Shiming TANG　Junmai CAO　Yanyun CHEN　Yulong MA

Abstract[Objectives] This study was conducted to screen different extraction methods for protein from potato tubers. [Methods]The bud eye， lateral and pith parts of Qingshu 168 and Kexin 1 were used as test materials， and were extracted by acetone extraction， trichloroacetic acid （TCA） extraction， salt extraction， alcohol extraction and phenol extraction， respectively. The protein contents of different parts were determined by spectrophotometry. A 2-factor randomized block design was used to study the effects of varieties， extraction methods， sampling parts and combinations of different factors on protein content. [Results] The protein contents of different varieties were different， and Qingshu 168 was significantly higher than Kexin 1 （P<0.05）， reaching 128.0 mg/L. The protein contents obtained by different extraction methods were different. Acetone extraction， salt extraction and TCA extraction showed no significant differences in the protein content， but they were significantly higher than phenol extraction and alcohol extraction （P<0.05）. The protein contents obtained by the former three were in the range of 153.3-159.7 mg/L. The protein content in the bud eye part of potato tubers was significantly higher than those in the lateral and pith parts （P<0.05）， reaching 140.7 mg/L. There were significant differences among different combinations of varieties， extraction methods and sampling parts （P<0.05）. The comprehensive comparison showed that the salt extraction method was suitable for the bud eye part of Qingshu 168， and the TCA extraction method was suitable for the bud eye part of Kexin No.1. [Conclusions]This study provides a reference for potato protein extraction and further research.

Key wordsPotato tuber; Variety; Extraction method; Part; Protein content

As a perennial herb， potato can be grown annualty or biannually. It can be used as both grain and vegetable， and can also be made into biofuel and feed. Potatoes have the functions of harmonizing the stomach， regulating middle jiao， strengthening the spleen and invigorating qi， and also have therapeutic effects on gastric ulcer， habitual constipation， collagen disease， hot cough and skin eczema. Meanwhile， potatoes are also a nutritional health food. Potato resources are very rich， and it has been reported that the nutritional value of potato protein powder is significantly better than that of soybean meal[1]. Potatoes are a high-quality natural nitrogen source and have broad development prospects. Potato protein can be well absorbed by the human body and is a complete protein[2]. Its protein titer is comparable to that of eggs and casein， and its nutritional value is better than that of protein extracted from grains or beans[3]. Potato protein is closest to animal protein and is a good health food[4]， and its net digestibility of protein is also very high[5]. The use value of potato protein is 71%， which is 21% higher than that of cereals. Potato protein contains 18 kinds of amino acids required by the human body and a variety of trace elements[6]. In recent years， the proteomic study of potatoes tubers has helped us to understand the mechanism of tuber growth and development， and also contributed to the improvement of potato tuber yield and quality traits[7]. Therefore， carrying out research on potato protein extraction methods is of great significance to the conduction of other research work. There are few reports at home and abroad about the protein extraction methods of potato tubers. Li et al.[8] carried out a study on the optimization of potato tuber protein extraction process and the determination of molecular weight. Li et al.[9] studied the construction of a calibration model for potato protein content based on near-infrared spectroscopy. However， because the protein content of different varieties of potato is very different， and it is affected by ambient temperature， maturity of potato tubers and different parts on potato tubers， the results are also different. For different materials， parts suitable for their characteristics for protein extraction should be used[8]. Aiming at these problems， in this study， different extraction methods were screened for different parts of different potato varieties， hoping to provide reference for potato protein extraction and further research.

Materials and Methods

Experimental materials

The potato varieties used in this experiment were Kexin 1 and Qingshu 168， which were provided by Ningxia Guyuan Tianqi Potato Industry Co.， Ltd.

Experimental methods

Experimental designExperiment 1 adopted a 2-factor randomized block design. Factor 1 was the variety （V）， involving Kexin 1 as V and Qingshu 168 as V. Factor 2 was the extraction method （T）， divided into 5 levels， namely the acetone extraction method T， the salt extraction method T， the trichloroacetic acid （TCA） extraction method T， the phenol extraction method T， and the alcohol extraction method T， respectively. The experiment was repeated 3 times for a total of 10 treatments.

Experiment 2 adopted a 2-factor randomized block design. Factor 1 was the extraction method （T）， divided into 5 levels：
T-T. Factor 2 was the sampling part （P）， divided into 3 treatments， namely the bud eye part P， the lateral part P， and the pith part P. The experiment was repeated 3 times for a total of 15 treatments.

Experiment 3 adopted a 2-factor randomized block design， Factor 1 was the variety （V）， including V and V. Factor 2 was sampling location （P）， divided into 3 treatments：
P， P， and P. The experiment was repeated 3 times for a total of 6 treatments.

Extraction parts of potato proteinBud eye：
The bud eyes are arranged in a spiral shape on tubers， dense at the top， and thin at the base. When extracting， the bud eye part was taken 0.7 cm around the bud eyes with a depth of about 0.6 cm. Lateral part：
It refers to the vascular bundle loops， about 0.6 cm in thickness from the cortex inward. Pith part：
It refers to the central part of a tuber. It can be clearly seen on the cross section of a tuber， as the center of the tuber is the pith， which is surrounded by open vascular bundle loops.

Extraction methods of potato proteinThe TCA extraction method and the acetone extraction method adopted the methods of Wang et al.[10]. The phenol extraction， salt extraction and alcohol extraction were respectively reported by Hurkman et al.[11-13].

Determination of protein content in samplesThe protein content was determined by the method introduced by Guo et al.[14]. Bovine serum albumin （BSA） was used to draw a standard curve by measuring the absorbance at 595 nm. The standard curve was drawn using drawing software with the protein concentration as the ordinate （y） and D as the abscissa （x） as， as shown in Fig. 1.

Statistical analysisThe data obtained from the experiments were processed by Excel 2003， and then SPSS 17.0 software was used for two-factor analysis of variance.

Results and Analysis

Effects of varieties on potato protein content

It can be seen from Table 1 that the protein contents of different varieties were different， and Qingshu 168 was significantly higher than Kexin 1.

Effects of extraction methods on potato protein content

It can be seen from Table 2 that different extraction methods all affected the protein content. There were no significant differences among the TCA method， acetone extraction method and salt extraction method， but they were significantly higher than the phenol extraction method and alcohol extraction method， and there was no significant difference between the latter two.

Effects of different parts on potato protein content

It can be seen from Table 3 that different sampling parts affected the protein content. The protein content in bud eyes was significantly higher than those in the pith and lateral parts， but there was no significant difference between the latter two.

Effects of varieties and extraction methods on protein content from potato tubers

It can be seen from Table 4 that the TCA extraction method was the best for variety Kexin 1， and the salt extraction method was the best for Qingshu 168， and the acetone extraction method was the second for both. The protein contents extracted by the phenol extraction and alcohol extraction were the lowest.

Effects of extraction methods and sampling parts on protein content from potato tubers

As can be seen from Table 5， the extraction methods suitable for bud eyes were the TCA extraction method and salt extraction method， followed by the acetone extraction method. The extraction methods suitable for the lateral part were salt extraction， acetone extraction， followed by TCA extraction. The extraction method suitable for the pith part was acetone extraction， followed by TCA extraction and salt extraction. The worst methods suitable for the extraction of the three parts were phenol extraction and alcohol extraction.

Effects of varieties and sampling parts on protein content from potato tubers

It can be seen from Table 6 that the protein contents of different potato sampling parts and varieties showed a certain regularity. The differences in protein content between different parts of potato tubers and different varieties basically reached a significant level. Both Qingshu 168 and Kexin 1 had the highest contents in the bud eye part and the lowest in the pith part. Therefore， the best sampling part for these two varieties was bud eyes.

Discussion

In this study， the bud eye， lateral and pith parts of Qingshu 168 and Kexin 1 were used as test materials， which were extracted for protein by acetone extraction， TCA extraction， salt extraction， alcohol extraction and phenol extraction. The results showed that the protein contents of different varieties were different. Qingshu 168 was significantly higher than Kexin 1. The protein content was different between different extraction methods. There were no significant differences among the TCA method， acetone extraction method and salt extraction method， but they were significantly higher than the phenol extraction method and alcohol extraction method， and there was no significant difference between the latter two. Different sampling parts affected the protein content. The protein content in bud eyes was significantly higher than those in the pith and lateral parts， but there was no significant difference between the latter two. The TCA extraction method was the best for variety Kexin 1， and the salt extraction method was the best for Qingshu 168， and the acetone extraction method was the second for both. The protein contents extracted by the phenol extraction and alcohol extraction methods were the lowest. It could be seen that the extraction methods suitable for bud eyes were the TCA extraction method and salt extraction method， followed by the acetone extraction method; the extraction methods suitable for the lateral part were salt extraction， acetone extraction， followed by TCA extraction; and the extraction method suitable for the pith part was acetone extraction， followed by TCA extraction and salt extraction. Considering the operability of the comprehensive test， it is suggested that the potato protein should be extracted with acetone.

Li et al.[15] used five methods to extract protein from the tubers of potato Zhongshu No. 9， and determine the protein content by the Bradford method. The results showed that the crude protein content of the phosphate buffer solution extraction method was the highest， which was 105.3 mg/L， followed by 72.3 mg/L in the direct extraction method， and the phenol extraction method extracted the least protein， which was 10.3 mg/L; the best extraction method for protein purity was the TCA method， and the purity could reach 10.92%; and the second was the direct extraction method， by which the purity obtained was 7.66%; and the phenol extraction method was the lowest， and the purity obtained was only 1.32%. The above results comprehensively show that the direct extraction method has the best effect. Fan et al.[16] compared several methods such as the acetone precipitation method， TCA method， and phenol method with the roots of Rhizophora stylosa as the material. They found that the above methods were all not ideal， and the protein extracted had a low purity， and could not show clear bands， but the improved phenol method achieved good results[16]. Gu et al.[17] used woody plants as materials and compared the acetone sedimentation method， TCA method and direct extraction method. The acetone sedimentation method was considered to be the best， and the acetone extraction method was suggested for the extraction of potato protein.

In this study， the alcohol extraction method and the phenol extraction method were both less effective and the protein loss was higher. The results obtained by acetone extraction， salt extraction and TCA extraction were better than those of alcohol and phenol extraction， indicating that acetone extraction， salt extraction and TCA extraction may be more suitable methods for potato tubers. However， the disadvantage of these methods was that the protein liquids contained relatively more impurities， which might interfere with further in-depth research. Therefore， it is necessary to further explore a more suitable， simpler， more efficient， especially higher-purity， separation and purification method for potato tuber protein， so as to provide a basis for the utilization and in-depth study of potato tuber protein.

Studies have shown that with a solid-liquid ratio at 1 g∶10 ml， a soaking temperature at 35 ℃， a pH value of 9.8 and soaking time of 1 h， the yield of potato protein extracted by the salt extraction method was the highest， reaching 0.80 g/100 g[15]. The conditions of the salt extraction method in this study were substantially the same， and the final extracted protein content was 159.67 mg/L.

The TCA precipitation method and acetone precipitation extraction method are the most common extraction methods in proteomic analysis. These two methods have the advantages of few operation steps， short time consumption and less loss in the extraction process， and are simple and easy to implement. They have a wide range of applicability and can achieve a good separation effect in microbial materials and animal and plant tissues. However， the extracts obtained by precipitation contain a large amount of crude protein extracts and impurities， and it is necessary to remove impurities and require strong lysis buffer and mechanical assistance to extract purer proteins. The alcohol extraction method removes salt-soluble proteins by adding sodium chloride before adding 2-chloroethanol extraction， so the overall extracted protein content is very small， but the alcohol-extracted potato protein is only suitable for the electrophoresis analysis technology.

Phenol extraction is a protein extraction method that is very suitable for plant materials， especially difficult-to-extract materials， although the extracted protein content is low. Due to the existence of cell walls and numerous intracellular secondary metabolites in plant cells， protein extraction and purification are very difficult. Compared with the direct precipitation of TCA extraction and acetone extraction， phenol extraction can avoid the pollution of a large amount of tissue residues and water-soluble impurities， and can also remove salt ions. During the sample preparation process， soluble substances such as nucleic acids， salts， phenols， pigments and polysaccharides enter the aqueous phase， while proteins and lipids enter the phenolic phase， thereby being separated. The protein in the phenolic layer can be further purified by precipitation to remove salts. The method obtains less impurities and is very suitable for electrophoresis experiments. Although the TCA precipitation method can remove salt ions in protein samples well， the removal effect of impurities such as pigments and polysaccharides is not very obvious， and it is difficult to dissolve proteins after repeated precipitation， which will cause a lot of protein loss.  Firstly， polyvinylpyrrolidone adsorbents are added to samples， which can reduce the interference of secondary metabolites such as phenols， quinones and pigments in plants; and secondly， phenols can effectively remove soluble impurities in samples and also remove salt ions[18]. In addition， due to the lipid solubility of phenols， the phenol extraction method is very favorable for the extraction of membrane proteins， which are often closely related to the salt tolerance of plants[19].  Therefore， the modified phenol method is an effective method for extracting the root protein of R. stylosa. It is in agreement with Wang et al.[20] who found that the potato protein extracted by phenol extraction and two-step precipitation combining trichloroacetic acid/acetone precipitation and ammonium acetate/methanol precipitation had the protein purity and concentration and the number of protein spots significantly higher than those extracted by phenol extraction alone.

The salt-dissolving method uses a certain concentration of sodium chloride as the extractant.  Although the extracted protein content is high[21]， compared with other methods， the salt extraction method is complicated in operation， and factors such as pH value， temperature， solid-liquid ratio and extraction time need to be well controlled， and the use of dialysis bags is also inconvenient. When the salt ion concentration in samples solution exceeds 100 mmol/L， the electroendosmosis of samples is relatively high[22]， and the isoelectric focusing voltage cannot go up， which will seriously affect the isoelectric focusing. Therefore， it can be considered that， salts are one of the most common impurities in 2D electrophoresis.

Conclusions

The research can draw the following conclusions. ① The protein contents of different varieties were different， and Qingshu 168 was significantly higher than Kexin 1. ② The protein contents extracted by different extraction methods were different. There were no significant differences among the TCA method， acetone extraction method and salt extraction method， but they were significantly higher than the phenol extraction method and alcohol extraction method， and there was no significant difference between the latter two. ③ Different sampling parts affected the protein content. The protein content in bud eyes was significantly higher than those in the pith and lateral parts， but there was no significant difference between the latter two. ④ Combinations favorable for the protein extraction of the 2 varieties：
VT was better for the V variety， and VT was better for the V variety. For both varieties， the acetone extraction method was the best， and the phenol extraction method and the alcohol extraction method were the worst， so the last two methods were eliminated. ⑤ The extraction methods suitable for bud eyes were the TCA extraction method and salt extraction method， followed by the acetone extraction method. The extraction methods suitable for the lateral part were salt extraction， acetone extraction， followed by TCA extraction. The extraction method suitable for the pith part was acetone extraction， followed by TCA extraction and salt extraction. The worst methods suitable for the extraction of the three parts were phenol extraction and alcohol extraction， so the two methods were eliminated. ⑥ For the convenience of experimental operation， it is recommended to use acetone extraction method for potato protein extraction.

References

[1] SUN M， MU T， SUN H， et al. Digestibility and structural properties of thermal and high hydrostatic pressure treated sweet potato （Ipomoea batatas L.） protein[J]. Plant Foods for Human Nutrition， 2014， 69（3）：
270-275.

[2] JIANG HB， XU X， QIN R， et al. Review on extraction methods of potato protein[J]. Journal of Anhui Agricultural Sciences， 2019， 47（3）：
9-11. （in Chinese）.

[3] SINGH A， VANGA SK， NAIR GR， et al. Electrohydrod-ynamic drying（EHD） of wheat and its effect on wheat proteinconformation[J]. LWT-Food Science and Technology， 2015， 64（2）：
750-758.

[4] HOU FN， MU TH， SUN HN， et al. Evaluation of the protein nutrition quality of potato flours from different cultivars[J]. Food Science and Technology， 2015， 40（3）：
49-56. （in Chinese）.

[5] QIU XL， LI Y， BAI YM， et al. Distribution of protein content in diploid potato plants and their relations[J]. Chinese Potato Journal， 2006， 20（2）：
65-67. （in Chinese）.

[6] YANG J， YU HJ， BAI AZ， et al. Effect of HVEF drying and hot air drying on secondary structure of potato protein[J]. Storage and Process， 2020， 20（3）：
85-89. （in Chinese）.

[7] VAN GELDER WMJ， VONK CR. Amino acid composition of coagulable protein from tubers of 34 potato varieties and its relationship with protein content[J]. Potato Researeh， 1980， 23（4）：
427-434.

[8] LI L， CHENG JS， REN AX， et al. Optimization of potato protein extraction process and determination of the molecular weight of potato protein[J]. Journal of Shaoguan University， 2020， 41（12）：
1-7. （in Chinese）.

[9] LI Z， GAO HX， JIN P， et al. Establishment of potato protein content calibration model based on near infrared spectroscopy[J]. Chinese Potato Journal， 2021， 35（6）：
307-513. （in Chinese）.

[10] WANG GP， PENG FR， LI SP. Studies on changes of proteins in the leaves of Ginkgo biloba by SDS-PAGE and IFE-SDS-PAGE[J]. Journal of Nanjing Forestry University （Natural Sciences Edition）， 2006， 30（4）：
114-118. （in Chinese）.

[11] HURKMAN WJ， TANAKA CK. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis[J]. Plant Physiology， 1986， 81（3）：
802-806.

[12] ZHANG WH. Study on rheological properties and thermal stability of potato protein isolate solution[D]. Changchun：
Jilin Agricultural University， 2011. （in Chinese）.

[13] SHAO JZ， QIU CE， DING Y. Comparison of the effect of different extractants on hordein and gliadin extraction by A-PAGE[J]. Journal of Wuhan Botanical Research， 2003， 21（3）：
262-266. （in Chinese）.

[14] GUO AG， GUO ZK. Biochemical experiment techniques[M]. Beijing：
Higher Education Press， 2007. （in Chinese）.

[15] LI MM， JIANG JZ， WANG HX. Comparison of protein extraction methods from potato tuber[J]. Anhui Agricultural Science Bulletin， 2009， 15（23）：
37-38. （in Chinese）.

[16] FAN JX， DENG YC， HUANG X， et al. Improvement of protein extraction method from Rhizophora stylosa root and establishment of two-dimensional electrophoresis[J]. Journal of Hunan Agricultural University：
Natural Sciences， 2008， 34（5）：
550-553. （in Chinese）.

[17] GU RS， LIU QL. Comparison and optimization of the methods on protein extraction and SDS-PAGE in woody plants[J]. Chinese Bulletin of Botany， 1999， 16（2）：
171-177. （in Chinese）.

[18] WANG W， SCALI M， VIGNANI R， et al. Protein extraction for two-di-mensional electrophoresis from olive leaf， a plant tissue containing high levels of interfering compounds[J]. Electrophoresis， 2003， 24（14）：
2369-2375.

[19] PENG CZ. Isolation of salt tolerance related protein in Rhizophora apiculata BL. and MALDI-TQ MS identification[D]. Haikou：
South China University of Tropical Agriculture， 2005. （in Chinese）.

[20] WANG RR， WANG DX， ZHANG XW， et al. Comparison of two extraction methods of potato tuber protein based on proteomic analysis[J]. Journal of Gansu Agricultural University， 2019（1）：
89-95. （in Chinese）.

[21] CHEN SB， ZHANG HM， JIN H， et al. Optimization of sodium chloride extraction of peanut meal protein by response surface methodology[J]. Academic Periodical of Farm Products Processing， 2022（1）：
37-44. （in Chinese）.

[22] GORG A， WEISS W， DUNN MJ. Current two-dimensional electrophoresis technology for proteomics[J]. Proteomics， 2004， 4（12）：
3665-3685.

Editor：
Yingzhi GUANG Proofreader：
Xinxiu ZHU