5,水分50%,发酵产物的真蛋白质含量为24.34%。 ZHANG Y C, XUE B, DA L C, LI Q J, HE Y.Distillers Grains: Optimization of mixed bacterial solid-state fermentation conditions to produce protein feed and nutrient value analysis .Chinese Journal of Animal Nutrition, 2016, 28(11): 3711-3720. (in Chinese)https://doi.org/10.3969/j.issn.1006-267x.2016.11.042URL [本文引用: 1]摘要 本试验旨在优化白地霉、米曲霉、绿色木霉和枯草芽孢杆菌混菌固态发酵白酒糟开发为蛋白质饲料的条件,并评定其营养价值。将白地霉、米曲霉、绿色木霉和枯草芽孢杆菌按照1∶1∶1∶1混合后按10%接种到培养基中,采用L16(54)正交试验设计,共5个发酵条件,分别为基料、尿素、磷酸二氢钾、p H、水分,每个条件4个变量,共16组发酵条件。按条件配制好的混合物放置于(30±2)℃中培养72 h。对发酵前后真蛋白质、粗纤维含量进行极差分析确定最优条件,再比较最优条件发酵前后白酒糟营养水平和氨基酸组成的变化。结果显示:1)基料按照80%白酒糟、10%麸皮、5%玉米粉、5%菜籽粕配比,尿素添加量为1.5%,磷酸二氢钾添加量为0.7%,p H为5、水分为50%时发酵效果最好,为最优发酵条件。2)最优条件下发酵后白酒糟与发酵前相比,真蛋白质含量提高了57.85%(P0.01);粗纤维、酸性洗涤纤维、中性洗涤纤维、粗脂肪含量分别降低了42.39%、31.95%、27.73%、21.48%(P0.01);钙、磷含量分别提高了16.67%和68.18%(P0.01);总氨基酸含量提高了24.47%,其中赖氨酸、蛋氨酸、苏氨酸、缬氨酸、亮氨酸、异亮氨酸和脯氨酸含量分别提高了109.68%、38.09%、39.39%、71.43%、28.93%、10.87%和3.70%。综上可得,利用白地霉、米曲霉、绿色木霉和枯草芽孢杆菌混菌发酵白酒糟的最佳条件是:基料组成80%白酒糟、10%麸皮、5%玉米粉、5%菜籽粕,尿素1.5%,磷酸二氢钾0.7%,p H 5,水分50%,发酵产物的真蛋白质含量为24.34%。
[15]	GABERT V M, SAUER W C, SCHMITZ M, AHRENS F, MOSENTHIN R.The effect of formic acid and buffering capacity on the ileal digestibilities of amino acids and bacterial populations and metabolites in the small intestine of weanling pigs fed semipurified fish meal diets .Canadian Journal of Animal Science, 1995, 75(4): 615-623.https://doi.org/10.4141/cjas95-091URL [本文引用: 1]摘要 An experiment was carried out to examine the effects of supplementation of formic acid to semipurified diets containing fish meal for weanling pigs with low and high buffering capacity on nutrient digestion and bacterial populations and metabolites in the small intestine. Twelve barrows, weaned at 21 d, were fitted with a simple T-cannula at the distal ileum. The BW of the pigs at weaning and at the conclusion of the experiment were 7.8 and 13.8 kg, respectively. The pigs were fed four corn starch-based fish meal diets according to a two-period change-over design. The diets were formulated to contain 18% CP. For two of the diets, referred to as NCaP, fish meal was the calcium and phosphorus source. For the remaining two diets, referred to as HCaP, the levels of calcium and phosphorus were doubled (compared with the NCaP diets) by the addition of calcium carbonate and dicalcium phosphate. The diets were supplemented with (+NCaP and +HCaP) or without (-NCaP and -HCaP) 1% (wt/wt) formic acid. The buffering capacity of the HCaP diets increased (P 0.05) the apparent ileal digestibilities of AA. The pH, ammonia and VFA concentrations, bacterial populations in ileal digesta and the incidence of diarrhea were also not affected (P > 0.05). Therefore, the supplementation of 1% (wt/wt) formic acid to corn starch-based fish meal diets with low and high buffering capacity for weanling pigs (7.8-13.8 kg) does not affect nutrient digestibilities and bacterial populations in the small intestine.
[16]	王岁楼, 章银良, 王平诸. 红酵母RY-98产类胡萝卜素培养基的优选及其发酵生理学研究 . 生物技术, 2000, 10(4): 24-27.URL [本文引用: 3] WANG S L, ZHANG Y L, WANG P Z.Studies on the selection of carotenoids fermentation medium for RY- 98 strain of Rhodotorula and its fermentative physiology. Biotechnology, 2000, 10(4): 24-27. (in Chinese)URL [本文引用: 3]
[17]	唐棠. 红酵母Y-5菌株产类胡萝卜素发酵条件的研究[D] . 雅安: 四川农业大学, 2011. [本文引用: 1] TANG T.Optimization of fermentation condition for production of carotenoid by Rhotlotorula Y-5 [D] . Ya’an: Sichuan Agricultural University, 2011. (in Chinese) [本文引用: 1]
[18]	张坤生, 连喜军, 李红, 任云霞. 红酵母高产β-胡萝卜素营养因子的选择 . 食品工业科技, 2004, 25(7): 60-62.https://doi.org/10.3969/j.issn.1002-0306.2004.07.024URL [本文引用: 1]摘要 研究了红酵母生产β-胡萝卜素的最优碳源、氮源及其浓度选择和金属离子对红酵母生长和β-胡萝卜素生成的影响,分析了各营养因子对生物量β-胡萝卜素产量的影响,确定了2%葡萄糖,2%蛋白胨和0.01%Mn2+离子的最佳营养因子. ZHANG K S, LIAN X J, LI H, REN Y X.Nutrition factors selection for β-carotene of red yeast .Science and Technology of Food Industry, 2004, 25(7): 60-62. (in Chinese)https://doi.org/10.3969/j.issn.1002-0306.2004.07.024URL [本文引用: 1]摘要 研究了红酵母生产β-胡萝卜素的最优碳源、氮源及其浓度选择和金属离子对红酵母生长和β-胡萝卜素生成的影响,分析了各营养因子对生物量β-胡萝卜素产量的影响,确定了2%葡萄糖,2%蛋白胨和0.01%Mn2+离子的最佳营养因子.
[19]	梁晓华, 杨莺莺, 杨铿, 陈永青. 海洋红酵母Y2发酵产类胡萝卜素条件的研究 . 中国微生态学杂志, 2011, 23(12): 1068-1073.URL [本文引用: 2]摘要 目的 对海洋红酵母Y2高产类胡萝卜素的发酵条件进行优化.方法 在摇瓶条件下,研究培养基成分和培养条件对海洋红酵母Y2生长和类胡萝卜素合成的影响,同时进行海洋红酵母Y2发酵过程的动态分析.结果 海洋红酵母Y2优化培养基组合为葡萄糖45 g/L,蔗糖15 g/L,酵母粉5 g/L,蛋白胨2.5 g/L,磷酸二氢钾1 g/L,磷酸二氢钠3 g/L,硫酸镁7.5 g/L,氯化钾3 g/L,氯化钠5 g/L.最适培养参数为:温度20℃,培养基初始pH为5,接种量为10％,250 mL摇瓶装液量为10～50 mL.类胡萝卜素的合成主要集中在对数生长期和稳定期.海洋红酵母Y2最适收获时间为72 h.种龄以36 h为宜.结论 利用优化培养基,在最适条件下培养海洋红酵母Y2,类胡萝卜素产量达到4.97 mg/L,比基础培养基提高了60.32％. LIANG X H, YANG Y Y, YANG K, CHENG Y Q.Production of carotenoid by marine red yeast Y2 .Chinese Journal of Microecology, 2011, 23(12): 1068-1073. (in Chinese)URL [本文引用: 2]摘要 目的 对海洋红酵母Y2高产类胡萝卜素的发酵条件进行优化.方法 在摇瓶条件下,研究培养基成分和培养条件对海洋红酵母Y2生长和类胡萝卜素合成的影响,同时进行海洋红酵母Y2发酵过程的动态分析.结果 海洋红酵母Y2优化培养基组合为葡萄糖45 g/L,蔗糖15 g/L,酵母粉5 g/L,蛋白胨2.5 g/L,磷酸二氢钾1 g/L,磷酸二氢钠3 g/L,硫酸镁7.5 g/L,氯化钾3 g/L,氯化钠5 g/L.最适培养参数为:温度20℃,培养基初始pH为5,接种量为10％,250 mL摇瓶装液量为10～50 mL.类胡萝卜素的合成主要集中在对数生长期和稳定期.海洋红酵母Y2最适收获时间为72 h.种龄以36 h为宜.结论 利用优化培养基,在最适条件下培养海洋红酵母Y2,类胡萝卜素产量达到4.97 mg/L,比基础培养基提高了60.32％.
[20]	刘卉琳. 红酵母合成类胡萝卜素及体外转化维生素A的研究[D] . 长沙: 湖南农业大学, 2012. [本文引用: 2] LIU H L.Studies on production of carotenoid by Rhodotorulla mucilaginosa and its conversion to vitamin A in vitro [D] . Changsha: Hunan Agricultural University, 2012. (in Chinese) [本文引用: 2]
[21]	丛莉. 海洋红酵母产类胡萝卜素的研究[D] . 青岛: 中国海洋大学, 2006. [本文引用: 1] CONG L.Investigation on carotenoids from Marine Rhodotorula sp. [D] . Qingdao: Ocean University of China, 2006. (in Chinese) [本文引用: 1]
[22]	TINOI J, RAKARIYATHAM N, DEMING R L.Simplex optimization of carotenoid production by Rhodotorula glutinis using hydrolyzed mung bean waste flour as substrate. Process Biochemistry, 2005, 40(7): 2551-2557.URL [本文引用: 1]
[23]	王增煌, 王文策, 翟双双, 谢强, 左鑫, 杨琳. 香蕉茎叶粉固态发酵条件优化及鹅对其养分利用率的研究 . 动物营养学报, 2017, 29(4): 1283-1293.URL [本文引用: 1] WANG Z H, WANG W C, ZHAI S S, XIE Q, ZUO X, YANG L.Study on optimal solid state fermentation conditions of banana stems and leaves powder and its nutrient utilization for geese .Chinese Journal of Animal Nutrition, 2017, 29(4): 1283-1293. (in Chinese)URL [本文引用: 1]
[24]	赵紫华, 韩雪, 王丽丽, 郭晶晶, 张率, 韩萌, 荀静娜. 高产β-胡萝卜素的红酵母培养条件的优化 . 食品与发酵科技, 2014, 50(4): 34-38.https://doi.org/10.3969/j.issn.1674-506X.2014.04-009URL [本文引用: 1]摘要 本实验以红酵母菌株PTH-1、SG-2、TR-3、SG-4、TR-5、TR-6为实验材 料,探讨了不同碳源、氮源、pH值、培养温度对红酵母生物量及产β-胡萝卜素的影响;在单因素优化实验基础上进行正交试验,实验结果表明产β-胡萝卜素最 优条件为：葡萄糖8%,蛋白胨1.5%,酵母浸粉0.5%,磷酸二氢钾0.1%,硫酸镁0.1%,pH 5.5,30℃;菌株经过84h摇瓶培养后各菌株的生物量、β-胡萝卜素产量都有了不同程度的提高。其中,菌株TR-6生物量和β-胡萝卜素产量分别达到 了12.31g/L和567.42μg/g,比优化前提高了89.3%和16.51%,达到了较好的效果,其他菌株生物量和β-胡萝卜素也有了较大程度的 提高;本实验结果为后续应用研究提供了良好的数据平台。 ZHAO Z H, HAN X, WANG L L, GUO J J, ZHANG L, HAN M, XUN J N.The fermentation technology optimization with high β-carotene by red yeast .Food and Fermentation Technology, 2014, 50(4): 34-38. (in Chinese)https://doi.org/10.3969/j.issn.1674-506X.2014.04-009URL [本文引用: 1]摘要 本实验以红酵母菌株PTH-1、SG-2、TR-3、SG-4、TR-5、TR-6为实验材 料,探讨了不同碳源、氮源、pH值、培养温度对红酵母生物量及产β-胡萝卜素的影响;在单因素优化实验基础上进行正交试验,实验结果表明产β-胡萝卜素最 优条件为：葡萄糖8%,蛋白胨1.5%,酵母浸粉0.5%,磷酸二氢钾0.1%,硫酸镁0.1%,pH 5.5,30℃;菌株经过84h摇瓶培养后各菌株的生物量、β-胡萝卜素产量都有了不同程度的提高。其中,菌株TR-6生物量和β-胡萝卜素产量分别达到 了12.31g/L和567.42μg/g,比优化前提高了89.3%和16.51%,达到了较好的效果,其他菌株生物量和β-胡萝卜素也有了较大程度的 提高;本实验结果为后续应用研究提供了良好的数据平台。
[25]	KAISER P, SURMANN P, VALLENTIN G, FUHRMANN H.A small-scale method for quantitation of carotenoids in bacteria and yeasts .Journal of Microbiological Methods, 2007, 70(1): 142-149.https://doi.org/10.1016/j.mimet.2007.04.004URLPMID:17509707Magsci [本文引用: 1]摘要 Microbial carotenoids are difficult to extract because of their embedding into a compact matrix and prominent sensitivity to degradation. Especially for carotenoid analysis of bacteria and yeasts, there is lack of information about capability, precision and recovery of the method used. Accordingly, we investigated feasibility, throughput and validity of a new small-scale method using Micrococcus luteus and Rhodotorula glutinis for testing purposes. For disintegration and extraction, we combined primarily mild techniques: enzymatically we used combinations of lysozyme and lipase for bacteria as well as lyticase and lipase for yeasts. Additional mechanical treatment included sonication and freeze-thawing cycles. Chemical treatment with dimethylsulfoxide was applied for yeasts only. For extraction we used a methanol-chloroform mixture stabilized efficiently with butylated hydroxytoluene and alpha-tocopherol. Separation of compounds was achieved with HPLC, applying a binary methanol/ tert-butyl methyl ether gradient on a polymer reversed C30 phase. Substances of interest were detected and identified applying a photodiode-array (PDA) and carotenoids quantitated as all-trans-尾-carotene equivalents. For evaluation of recovery and reproducibility of the extraction method, we used 尾-8鈥-apo-carotenal as internal standard. The method provides a sensitive tool for the determination of carotenoids from bacteria and yeasts and also for small changes in carotenoid spectrum of a single species. Corequisite large experiments are facilitated by the high throughput of the method.
[26]	DOMYOUNG K, EUNJI C, JIWON K, YONGWOOG L, HWAJEE C.Production of cellulases by Penicillium sp in a solid-state fermentation of oil palm empty fruit bunch. African Journal of Biotechnology, 2014, 13(1): 145-155.https://doi.org/10.5897/AJB12.2970URL [本文引用: 1]摘要 To use oil palm empty fruit bunch (EFB) for cellulase production, a novel fungus was isolated from moistened EFB. This fungus was classified as Penicillium sp. by sequence analysis of the internal transcribing space and it was named Penicillium sp. GDX01. The Penicillium sp. strain secreted cellulases under solid-state fermentation of EFB. The fermentation conditions were optimized for maximal enzyme production. Of the different substrates tested, both EFB and rice straw gave the maximum production of filter paper activity (FPase). Five percent yeast extract and 40-50% initial moisture content were found to be optimal for enzyme production. In addition, the pretreatment of EFB with NaOH before fermentation inhibited the cellulase production of Penicillium sp. GDX01. Saccharification of pretreated EFB by cellulases from Penicillium sp. GDX01 resulted in a more than 80% release of glucose during a 72 h incubation, which is a better result than when using Celluclast 1.5L without the addition of 尾-glucosidase. Our results show that the cellulases produced by Penicillium sp. GDX01 are more efficient at the saccharification of EFB than Celluclast 1.5 L.聽Key words: Cellulase, oil palm empty fruit bunch, solid-state fermentation, Penicillium.
[27]	MAURYA D P, SINGH D, PRATAP D, MAURYA J P.Optimization of solid state fermentation conditions for the production of cellulase by Trichoderma reesei. Journal of Environmental Biology, 2012, 33(1): 5-8.https://doi.org/10.1038/emboj.2010.40URL [本文引用: 1]摘要 Cellulase production studies have been carried out using the fungal strain Trichoderma reesei NCIM 992 by using three different lignocellulosic materials by solid state fermentation (SSF). The effect of basic fermentation parameters (pH, temperature, moisture content, particle size of substrate and moistening agent) on enzyme production was studied. Maximum cellulase production was 2.63 U ml-1 using wheat bran as substrate. The optimal conditions for cellulase production for wheat bran were found to be: initial moisture content-70%, initial medium pH-5.0, temperature-30oC, moistening agents (MSS) and particle size of substrate (500 &mgr;m).The optimal incubation time for production was six days. Results indicate the scope for further optimization of the production conditions to obtain higher cellulase titres using the strain under SSF.
[28]	AKSU Z, EREN A T.Carotenoids production by the yeast Rhodotorula mucilaginosa: Use of agricultural wastes as a carbon source. Process Biochemistry, 2005, 40(9): 2985-2991. [本文引用: 1]
[29]	张闯. 红酵母发酵生产类胡萝卜素的研究[D] . 大连: 大连工业大学, 2011. [本文引用: 1] ZHANG C.Preparation of carotenoids by fermention of Rhodotorula [D] . Dalian: Dalian Polytechnic University, 2011. (in Chinese) [本文引用: 1]
[30]	ZHANG X Y, YANG Z H, LIANG J, TANG L, CHEN F.Detoxification of Jatropha curcas seed cake in solid-state fermentation of newly isolated endophytic strain and nutrition assessment for its potential utilizations .International Biodeterioration & Biodegradation, 2016, 109: 202-210.https://doi.org/10.1016/j.ibiod.2016.02.001URL [本文引用: 1]摘要 61The zxy-12 strain was isolated, characterized and could efficiently biodegrade PEs or derivatives and curcin in JSC.61The solid-state fermentation parameters was analyzed and optimized.61HPLC method was established for the quantitative analysis of PEs or derivatives in JSC.61The nutritional compositions were determined.61JSC is a good-quality protein source for potential utilization in animal feeding and food applications.
[31]	王建军. 混菌固态发酵黄酒糟生产蛋白饲料的研究[D] . 杭州: 浙江大学, 2007. [本文引用: 1] WANG J J.Production and application of protein feed from yellow rice wine lees using mixed strains combinations [D] . Hangzhou: Zhejiang University, 2007. (in Chinese) [本文引用: 1]
[32]	孙展英. 马铃薯渣固态发酵工艺研究及生物学价值评定[D] . 保定: 河北农业大学, 2014. [本文引用: 1] SUN Z Y.Study on technology of solid-State fermentation and evaluation of biological value of potato residue [D] . Baoding: Hebei Agricultural University, 2014. (in Chinese) [本文引用: 1]