毛竹笋中4种蛋白的提取与功能性质研究

杨金来，杨慧敏，吴良如，John Michael CARNEY，Charles-Henri LIGNET，WU CAO，潘雁红

（1.国家林业和草原局竹子研究开发中心，浙江 杭州310012；2.浙江省竹子高效加工重点实验室，浙江 杭州310012；3.玛氏公司，麦克莱恩，弗吉尼亚州22101，美国）

Recently，plant proteins have received renewed attention due to their increasing application as functional food in the food industry.Thus，many researchers have extracted proteins from a broad range of plants and studied their functional properties，such as African yam bean seed（Sphenostylis stenocarpa）［1］，lupin（Lupinus luteus）［2］，leguminous tuberous roots（Pachyrhizus ahipa）［3］， pea［4］， arthrospira platensis［5］， hazelnut（Corylus avellanaL.）［6］，etc.

Bamboo is another useful plant which is widely distributed in both temperate and tropical regions of the world and offers a rich yield of bamboo shoots［7］.In addition to being delicious，bamboo shoots are rich in nutrient components of fiber，proteins，amino acids，carbohydrates，minerals，and phytosterols.They are low in fat and sugars［8］，making bamboo shoots an excellent health food.The effective utilization of bamboo shoots is a very meaningful goal because it can increase their value［9］.For this reason，there has been a significant increase in research efforts in this field.A recent study on bamboo fiber showed that it could lowered cholesterol levels and improved bowel functions［10］.Six novel nucleosides and two unique amino acids were also isolated and identified from bamboo shoots of Phyllostachys prominens［11］.At the same time，two water-soluble polysaccharides were extracted from bamboo shoots（Phyllostachys praecox）using hot-water extraction［12］，and aβ-pyran polysaccharide from the bamboo shoot shell showed natural anti-diabetic activity［13］.Carboxymethylcellulose was prepared from bamboo shoot cellulose via chemical modification［14］.The volatile aromatic components were obtained from spring and winter shoots of Phyllostachys heterocycla var.pubescens，the results showed that the fresh green aroma ingredients were methoxy-phenyl oxime，byn-hexanol and 3Z-hexenal［15］.

The eighth national forest resources inventory results shows that Moso bamboo covered an area of 4.43 million hm2in China，providing abundant Moso bamboo shoots［16］.So，how to make the most of the shoots to provide high value-added products are urgently needed.At the same time，little attention has been paid to the different kinds of proteins that can be extracted from Moso bamboo shoots.In this paper，albumin，globulin，gliadin and alkali soluble protein were gradually isolated from Moso bamboo shoots，and their functional properties were fully studied for future use as functional food.

1 Experimental

1.1 Materials and instruments

Moso bamboo shoots were collected from Yuhang district，Hangzhou city，they were stored at 4℃until used for protein extraction.Reagents and solvents were purchased from commercial suppliers and used without further purification.The UV-visible absorption spectra were measured on a UV-2550 spectrophotometer using a quartz cuvette with a 1-cm path length.Amino acids were analyzed on A300 type amino acids analyzer.The elemental analysis of proteins was characterized by elemental analysis（Elementar vario EL cube）.All pH measurements were made with a Model PHS-25C pH meter at room temperature.

1.2 Preparation of protein isolate

The albumin，globulin，gliadin and alkali soluble protein fractions were fractionated according to the reported method with some modifications［17］，as shown in Fig.1.

First，the smashed Moso bamboo shoots were mixed with distilled water at 1∶10（w／v）.The mixture′s pH was adjusted to neutral using 1 M NaOH solution followed by stirring for 120 min at 30℃.After that，the suspension was centrifuged at 4 000×g for 20 min to provide residue 1.This collected supernatant was adjusted with 1 M HCl solution to precipitate albumin at the isoelectric point（pI）.Then，the albumin precipitation was collected by a way of centrifuging at 4 500×g for 15 min，and the ions of precipitation were removed by a dialysis method at the temperature of 4℃.Finally，the resulting albumin powder was obtained after treating the precipitation by a freeze drying process.

Fig.1 Technological process of classification extract of Moso bamboo shoots

Then residue 1 was mixed with 3% NaCl water solution at 1∶10（w／v），and the mixture was stirred for 120 min at 30℃.After that，the supernatant and residue 2 were separated by centrifuge at 4 000×g for 20 min.Globulin precipitation of Moso bamboo shoots occurred by centrifugation at 4 500×g for 15 min after the supernatant was adjusted to the pI of globulin by weak acids and bases.After the ions were removed through dialysis at 4℃，the precipitate was freeze dried to produce globulin powder.

After residue 2 was washed by distilled water with 2-3 times，it was mixed with 75% ethanol at 1∶10（v／v）.The supernatant and residue 3 were separated by centrifugation at 4 000×g for 20 min after the above mixture was stirred for 120 min at 45℃.The ethanol in the supernatant was removed by the reduced pressure distillation at 40℃，allowed to let stand for a period of time，then the residual solution was adjusted to the pI to precipitate the gliadin.After centrifugation at 4 500×g for 15 min，the precipitation was transferred into a dialysis bag to remove ions at a temperature of 4℃.A gliadin powder was produced after the precipitation was through a freeze drying process.

To obtain alkali soluble protein，residue 3 was washed with distilled water for 2-3 times.Then the residue was mixed with 0.01 M NaOH solution at 1∶10（v／v）to provide a mixture，which was adjusted to a pH of 9.0 and extracted for 120 min at 30℃.After that，the supernatant and residue 4 were separated by centrifugation at 4 000×g for 20 min.The supernatant was adjusted to the pI to provide alkali soluble protein precipitation.A new precipitation was collected through centrifugation at 4 500×g for 15 min，and it was put into a dialysis bag to remove ions at 4℃.As a result，alkali soluble protein powder of Moso bamboo shoots was produced via freeze drying.

1.3 Functional properties

1.3.1 Protein solubility（PS）

A 200 mg sample of albumin，globulin，gliadin or alkali soluble protein was dispersed in 20 mL of distilled water and the solution′s pH was adjusted to 7.0，using HCl（0.1 M）or NaOH（0.1 M）.The mixture were stirred for 45 min using a magnetic stirrer，then centrifuged at 3 000×g for 15 min（4℃）.The supernatant was removed as sample solutions of the four proteins.PSwas a percentage ratio of supernatant protein content to the total protein content.

1.3.2 Water and oil holding capacity

Protein powder（0.1 g）was dispersed in 5 mL distilled water（or pure soybean salad oil）in a 10 mL centrifuge tube.The dispersions were fully mixed，allowed to stand for 6 h and then centrifuged at 2 000×g for 30 min at room temperature.After that，the supernatant was removed to measure volume accurately，then the water and oil holding capacity were calculated as follow：WHC（%）＝（5 mL-the volume of supernatant after centrifugation）／0.1 g×100 OHC（%）＝（5 mL-the volume of supernatant after centrifugation）／0.1 g×100

1.3.3 Emulsifying capacity（EC）and emulsion stability（ES）

A 2% （W／V）protein solution was prepared by 0.4 g accurate weight protein and 20 mL distilled water，and it was adjusted to a pH of 7.0 with 0.01 M hydrochloric acid or sodium hydroxide solution，then a 20 mL volume of soybean salad oil at room temperature was added and shaken well.Water-oil emulsion was fully formed after the mixture was stirred a high speed of 2-3 mL／s.Water-oil emulsion（8 mL）was respectively added to three scale centrifuge tubes，centrifuged at 2 500×g for 30 min.After that，the volumes of emulsion layers were measured immediately，emulsifying capacity was calculated as follow：

EC（%）＝The height of the emulsion layer／The total height of the emulsion×100

At the same time，the above emulsion sample was put into a 80℃water bath for 30 minutes，took out and cooled，then the height of emulsion layer was measured immediately.Emulsion stability was calculated as follow：

ES（%）＝The height of the emulsion layer after heating／The height of the emulsion before heating×100

1.3.4 Foaming capacity（FC）and foam stability（FS）

Albumin，globulin，gliadin and alkali soluble protein were separately dissolved in 50 mL distilled water to make a 20 mg·mL-1solution，homogenized at 10 000 rpm for 3 min，FC and FSwere calculated as follows：

where V1is the initial volume and V2is the volume after foam formation.FSwas evaluated measuring the total volume 30 min after foam formation（V3）.

1.3.5 Amino acid analysis

Dried samples（20 mg）of albumin，globulin，gliadin and alkali soluble protein were separately digested using 8 mL HCl（6 M）at 110℃for 22 h under nitrogen atmosphere.The hydrolysate solutions were cooled at room temperature and impurities removed by filter.The solution′s volumes were adjusted to a final volume of 50 mL with double distilled water.An aliquot of 1 mL was removed from every sample solution and freeze dried.To this freeze dried sample was added 1 mL 0.02 M HCl solution，shaken well，and centrifuged at 14 000×g for 15 min.After that，the supernatants were analyzed on an amino acid analyzer after filtration（0.45μm membrane）.

2 Results and discussion

2.1 Protein extraction

Four kinds of albumin，globulin，gliadin and alkali soluble protein were successfully extracted from Moso bamboo shoots by Osborne method（Fig.1 ）.The gross yield of albumin，globulin，gliadin and alkali soluble protein concentrate were 1.42%，0.33%，0.11% and 0.12% （w／w of fresh shoot）.

2.2 Amino acid ingredients

The amino acid ingredients of four proteins from Moso bamboo shoots were also studied，the results were shown in Tab.1.Comparing with the four proteins，there were 17 amino acids in albumin，globulin，gliadin or alkali soluble protein，no difference in amino acid composition，only difference in content.It was known that the essential amino acids for human body were threonine，methionine，isoleucine，leucine，phenyalanine，lysine，histidine，tryptophan and valine，except for tryptophan，all of these amino acids were present in the four proteins of Moso bamboo shoots.

Tab.1 Amino acid composition of Moso bamboo shoots g／100 g protein

2.3 Hydrophobicity

To obtain the hydrophobicity of albumin，globulin，gliadin or alkali soluble protein，a relationship of fluorescence intensity and concentration of every protein was provided in Fig.2.The index of surface hydrophobicity was evaluated by the slope of the initial curve，and the hydrophobicity increased with an rising value of the slope.Fig.2 showed the results of hydrophobicity of four proteins，gliadin＞alkali soluble protein＞globulin＞albumin.

2.4 Solubility

Fig.3 showed the results of solubility of albumin，globulin，gliadin or alkali soluble protein at neutral condition.Among them，albumin had the highest solubility，globulin for the second，because they were water soluble proteins.Differently，the gliadin′s solubility was the worst of the four proteins，and the solubility of alkali soluble protein was also bad.The reason might be that the two proteins had a high hydrophobicity.

2.5 WHC and OHC

Water and oil holding capacity is very important for food industry.WHC has an effect on food quality and taste，and OHC could keep grease and flavors.Thus，WHC and OHC had been fully studied，as shown in Fig.4.

Fig.2 The relationship of fluorescence intensity and concentration of four proteins

From Fig.4 a，it could be seen that globulin had the best water holding capacity（5.0 mL·g-1）of the four proteins，and the second was albumin with a water absorption volume of 4.2 mL·g-1.However，the water absorption volume of gliadin or alkali soluble protein was less than 3 mL·g-1.Fig.4 b showed the results of oil holding capacity of albumin，globulin，gliadin and alkali soluble protein，their strength of oil absorption capacity was easy to see，albumin＞gliadin＞globulin＞alkali soluble protein.Physical entrapment was a major contribution to the OHC， so scanning electron microscope（SEM）of four proteins was finished for further analysis（Fig.5 ）.The surface of albumin and gliadin were both porous and rough with small pore size and large surface area，which had good physical entrapment function，thus led to high oil absorption volume.At the same time，alkali soluble protein had a relative flat area and the structure was dense，so it gave a low oil absorption capacity.The results could explain the oil holding capacity which agreed with that of Fig.4 b.

Fig.3 Solubility of albumin，globulin，gliadin or alkali soluble protein

Fig.4 WHC（a）and OHC（b）of albumin，globulin，gliadin and alkali soluble protein

2.6 EC and ES

To explore the emulsifying properties of albumin，globulin，gliadin and alkali soluble protein，EC and ESwere determined，as shown in Fig.6.The emulsification ability of the four proteins was further obtained，albumin（35.8%）＞globulin（29.2%）＞gliadin（22.1%）＞alkali soluble protein（14.2%）.The results were connected with the extraction sequence of four proteins，and the emulsification potential，which decreased along with the protein extraction efficiency from first to last.Meanwhile，except alkali soluble protein（65.1%），the emulsifying stability of other proteins changed very little with a more than 72%stability after bathing in 80℃water for 30 minutes.That was to say that the four proteins all exhibited good emulsifying stability.

Fig.5 SEM of albumin（a），globulin（b），gliadin（c）and alkali soluble protein（d）

2.7 FC and FS

Foaming capacity（FC）and foam stability（FS）are also important functional properties for a protein.Thus FC and FSwere further studied，the results were showed in Fig.7.The foamability was also connected with the extraction sequence of four proteins，and it decreased along with the protein extracted from beginning to end， which agreed with that of emulsibility（Fig.6 ）.That is to say that albumin had the best foamability and the final extracted protein（alkali soluble protein） offered the lowest foamability.For foam stability，globulin offered a maximal FC value，followed by alkali soluble protein，and the worst foam stability appeared at gliadin.

Fig.6 ECand ESof albumin，globulin，gliadin and alkali soluble protein

3 Conclusion

Four proteins were gradually isolated from Moso bamboo shoot.The four proteins all had 17 amino acids，among them，8 amino acids are essential for human nutrition.Gliadin had the best hydrophobicity，followed by alkali soluble protein，globulin，and albumin.The solubility of albumin was the best while another protein of gliadin only had a bad solubility at a neutral condition.At the same time，albumin and globulin possessed a high water holding capacity of 4.2 and 5.0 mL·g-1while albumin and gliadin offered a good oil absorption volume of 3.6 and 3.2 mL·g-1.Furthermore，albumin，globulin，gliadin and alkali soluble protein finally gave emulsification ability of 35.8%，29.2%，22.1%，14.2%，and they all offered a stability of more than 65.1%.Albumin had the best foamability which reached to 67.7%，and it also provided 48.6% foam stability.The proteins from natural Moso bamboo shoots could be further used in functional food，then added the value of bamboo shoots.

Fig.7 FC and FSof albumin，globulin，gliadin and alkali soluble protein