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Article

An Efficient Approach for β-Cyclodextrin Production from Raw Ginkgo Seed Powder Through High-Temperature Gelatinization and Enzymatic Conversion

by
Xuguo Duan
*,
Yucheng Fan
,
Qianqian Liu
and
Yucheng Ding
College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Catalysts 2025, 15(2), 108; https://doi.org/10.3390/catal15020108
Submission received: 25 December 2024 / Revised: 18 January 2025 / Accepted: 19 January 2025 / Published: 23 January 2025
(This article belongs to the Section Biocatalysis)
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Abstract

:
Ginkgo seeds, which are abundant in starch, remain significantly underutilized, contributing to substantial resource waste and environmental pollution. This study investigates the production of β-cyclodextrin (β-CD) from ginkgo seeds utilizing β-cyclodextrin transferase. The research introduces a comparative analysis of two distinct pretreatment schemes for ginkgo seed powder, of which scheme B, which incorporates high-temperature gelatinization at 90 °C, emerges as particularly effective. This approach not only reduces the viscosity of the starch but also eliminates gel formation, leading to a homogeneous distribution of short-chain starch particles. This is evidenced by a notable transition in X-ray diffraction patterns from type A to type B, indicating a fundamental change in the starch structure. Furthermore, the study achieves a significant milestone in process optimization, resulting in an impressive cyclodextrin conversion rate of 72.63%. This represents a substantial 1.9-fold increase compared to the initial conversion rate prior to optimization. The research highlights the critical role of temperature in modifying starch structure and emphasizes the essential function of β-CGTase in this transformation. These findings are not only noteworthy for revealing the untapped industrial potential of ginkgo seed powder but also for demonstrating its practical application in β-CD production. This study offers valuable insights and a scientific basis for the development and utilization of ginkgo seeds across various industries, potentially opening new avenues for the sustainable use of this abundant resource.

1. Introduction

Ginkgo biloba, an indigenous tree species, has gained significant recognition worldwide. The annual output value of the Ginkgo biloba growing and processing industry is about $1.7 billion, highlighting its importance as an agricultural and forestry resource. However, it is worth noting that despite its immense potential, the utilization of ginkgo biloba has been largely limited, with applications primarily focused on ginkgo leaf extract [1]. Relatively few studies have explored the development and utilization of ginkgo biloba fruits, which primarily consist of the seed coat (or exocarp) and the kernel (seeds or endosperm) [2]. This represents a missed opportunity for further exploration and exploitation of the diverse applications of ginkgo biloba. In recent years, the cultivation area for ginkgo biloba has been expanding steadily. With this expansion, there has been a notable increase in ginkgo seed production. However, this surge in production not only lowers the value of the commodity but also leads to resource wastage and environmental degradation due to insufficient processing capacity. Regarding ginkgo seeds, they are rich in starch content. However, there is a lack of comprehensive research on the further processing of ginkgo seeds and ginkgo starch [3]. Consequently, a significant amount of ginkgo seeds, estimated at 60, 000 tons annually, remains unused, resulting in considerable waste [4]. To address these challenges and unlock the full potential of ginkgo seeds, it is essential to promote deeper research and development in the processing and utilization of ginkgo seeds [5]. This will help minimize waste and maximize the economic and ecological benefits associated with ginkgo biloba cultivation.
The composition of the whole ginkgo seed includes approximately 60–70% starch, 10–20% protein, 2–4% lipid, and 0.8–1.2% pectin. The starch content in ginkgo seeds is comparable to that found in corn and wheat, indicating its significant potential for development [6]. Ginkgo seeds have been used as a traditional Chinese medicine and food for thousands of years, including in the treatment of cancer [7]. Despite the fact that the output of ginkgo seeds surpasses the current demand, their development remains limited [2,8]. Starch represents the most abundant carbohydrate in ginkgo seeds, yet its characteristics have not been extensively studied. Therefore, further investigation into the processing techniques of ginkgo seed starch is necessary to fully unlock its potential as an important agricultural and forestry product, as well as for other industrial applications [9].
Cyclodextrin is a cyclic oligosaccharide with diverse applications in various fields, including biomedicine, cosmetics, food, textiles, and chemistry. It is synthesized through cyclization of glucose polymers such as starch, glycogen, and maltooligosaccharides, by cyclodextrin glucosyltransferase (CGTase). Researchers have dedicated their efforts to studying the modification effects of cyclodextrin glucosyltransferase on starch, aiming to enhance resource utilization and minimize the costs associated with cyclodextrin production [10]. Currently, the main materials used for cyclodextrin production are grain starch (e.g., corn starch) and tuber starch (e.g., cassava starch, potato starch) [11]. Additionally, studies have investigated the development and utilization of non-food raw materials rich in starch, utilizing them to produce various types of cyclodextrin or other starch derivatives, such as jackfruit seeds and cassava residue. However, ginkgo, as a starch-rich crop that is overproduced and very underutilized, there was a notable absence of documented records regarding the production of cyclodextrins utilizing unprocessed ginkgo seed starch or ginkgo seed powder. Exploring the potential for developing and effectively utilizing unprocessed samples could prove to be a crucial step in advancing the industrialization of ginkgo seeds.
Taking all of the above into consideration, the purpose of the present study was to examine alterations in ginkgo starch modified by β-cyclodextrin glucosyltransferase and to assess the potential of raw ginkgo seed powder, in its raw form, as a substitute for starch in the synthesis of β-CD. By analyzing the intrinsic relationship between the structure, composition, and physicochemical properties of ginkgo starch and the yield of cyclodextrin, this study aims to identify the optimal conditions for the production of cyclodextrin from raw ginkgo seed powder. The findings will provide a scientific basis for the application research of ginkgo starch and the exploration of the key factors affecting β-CD production.

2. Results and Discussion

2.1. Extracellular Expression of β-CGTase in E. coli BL21(DE3) and B. subtilis WB600

In our previous study, we expressed β-cyclodextrin transferase (β-CGTase) from B. circulans [12] heterologously in E. coli BL21(DE3). However, the extracellular activity of β-CGTase was only 9.5 U/mL, and it tended to form inclusion bodies, limiting its potential applications in cyclodextrin production. E. coli and B. subtilis are widely recognized as bacterial hosts for recombinant protein expression. However, when it comes to the production of extracellular enzymes, B. subtilis has emerged as a superior alternative due to its enhanced performance and improved safety profile [13]. In this study, we constructed recombinant B. subtilis strains expressing β-CGTase with a signal peptide (SPamyE) and compared their enzyme activities and protein profiles with those of E. coli BL21(DE3). After 48 h of fermentation in a TB medium, we evaluated the enzyme activities of the recombinant E. coli BL21(DE3) and B. subtilis WB600. The β-CGTase activity of B. subtilis was 22.1 U/mL, 231.4% higher than that in E. coli. SDS-PAGE analysis of the fermentation broth of both strains revealed distinct protein bands corresponding to the expected β-CGTase enzyme (Figure 1).
To evaluate the applicability of enzymes from two distinct sources for conversion, a substrate consisting of 15% potato starch was employed in the synthesis of β-cyclodextrin. The conversion process was conducted for 18 h under standardized conditions of 30 °C and pH 5.5, with the aim of measuring cyclodextrin yield. When β-CGTase expressed in E. coli was employed for the conversion, the total conversion reached 49.9%, with β-cyclodextrin (β-CD) accounting for 76.6% of the product. Conversely, when β-CGTase expressed in B. subtilis was used, the total conversion significantly increased to 75.4%, with β-CD comprising 98.9% of the final product. This represents a remarkable increase of 151.1% in total conversion compared to the former scenario, with a higher proportion of β-CD in the product. Based on these findings, we selected B. subtilis expressing β-CGTase for further characterization and application in conversion processes.

2.2. Effect of Substrate and Pretreatment Methods on the Preparation of Cyclodextrins from Raw Ginkgo Seed Powder

2.2.1. Feasibility of β-Cyclodextrin Production from Raw Materials

Firstly, the starch content in raw ginkgo seed powder was analyzed, revealing that the raw ginkgo seed powder demonstrated a starch content of 62.41%, which is comparable to that of other plants, such as corn and wheat. Subsequently, the feasibility of cyclodextrin production from different raw materials was investigated using raw ginkgo seed powder, potato powder, and corn powder. Ginkgo starch, potato starch, and corn starch were used as the control group in this study. Enzymatic conversion experiments were conducted following the methodology detailed in Section 3.8 to evaluate the conversion results.
Table 1 illustrates the obtained conversion results. Within the control group utilizing starches, potato starch exhibited the highest conversion rate, significantly surpassing the rates achieved by other samples, achieving an impressive rate of 75.4%. As for the conversion group utilizing raw substrates, both raw ginkgo seed powder and crude corn powder exhibited successful cyclodextrin synthesis. Notably, the conversion rate of raw ginkgo seed powder slightly exceeded that of raw corn powder (31.3%), with a β-CD conversion rate of 38.9%. In contrast, raw potato powder proved unsuitable for direct conversion into cyclodextrins due to a severe oxidative browning reaction during the drying process. Consequently, raw potato should not be considered as a primary material for cyclodextrin synthesis. Considering the crucial role of potatoes and corn as food or feed resources, the development of alternative non-food resources could effectively alleviate the pressure on supplies. Therefore, raw ginkgo seed starch was chosen as the subject of subsequent investigations in this experiment.

2.2.2. Effect of Pretreatment Schemes on the Preparation of β-Cyclodextrin

Under both scheme A and scheme B conditions, the impact of enzyme-catalyzed cyclodextrins (CDs) production was studied. As depicted in Table 2, at 70 °C, the conversion rate of scheme A was remarkably low, with almost no β-CD produced. This can be attributed to the fact that the raw ginkgo seed starch did not reach the gelatinization temperature during pretreatment, and β-CGTase was directly added in scheme A, rendering the enzyme unable to act on starch particles [14]. On the other hand, in scheme B, due to the initial expansion of starch particles after a certain heating period [15], enzyme molecules could bind to them. However, even at this temperature (70 °C), the conversion rate of scheme B remained relatively low, reaching only 38.9%. The highest conversion rate of 64.0% was attained at 90 °C, which aligns with the observed viscosity changes in the samples. Regardless of the temperature, scheme B consistently exhibited higher conversion rates compared to scheme A. This is because the enzyme in scheme B was always added at its optimum liquefaction temperature, while the operation in scheme A was more direct and energy-saving [16]. Due to the complete inactivation of β-CGTase at 100 °C under scheme A conditions during simultaneous gelatinization and liquefaction, CD production experiments were not conducted at this temperature [17].
In this study, we found that the pretreatment regimen at different temperatures had a greater effect on the preparation of cyclodextrins. To determine the link between the two, the pretreated samples were further characterized to determine the changes in viscosity, straight-chain amylose content, structure, XRD patterns, and infrared spectra to confirm the mechanism of action [18].

2.3. Effect of Pretreatment Schemes on the Physicochemical Properties of Ginkgo Starch

2.3.1. Viscosity Analysis

To investigate the effect of pretreatment protocols on the viscosity of ginkgo starch, we determined the change in viscosity at different pasting temperatures by heating and analyzing the pretreated samples using a fast viscosity analyzer for both scheme A and scheme B (Figure 2).
When the gelatinization temperature was set at 70 °C, the viscosity of all schemes remained constant, reaching a substrate viscosity of 2315 cP (Figure 3). It is worth noting that the optimal liquefaction temperature for β-CGTase is 70 °C, while the initial gelatinization temperature of purified ginkgo biloba powder starch is approximately 69 °C [19]. These two temperatures are similar. However, the initial gelatinization temperature of raw ginkgo seed starch (starch in raw ginkgo seed powder) is approximately 79.6 °C (Figure 3A). This difference could be attributed to the elevated levels of protein, oil, and linear starch present in the raw powder, which increase the gelatinization temperature. Additionally, the high content of amylose can act as an inhibitor of expansion (starch granules swell), thereby preventing the expected enzymatic substrate conversion and starch gelatinization process from occurring at 70 °C as anticipated [20].
At 80 °C, the substrate viscosity of both treatment schemes began to decrease significantly. In scheme A, the viscosity of the substrate decreased to 978 cP, whereas in scheme B, it decreased to 630 cP (Figure 3C). It is important to note that this temperature is close to the gelatinization temperature of raw ginkgo seed starch. At this point, the starch formed a dense three-dimensional gel network, and the swelling of the amorphous phase (water-penetrated phase) accelerated the disruption of the crystalline region [21]. The enzyme molecules found it easier to act on the starch, but the destruction of the starch particle structure by β-CGTase was relatively slow, according to Wu [22]. As a result, the viscosity of the sample decreased noticeably but did not reach its minimum level, as reported by Benavent-Gil [23].
The viscosity of the substrate was significantly affected by the two schemes at 90 °C. Scheme A decreased the viscosity to 30 cP, while scheme B decreased the viscosity to −42 cP (Figure 4D). At 100 °C, the viscosity of scheme B decreased to −2 cP (Figure 3A), which was slightly lower than that at 90 °C. Both schemes were able to completely liquefy ginkgo seed powder, starting from 90 °C. However, the high temperature used in scheme A was found to be less conducive to the action of enzymes. On the other hand, scheme B maintained the highest liquefaction activity of β-CGTase, making it more suitable for subsequent transformation.
The changes in viscosity and conversion showed similar trends. The effect of viscosity change on cyclodextrin preparation was also tentatively explained. To fully swell the starch granules in ginkgo seed powder, high temperatures are necessary. When the temperature surpasses the gelatinization threshold, the crystal structure disintegrates, allowing for the adsorption and permeation of starch with enzyme molecules [24]. As shown in Figure 3B, at 70 °C, the initial swelling of ginkgo starch is time-consuming, leading to a limited viscosity increase. Therefore, the initial step involves raising the temperature to 90 °C (Figure 3D). This resulted in a starch viscosity of 2315 cP. After gelatinization, the temperature was rapidly decreased to 70 °C, and β-CGTase was introduced, causing a decline in viscosity from 2465 to −42 cP (Figure 3D). Upon complete liquefaction, during the cooling phase, the substrate’s viscosity only slightly increased, from −2 cP to 30 cP. At this point, the substrate is unable to form a three-dimensional gel network with high viscosity. Scheme B exhibited higher liquefaction activity of β-CGTase, making it more suitable for subsequent transformation. In general, high temperatures above 90 °C effectively reduce viscosity, enabling better access of enzyme molecules to the liquid phase for further transformation [25]. Furthermore, it indicates the potential for using high-temperature-resistant enzymes in the direct liquefaction of ginkgo seed starch, thereby further reducing industrial production costs [26].

2.3.2. Starch Structure Morphology Analysis

The scanning electron microscope was used to observe the surface structure and molecular chain entanglement of ginkgo seed starch particles. When the samples were pretreated at 70 °C, the surface structure of starch granules did not obviously change for either scheme A or scheme B (Figure 4A,B). The ginkgo starch granules still retained their integrity, which is consistent with the results of Yi Zheng et al. [27]. Ginkgo starch has a high resistance to swelling and rupture. It is difficult to destroy the structure of the crystalline region by short-time heating, and it is difficult for the directly added enzyme molecules to bind to it. Further, the corresponding properties, such as viscosity and structure, are only slightly changed in this state. However, when the starch granules from scheme B were heated to the gelatinization temperature for 30 min, they initially became entangled [28] (Figure 4B), resulting in slightly higher viscosity compared to the starch granules from scheme A (Figure 4A).
On the other hand, when the samples were heated to 80 °C and reacted with β-CGTase at a constant temperature for 30 min (scheme A, 80 °C), the swelling of the starch granules and the inhalation of water molecules caused the molecular chains on the surface of the starch granules to begin to tangle. The gel network formed by amylose between amylose chains or between amylose and amylopectin chains was disrupted by β-CGTase, leading to the exposure of the amylose structure (Figure 4C). In contrast, when the sample was gelatinized at 80 °C, cooled to 70 °C, and β-CGTase was added (scheme B, 80 °C), β-CGTase acted at its optimal temperature, causing the straight-chain starch to be broken down and the network structure on the surface of the sample to disappear, forming a smooth starch surface (Figure 4D) [29].
Once the raw ginkgo seed starch was completely gelatinized at 90 °C, a three-dimensional gel network was formed [30]. In scheme A at 90 °C, the higher temperature gradually deactivated β-CGTase, preventing the sample from forming a smooth surface (Figure 4E). However, after cooling and adding β-CGTase (scheme B, 90 °C), the starch granules were acted upon by β-CGTase, which catalyzed the formation of cyclosextrins from the starch molecules [31] (Figure 4F). This indicated that the enzyme was fully effective at this stage, and the starch molecules were converted into cyclodextrins. At this point, the viscosity was the lowest, as the formation of cyclodextrins reduced the entanglement of starch chains. This also resulted in the most pronounced conversion effect, as evidenced by the significant reduction in starch granule size [32].
In scheme B at 100 °C, the temperature exceeded the gelatinization temperature of ginkgo seed starch. As a result, the crystal swelled, causing the starch particles to expand and break (Figure 4G), leading to the formation of numerous bubble-like structures on the originally smooth surface. Regardless of the pretreatment process, higher temperatures were required to convert ginkgo starch particles into a short-chain structure [33], weaken the entanglement between molecular chains, reduce viscosity, and facilitate cyclodextrin conversion [34].

2.3.3. Characterization of Amylopectin and Amylose Composition

The change in amylose and amylopectin content is a significant factor contributing to the viscosity alteration of the substrate. The contents of the amylopectin-I2 complex and amylose-I2 complex were determined at 500 nm and 600 nm, respectively [35]. In the pretreatment scheme, the amylose content exhibited a peak-shaped fluctuation with increasing temperature. At 70 °C, it was observed that scheme A did not induce any noticeable changes in the starch structure [35]. Additionally, it has been demonstrated that β-CGTase can partially catalyze the degradation of linear starch molecules, resulting in a lower content of linear starch under these conditions [36]. Based on the standard curve, the highest amylose content was observed in scheme A at 80 °C, which was 59.66% (Figure 5B), aligning with the electron microscope results where more amylose structures were clearly visible [37]. During this period, most of the ginkgo starch was converted into amylose by β-CGTase [38]. As the temperature increased, the amylose content decreased to 28.06%. In the process of starch liquefaction, enzymes preferentially degrade amylose in the continuous liquid phase or suspended gel phase [39]. When β-CGTase interacts with amylose, the structure of amylose is modified and decomposed into shorter chains. With the reduction in amylose content, the viscosity of the substrate also decreases [40].

2.3.4. XRD Analysis

The crystal structure of starch has an important influence on the physicochemical properties of starch. To study the effect of β-cyclodextrin glucosyltransferase on the crystal structure of ginkgo starch samples after thermal expansion, the XRD of the pretreated samples was analyzed in Figure 6C. Ginkgo starch is typical of an A-type crystal structure, with diffraction peaks appearing at the angles of 2θ of 15°, 17°, 18°, and 23° [41]. From the analysis of XRD patterns, the crystal structure of the pretreatment scheme A did not change at 70 °C and 80 °C, but the intensity of the diffraction peaks gradually weakened with increasing temperature. When heated to 90 °C, the A-type crystal structure was completely destroyed, and the remaining characteristic peaks disappeared, leaving only a single diffraction peak. This may be due to the destruction of the spatial structure by the sustained high temperature and the hydrolysis of β-CGTase, leading to the formation of a large number of amorphous zones in the sample. This also explains why the viscosity of the matrix of the pretreated samples decreased rapidly after 90 °C. In contrast, the samples of pretreatment scheme B at 90 °C and 100 °C formed continuous and sharp diffraction peaks at 6.2°, 7.2°, 12°, 15.5°, 17.5°, and 18.8°, which changed from an A-type to B-type crystal structure. This may be attributed to the fact that, at this time, the α-1,4-glycosidic bond of the starch was destroyed, and a large number of straight-chain and branched-chain starch were decomposed into short-chain starch molecules or glucose units, which were modified by β-CGTase [36]. The β-CGTase modification and rearrangement formed an ordered network structure (Figure 4F). This is also consistent with the study of Dong-Hui Geng et al. [42]. At this point, β-CGTase fully utilized its cyclization activity to act on glucose units, making more efficient use of the limited enzyme activity. This also explains mechanistically why the highest conversion of cyclodextrins was achieved in the samples prepared from scheme B at 90 °C.

2.3.5. FTIR Analysis

FTIR spectroscopy is effective in responding to structural changes in starch molecular chain images, crystallinity, and other structural changes. No new absorption peaks were generated in the pretreated samples; only shifts in the absorption peaks occurred. This indicates that the high temperature and enzyme action did not destroy or form chemical groups between the starches (Figure 5D). The broad absorption peak between 3403 cm−1 and 2927 cm−1 of the raw ginkgo seed powder was caused by the O-H stretching vibration, and the absorption peak at 3403.1 cm−1 of the pretreated samples gradually became narrower. This change could be attributed to the formation of a large number of short straight-chain hydroxyl groups during the high-temperature enzymatic treatment. Luane de Oliveira Maior’s study reported that a large number of hydroxyl groups led to a reduction in the swelling of the granules during the pasting process, preventing the formation of a starch paste from the available free water. This also explains why the samples started to decrease in viscosity after pretreatment [43]. In addition, the removal of water molecules may also have led to the narrowing of the absorption peak, further increasing the hydroxyl content [44]. In the infrared spectrum, 700–1300 cm−1 are the absorption peaks commonly associated with the crystalline region of starch, with C-O stretching vibrations in the region of 1000–1300 cm−1 and C-H bending vibrations in the region of 700–1000 cm−1, where 1159 cm−1 is the peak of the glucose C-O-C bond and the whole glucose ring, while 930 cm−1 and 780 cm−1 are the vibrational modes of the α-1,4 glycosidic bond and C-C stretching, respectively [45]. The absorption peaks of the pretreated samples at these three locations were slowly shifted with increasing temperature. This indicated that the destruction of the crystalline region by β-CGTase was slowly carried out, and increasing the temperature could accelerate the destruction of the ordered structure and the stretching vibration of the C-C bond [46]. This facilitated the binding of β-CGTase to starch molecules, which continuously exerted the debranching effect and caused local bending, folding, or breaking of the α-1,4 glycosidic bonds. The α-1,4 glycosidic bond was bent, folded, or broken locally, destroying the double helix structure of starch, and the long-chain starch molecules were hydrolyzed to produce short-chain starch and glucose units of different lengths. This was also consistent with the XRD results, and the higher temperature was more conducive to the preparation of cyclodextrins in the subsequent stage.

2.4. Optimization of β-Cyclodextrin Conversion Process

To optimize the production of raw ginkgo seed powder, scheme B was employed at 90 °C to optimize the pretreatment process parameters and transformation conditions.

2.4.1. Effect of Substrate Concentration

The pretreatment process of scheme B was further refined, with a particular emphasis on the impact of varying substrate concentrations on the conversion rate of CDs from pre-treated starch. Prior research has indicated that the conversion rate of β-cyclodextrin tends to decline as the substrate concentration increases during starch preparation. While a lower substrate concentration may yield a higher conversion rate, it is not conducive to industrial production due to the low output, which in turn results in increased comprehensive costs.
To ascertain the optimal substrate concentration for industrial production and to optimize its value, cyclodextrin production was examined under specified technological conditions with different substrate concentrations. The results, as depicted in Figure 6A, reveal that at a substrate concentration of 5%, the total conversion rate reaches 70.8%. However, the actual yield of β-CD is the lowest, amounting to only 21.2 g/L, approximately 30% of the highest actual yield. Conversely, when the substrate concentration reaches 20%, the highest achievable yield of β-CD is observed at 68.8 g/L, albeit with a comparatively lower conversion rate of 55.5%.
Considering the principles of resource conservation and cost reduction in industrial production, a substrate mass concentration of 15% was selected for subsequent experiments. This concentration yields an actual output of 57.6 g/L and a conversion rate of 62.1%. This choice strikes a balance between maximizing yield and maintaining a reasonably high conversion rate, thereby optimizing the overall efficiency and economic viability of the process. It is crucial to find such a balance to ensure that the production process is not only effective in terms of conversion but also practical and cost-effective for large-scale industrial applications.

2.4.2. Effect of Liquefaction Time

Following the optimization of substrate concentration, six different liquefaction times were employed to determine the optimal reaction time for liquefying ginkgo starch prior to conversion. These times included 5 min, 10 min, 15 min, 30 min, 60 min, and 120 min. The conversion rates were measured, and the results are presented in Figure 6B.
The data indicate that the conversion rate is highest between 30 and 60 min, with a peak conversion rate of 69.1% achieved at 60 min. This optimal liquefaction time for raw ginkgo starch is notably longer than the liquefaction time of potato starch, which has been reported to be 10 min in previous studies [4]. This discrepancy can be attributed to the long amylopectin chains present in ginkgo seed starch, as well as the compact crystal structure and other impurities found in ginkgo seed powder. These factors necessitate a longer time for β-CGTase to effectively hydrolyze the starch.
The selection of a 60-min liquefaction time for further investigation is based on these findings. This extended time allows for more complete hydrolysis of the starch, which is essential for achieving higher conversion rates. The longer liquefaction time also suggests that the ginkgo starch requires more extensive processing to overcome its structural and compositional challenges. This insight is valuable for optimizing the production process, as it highlights the need for sufficient time to ensure that the starch is fully liquefied and ready for the subsequent conversion to cyclodextrins. This approach not only maximizes the conversion efficiency but also ensures that the process is robust and reliable for industrial-scale operations.

2.4.3. Effect of the Amount of Enzyme Added

β-CGTase is a multifunctional enzyme with four activities: cyclization, coupling, disproportionation, and minhydrolysis [47]. These activities coexist and interact with each other in the reaction system, and their interaction is influenced by the amount of β-CGTase added to the system, thereby affecting the conversion rate [48].
To determine the optimal enzyme dosage in the pretreatment process, various amounts of enzyme were tested, namely 1 U/g, 2.5 U/g, 5 U/g, 7.5 U/g, and 10 U/g of the substrate. As depicted in Figure 6C, the conversion rate of cyclodextrin was the lowest when the enzyme dosage was 1 U/g substrate, indicating poorer results compared to other conversions. This discrepancy may be attributed to the low degree of liquefaction of starch, which is not conducive to the subsequent starch cyclization reaction. As the enzyme dosage increased, both the conversion rate and the yield gradually increased. However, when the enzyme dosage reached 10 U/g for liquefaction, the conversion rate decreased. This decline might be due to the saturation of hydrolysis activity at this point, leading to enhanced disproportionation and coupling activities that converted the generated cyclodextrin into other products. When the enzyme dosage was 7.5 U/g, the conversion rate and the yield were 70.1% and 65.7 g/L, respectively. Consequently, subsequent research was conducted using an enzyme dosage of 7.5 U/g for liquefaction.
Cyclization activity plays a crucial role in the conversion of cyclodextrin. Therefore, the amount of β-CGTase added during the cyclization stage also has a significant impact on the conversion rate, ultimately affecting the overall conversion efficiency. To investigate the optimal amount of cyclization enzyme for Ginkgo starch transformation, the β-CGTase dosage was controlled between 7.5 U/g and 40 U/g of the substrate. As the enzyme dosage increased, both the conversion rate and the yield gradually increased (Figure 6D). The highest conversion rate, reaching 72.6%, was obtained when using a cyclization enzyme dosage of 20 U/g of substrate. This condition also resulted in a yield of 67.9 g/L. However, when the cyclization enzyme dosage exceeded 20 U/g of the substrate, the conversion efficiency significantly decreased, possibly due to the saturation of cyclization activity. Meanwhile, the enhanced hydrolysis, disproportionation, and coupling activities resulted in the conversion of cyclodextrin into other products. Taking all factors into consideration, the optimum cyclization enzyme dosage for β-CGTase is determined as 20 U/g of substrate.

3. Materials and Methods

3.1. Materials

E. coli JM109, E. coli BL21 (DE3), and Bacillus subtilis WB600 were employed as host organisms for gene cloning and expression, respectively. E. coli expression vector pET-20b(+) was purchased from Novagen (Madison, WI, USA). B. subtilis expression vector Cut [13] was previously constructed and preserved in our laboratory. Ginkgo seeds were kindly provided by Pizhou Hanshang Co. Ltd. (Xuzhou, China). α-CD, β-CD, and γ-CD were purchased from Sangon Biotech Co. (Shanghai, China). Cyclohexane, potato starch (15% moisture content, 20% amylose), and corn starch (10.5% moisture content, 28% amylose) were purchased from Sinopharm Group Co. Ltd. Ginkgo starch (13% moisture content, 33% amylose) was previously extracted and preserved in the laboratory. TB medium: 24.0 g/L yeast powder, 12.0 g/L tryptone, 16.4 g/L K2HPO4–3H2O, 2.3 g/L KH2PO4, and 5.0 g/L glycerol. All other chemicals used were of reagent grade, unless otherwise stated.

3.2. Construction of Expression Vector

The gene derived from Bacillus circulans 251, as documented by Knegtel et al., was successfully expressed in both E. coli and B. subtilis. The β-CGTase gene (NCBI No.X78145.1) was amplified via polymerase chain reaction (PCR) using B. circulans genomic DNA as a template [12]. The correct β-CGTase gene fragment was ligated to a linearized pET-20b(+) vector using the In-Fusion cloning system and then transformed into E. coli JM109 receptor cells. Positive clones were selected and cultured for plasmid extraction. The plasmid was sequenced to verify its correctness. The plasmid with the correct sequence was transformed into E. coli BL21 (DE3) via heat shock and cultured overnight at 37 °C on LB solid medium containing 100 mg/L ampicillin. The recombinant strain E. coli BL21(DE3)/pET-20b-β-CGT was obtained by selecting positive transformants.
Following a similar procedure as described above, we proceeded with the integration of the gene fragment into the vector pCut, ultimately yielding the recombinant vector pCut-β-CGT. By employing B. subtilis WB600 harboring the plasmid pCut-β-CGT, the production of CGTases was accomplished.

3.3. Production and Assay of β-CGTase

The fermentation conditions in shake flasks were as follows. To initiate the seed culture, a single colony of E. coli BL21 (DE3) containing plasmid pET-20b-β-CGT was transferred into 10 mL of LB liquid medium supplemented with 100 mg/L ampicillin. The seed culture was incubated at 37 °C with a rotation speed of 200 r/min for 8–10 h. For the actual shake-flask fermentations, 50 mL of TB medium supplemented with 100 mg/L ampicillin was inoculated with 2.5 mL of the aforementioned seed culture. This mixture was then incubated at 30 °C with a rotation speed of 200 r/min for a duration of 48 h.
In a similar manner, a single colony of B. subtilis WB600 containing plasmid pCut-β-CGT was transferred into 10 mL of LB liquid medium supplemented with 30 mg/L tetracycline. The seed culture was incubated at 37 °C with a rotation speed of 200 r/min for 8–10 h. For the shake flask fermentations, 50 mL of TB medium supplemented with 30 mg/L tetracycline was inoculated with 2.5 mL of the seed culture and incubated at 37 °C with a rotation speed of 200 r/min for 72 h.
The resulting supernatant was obtained by centrifuging the fermentation broth at 8000× g for 20 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12% gel) was employed for enzyme confirmation.

3.4. Assay Activity of β-CGTase

The cyclodextrin formation activities were assessed using the phenolphthalein method [49]. A 1% soluble starch solution was prepared as the substrate in a 25 mmol/L Na2HPO4-KH2PO4 buffer at pH 5.5. Two milliliters of the substrate solution were incubated at 50 °C for 10 min. Then, 0.1 mL of the appropriately diluted crude enzyme solution was added. The reaction was carried out precisely for 10 min. To terminate the reaction, 0.2 mL of 0.6 mol/L HCl was added. Subsequently, the pH was adjusted to 10.0 by adding 0.2 mL of 0.6 mol/L Na2CO3 solution. The absorbance value was measured at 550 nm at 25 °C after developing the color for 15 min with the addition of 0.2 mL of 1.2 mmol/L phenolphthalein solution. The β-CGTase activity unit was defined as the amount of enzyme required to produce 1 μmol β-CD in 1 min under the given reaction conditions.

3.5. Preparation of Raw Ginkgo Seed Powder

To prepare raw ginkgo seed powder, fresh ginkgo nuts with shells were first dried in an oven at 75 °C for 5 h. After drying, the shells, inner seed coats, and cores were removed. The remaining shelled ginkgo nut was then ground into a fine powder using a grinder. The powder was passed through a 100-mesh sieve to remove any larger particles and ensure a consistent size distribution. Finally, the sealed powder was placed in a cool and dry place.

3.6. Determination of Moisture and Starch Content of Raw Ginkgo Seed Powder

The moisture content of the raw ginkgo seed powder was determined by drying it to a constant weight in an air oven at 105 °C for 24 h. The starch content of the ginkgo seed powder was determined using an acid hydrolysis protocol. After removing fat and soluble sugars, the starch was hydrolyzed with acid to reduce monosaccharides, which were then determined as reducing sugar and converted to starch. Then, it was measured as reducing sugar and converted to starch [18].

3.7. Pretreatment of Raw Ginkgo Seed Powder

In this experiment, we investigated the effect of temperature change on the pretreatment of raw ginkgo seed powder and the possibility of producing β-CD directly under high-temperature conditions using two pretreatment schemes: direct addition of β-CGTase after high-temperature pasting and addition of β-CGTase after cooling the paste. A suspension of raw ginkgo seed powder (containing 15% w/v) was prepared in a 25 mM Na2HPO4-KH2PO4 buffer (pH 5.5) and uniformly stirred at a speed of 200 rpm. Two different pretreatment schemes were employed for the raw ginkgo seed powder, as illustrated in Figure 2. In scheme A, the enzyme solution was directly added to the suspension, which was then heated to various temperatures (70 °C, 80 °C, 90 °C) for a period of 30 min. In contrast, scheme B first heated the suspension to the specified temperature (70 °C, 80 °C, 90 °C, and 100 °C) for stirring and heat treatment for 30 min to achieve complete expansion. After the temperature was cooled to 70 °C, β-CGTase was added, and the reaction was carried out for 30 min. Both schemes involved varying the temperature and time of enzyme addition while utilizing the same amount of enzyme (5 U/g) for pretreatment. The preprocessed ginkgo seed powder solution was used for subsequent enzymatic transformation and analysis detection.

3.8. Enzymatic Conversion of Raw Ginkgo Seed Powder to β-Cyclodextrin

To prepare β-cyclodextrin, the preprocessed raw ginkgo seed powder solution (as described in Section 3.7) was initially cooled to 30 °C. Subsequently, the β-CGTase enzyme at a concentration of 10 U/g and cyclohexane as the complexing agent at a concentration of 5% (v/v) were added for the reaction. The reaction mixture was allowed to react for 18 h.
After the reaction was completed, the sample was boiled for 15 min to deactivate the enzyme. Distillation under reduced pressure was then performed to remove the organic reagent cyclohexane. The resulting solution was diluted several times and mixed with an equal volume of acetonitrile to precipitate any impurities. The supernatant was separated from the solid particles through centrifugation. The content of cyclodextrins (α-, β-, γ-CD) in the supernatant was determined using high-performance liquid chromatography (HPLC).

3.9. Rapid Visco Analyzer (RVA) Analysis

The preprocessed raw ginkgo seed powder solution (as described in Section 3.7) was freeze-dried using a Labconco freeze dryer (Kansas City, MO, USA) and subsequently ground into a fine powder that could pass through a 100-mesh sieve. This grinding process was conducted using a mortar from Sinopharm (Shanghai, China).
For the RVA analysis, freeze-dried powder samples weighing 3 g were mixed with 25 mL of water in a canister and heated in a Rapid Visco Analyzer (RVA) manufactured by Perten (Stockholm, Sweden). The pasting procedure followed the outlined profile, with an initial temperature of 50 °C for 1 min. Subsequently, the temperature was gradually increased to 95 °C over a period of 3.7 min and maintained at this temperature for 5 min during the heating phase. In the cooling phase, the temperature was gradually reduced to 50 °C over a period of 3.8 min and held at this temperature for 2 min.
Throughout the analysis, the paddle rotation speed was maintained at 160 rpm. Changes in viscosity during the heating, cooking, and cooling phases were recorded, and various parameters, including gelatinization temperature, peak viscosity, final viscosity, breakdown viscosity, and setback viscosity, were noted.

3.10. Scanning Electron Microscopy (SEM)

For SEM analysis, the powder samples were affixed onto a specimen holder using copper tape and subsequently coated with gold in a vacuum evaporator. The structural properties of the samples were assessed using SEM (Quanta 200, FEI, Portland, OR, USA) at an accelerating voltage of 15 kV.

3.11. Amylopectin and Amylose Composition Analysis

The content of amylose and amylopectin was determined using a modified iodine scheme, as described by Shi et al. [50]. Samples weighing 0.25 mg were dissolved in 5 mL of 1 M NaOH and heated in a boiling water bath for 30 min. The solution was then diluted to a final volume of 25 mL with water. Subsequently, 0.2 mL of 1.2 M HCl and 2 mL of iodine solution (0.02% I2 and 0.2% KI) were added to 1 mL of the prepared sample. The mixture was further diluted with 6.3 mL of deionized water and thoroughly dispersed. The absorbance of the resulting solution was measured using a microplate reader (Molecular Devices, San Jose, CA, USA). The ratio of amylose to the total starch mass was calculated based on a standard curve.

3.12. X-Ray Diffraction (XRD)

Diffraction patterns of the samples were evaluated using an Ultima IV X-ray diffractometer (Tokyo, Japan). The samples were scanned at a voltage of 3 kV, a current of 20 mA, at 10°/min, and at a 2θ angle ranging from 5° to 45°.

3.13. Fourier Transform Infrared (FTIR) Spectroscopy

The Fourier Transform Infrared (FTIR) spectra were acquired using a VERTEX 80V spectrometer (Bruker, Karlsruhe, Germany). Samples were mixed with KBr in a 1:100 ratio, thoroughly mixed, ground, and compressed into thin slices for spectral analysis. The spectral measurement spanned the range of 400 to 4000 cm−1, with a resolution of 4 cm−1.

3.14. Determination of Cyclodextrin Content

The contents of cyclodextrins (α-, β-, γ-CD) in the enzyme reaction samples were determined using high-performance liquid chromatography (HPLC). Prior to analysis, the samples were filtered through a 0.45 μm organic membrane (MAISINUO, Nantong, China). The analysis was performed using a Hypersil APS-2 amino HPLC column (250 mm × 4.6 mm, 5 μm, Thermo Fisher Scientific, Waltham, MA, USA) with a mobile phase consisting of acetonitrile/water (70:30, v/v). This method allowed for the identification and quantification of the different components present in the enzyme reaction samples.

3.15. Statistical Analysis

Data were analyzed with SPSS 26.0 (SPSS Inc., Chicago, IL, USA) and presented as the mean ± standard deviation of three replicates. Single-factor analysis of variance (ANOVA) was used to test the difference in mean values at p < 0.05 level. The graphics were prepared with Origin 2021 (OriginLab Corporation, Northampton, MA, USA).

4. Conclusions

In conclusion, this study successfully demonstrated the potential of ginkgo biloba seeds as a cost-effective and sustainable raw material for β-cyclodextrin (β-CD) production. By employing β-cyclodextrin transferase (β-CGTase) from B. circulans, we explored and optimized the pretreatment and conversion processes. Our results showed that scheme B, involving high-temperature gelatinization at 90 °C, significantly improved the physicochemical properties of ginkgo starch, making it more suitable for cyclodextrin preparation. This approach not only enhanced the pasty shape, morphology, and amylose content but also altered the relative crystallinity of the starch, leading to a more efficient conversion process.
Furthermore, the optimized process conditions resulted in a remarkable β-CD conversion rate of 72.6%, which is 1.9 times higher than the initial value before optimization. This significant improvement in yield highlights the economic and industrial viability of using ginkgo seed powder for β-CD production. The study not only provides a scientific basis for the application of ginkgo starch in cyclodextrin production but also offers valuable insights into the sustainable development and utilization of ginkgo seeds. This research paves the way for further exploration of the potential of ginkgo seeds in various industrial applications, contributing to resource conservation and environmental sustainability.

Author Contributions

X.D.: Conceptualization, Methodology, Project administration, Investigation, Formal analysis, Visualization, Writing—review and editing, Funding Acquisition. Y.F.: Writing—original draft, Methodology, Formal analysis, Data curation, Validation, Visualization. Q.L.: Investigation, Resources. Y.D.: Investigation, Formal analysis. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a grant from the University-Industry Cooperation Research Project in Jiangsu (BY2022806).

Data Availability Statement

Data will be made available on request.

Conflicts of Interest

No conflicts of interest are declared for any of the authors.

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Catalysts 15 00108 g001
Figure 1. SDS-PAGE analysis of β-CGTases. M, Molecular weight markers; (1). β-CGTase expressed in B. subtilis; (2) β-CGTase expressed in E. coli.
Figure 1. SDS-PAGE analysis of β-CGTases. M, Molecular weight markers; (1). β-CGTase expressed in B. subtilis; (2) β-CGTase expressed in E. coli.
Catalysts 15 00108 g001
Catalysts 15 00108 g002
Figure 2. Flow chart of different pretreatment schemes of ginkgo seed powder. (A) Preparation of raw ginkgo seed powder. (B) Pretreatment of raw ginkgo seed powder. (C) Enzymatic conversion of ginkgo seed powder to β-cyclodextrin.
Figure 2. Flow chart of different pretreatment schemes of ginkgo seed powder. (A) Preparation of raw ginkgo seed powder. (B) Pretreatment of raw ginkgo seed powder. (C) Enzymatic conversion of ginkgo seed powder to β-cyclodextrin.
Catalysts 15 00108 g002
Catalysts 15 00108 g003
Figure 3. Viscosity changes under different pretreatment schemes. (A) Gelatinization curve of ginkgo seed powder and scheme B pretreatment at 100 °C. (B) Samples pretreated by schemes A and B at 70 °C. (C) Samples pretreated by schemes A and B at 80 °C. (D) Samples pretreated by schemes A and B at 90 °C.
Figure 3. Viscosity changes under different pretreatment schemes. (A) Gelatinization curve of ginkgo seed powder and scheme B pretreatment at 100 °C. (B) Samples pretreated by schemes A and B at 70 °C. (C) Samples pretreated by schemes A and B at 80 °C. (D) Samples pretreated by schemes A and B at 90 °C.
Catalysts 15 00108 g003
Catalysts 15 00108 g004
Figure 4. Scanning electron microscope image of pretreated ginkgo seed powder at 2000 times magnification. (A) Pretreat the sample with scheme A at 70 °C. (B) Pretreating the sample by scheme B at 70 °C. (C) Pretreating the sample by scheme A at 80 °C. (D) Pretreatment of samples by scheme B at 80 °C. (E) Pretreating the sample by scheme A at 90 °C. (F) Pretreating the sample by scheme B at 90 °C. (G) Pretreatment of samples by scheme B at 100 °C.
Figure 4. Scanning electron microscope image of pretreated ginkgo seed powder at 2000 times magnification. (A) Pretreat the sample with scheme A at 70 °C. (B) Pretreating the sample by scheme B at 70 °C. (C) Pretreating the sample by scheme A at 80 °C. (D) Pretreatment of samples by scheme B at 80 °C. (E) Pretreating the sample by scheme A at 90 °C. (F) Pretreating the sample by scheme B at 90 °C. (G) Pretreatment of samples by scheme B at 100 °C.
Catalysts 15 00108 g004
Catalysts 15 00108 g005
Figure 5. (A) The absorbance of amylose-I2 complex (600 nm) and amylopectin-I2 complex (500 nm). (B) Amylose content of the sample. (C) XRD spectra of the sample. (D) FT-IR spectra of the sample.
Figure 5. (A) The absorbance of amylose-I2 complex (600 nm) and amylopectin-I2 complex (500 nm). (B) Amylose content of the sample. (C) XRD spectra of the sample. (D) FT-IR spectra of the sample.
Catalysts 15 00108 g005
Catalysts 15 00108 g006
Figure 6. Effects of reaction conditions on the process of β-cyclodextrin conversion. (A) Substrate concentration. (B) Pretreatment time. (C) Pretreatment enzyme dosage. (D) Effect of cyclization enzyme dosage on β-cyclodextrin production.
Figure 6. Effects of reaction conditions on the process of β-cyclodextrin conversion. (A) Substrate concentration. (B) Pretreatment time. (C) Pretreatment enzyme dosage. (D) Effect of cyclization enzyme dosage on β-cyclodextrin production.
Catalysts 15 00108 g006
Table 1. Effect of substrate types on β-cyclodextrin conversion rate.
Table 1. Effect of substrate types on β-cyclodextrin conversion rate.
Substrate Typeβ-CD Conversion Rate Using Different Raw Materials (%)β-CD Conversion Rate Using Different Starches (%)
Ginkgo38.96 ± 1.5253.41 ± 1.37
Corn31.30 ± 3.2739.91 ± 2.56
Potato-75.40 ± 4.31
Table 2. Effect of pretreatment schemes and pretreatment temperature on β-cyclodextrin conversion rate.
Table 2. Effect of pretreatment schemes and pretreatment temperature on β-cyclodextrin conversion rate.
Temperature (°C)β-CD Conversion Rate by Scheme A (%)β-CD Conversion Rate by Scheme B (%)
701.57 ± 0.0738.97 ± 1.38
8051.27 ± 2.6556.47 ± 2.47
9053.43 ± 3.2164.04 ± 3.38
100-61.83 ± 2.97
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MDPI and ACS Style

Duan, X.; Fan, Y.; Liu, Q.; Ding, Y. An Efficient Approach for β-Cyclodextrin Production from Raw Ginkgo Seed Powder Through High-Temperature Gelatinization and Enzymatic Conversion. Catalysts 2025, 15, 108. https://doi.org/10.3390/catal15020108

AMA Style

Duan X, Fan Y, Liu Q, Ding Y. An Efficient Approach for β-Cyclodextrin Production from Raw Ginkgo Seed Powder Through High-Temperature Gelatinization and Enzymatic Conversion. Catalysts. 2025; 15(2):108. https://doi.org/10.3390/catal15020108

Chicago/Turabian Style

Duan, Xuguo, Yucheng Fan, Qianqian Liu, and Yucheng Ding. 2025. "An Efficient Approach for β-Cyclodextrin Production from Raw Ginkgo Seed Powder Through High-Temperature Gelatinization and Enzymatic Conversion" Catalysts 15, no. 2: 108. https://doi.org/10.3390/catal15020108

APA Style

Duan, X., Fan, Y., Liu, Q., & Ding, Y. (2025). An Efficient Approach for β-Cyclodextrin Production from Raw Ginkgo Seed Powder Through High-Temperature Gelatinization and Enzymatic Conversion. Catalysts, 15(2), 108. https://doi.org/10.3390/catal15020108

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