Improvement in extraction and sensory properties of soapnut extract by fermentation

Sapindus saponins are potential biosurfactants that can widely be used to replace many chemical cleaning products. This study aimed to investigate the water extraction of saponins from the pericarps of Sapindus mukorossi and enhance the sensory properties of the extract by yeast fermentation. Extraction conditions including temperature, solid-to-liquid ratio, extraction time, and number of extraction times were studied. A yield of 21.4% was obtained by 2 h of extraction at 80 o C with a solid-to-liquid of 1/6 (w/v) and two times. Fermentation was used to purify the Sapindus extract, inoculum amount and fermentation time were optimized. The fermentation by S. cerevisiae (2%) within 4 days signi ﬁ cantly improved the color and smell of aqueous extract, turbidity decreased by 75.6%, total sugar content decreased by nearly 50% and saponins content slightly decreased. These results could contribute to the development of industrial–scale production of Sapindus saponins.


INTRODUCTION
Sapindus mukorossi Gaertn (also known as soapnut) is a deciduous tree that is widely distributed in Asia's tropical and sub-tropical regions 1, 2 .Soapnut is tolerant to reasonably poor soil and each tree can produce 30-35 kg of fruit per year 3 .The major active components of its pericarp are saponins, which have been confi rmed to be effective natural nonionic surfactants and have very low toxicity, hemolysis, and skin irritability 4 .Sapindus saponins have also been proven to possess many pharmacological effects, including anti-microbial, anti-fungal, anti-dermatophytic, and anti-infl ammatory activities 5 .Therefore, having great potential in developing functional cosmetics and daily cleaning products 6, 7 .The natural surfactants were also proven to be readily biodegradable and have excellent performance for environmental remediation, as opposed to chemical and synthetic surfactants 8 .As a result, Sapindus saponins which are environmentally friendly surface-active agents, have raised great interest worldwide in terms of the replacement for chemical cleaning products.
Sapindus saponins can be extracted effi ciently by water due to their compatibility; hence water -an inexpensive and safe solvent has been investigated for Sapindus saponins extraction in many research 9 .However, the Sapindus water extract (SW) has some undesired sensory characteristics such as color, odor, and turbidity.The dark brown color, intensely sour odor, and higher turbidity during the storage time of the SW have limited its application in commercial cleaning products 10 .A method to decolorize the SW by hydrogen peroxide has been investigated in some research. 11Despite its quick effi ciency, the strong oxidizer partially affects the surface activities of the SW and raises safety concerns in daily uses.
Sapindus crude extract contains an abundance of carbohydrates, a few proteins, calcium, magnesium, iron, and other impurities 12 , which correlates with the undesired sensory properties of the SW 13, 14 .Enzymatic treatment and bacterial fermentation are effective methods in purifying fruit/plant extract, improving its quality and sensory characteristics 15, 16 .The enzymatic process is quick, productive, and targeted for certain substances, while the fermentation method is less expensive, resulted reducing various components and producing some by-products 17, 18 .Microbial fermentation to purify the Sapindus saponins after water extraction was a recently introduced method 19 .After purifi cation, Sapindus fermentation product (SWF) has been shown to enhance various properties, such as surface activities, microorganism inhibition, and anti-acne 9, 19, 20 .The fermentation process also showed signifi cant positive changes in sensory characteristics (color, odor, and turbidity) 21, 22 .Via the activities of bacteria, the dark brown color of the SW has been transformed into light yellow-brown, reducing the turbidity and creating a better smell.Therefore, fermentation is grasping attention as a potential method of purifying Sapindus saponins extract.
Traditional fermentation of Sapindus extract is also applied by many people to obtain the SWF as a multipurpose cleaner 23 .This method is inexpensive and straightforward, whereas spontaneous and uncontrolled fermentation has created a real challenge in achieving consistent quality of the fi nal products 24 .Therefore, some issues regarding standards and effi ciency should be carefully considered, primarily to conduct on a large scale.This study aimed to optimize the water extraction of Sapindus saponins, which could be used to develop more extensive manufacturing processes .Then yeast fermentation is employed to remove impurities and improve the sensory properties of Sapindus extract, and natural fermentation was also compared.

Extraction of Sapindus saponins
For large-scale production, pericarps of S. mukorossi were kept at their initial size.Sapindus pericarps were extracted with distilled water by maceration and stirring under specifi c conditions.The fi rst extract was crudely fi ltered through a gauze and the residue obtained was continuously extracted with the same procedure.Subsequently, the product solutions were combined and thoroughly mixed to obtain the Sapindus saponins extract (SW) for the fermentation process or fi ltered with fi lter aid powder (diatomite) for further analysis.The extraction conditions such as temperature, material-to-liquid ratio, and extraction time were investigated.

Yeast Activation
Before inoculation, 3 g of the S. cerevisiae dry yeast was rehydrated in 15 mL of distilled water and 15 mL of SW.The yeast solutions were activated for 30 min in a water bath at 35 o C and used for further fermentation.

Fermentation Method
The activated yeast was inoculated into 150 mL of SW obtained from the extraction process.The containers were sealed and incubated at room temperature (25-35 o C), away from direct sunlight.Then, the fermented extract was then autoclaved and fi ltered with fi lter aid powder to obtain the Sapindus fermentation product (SWF).Fermentation parameters such as time and inoculum amount were investigated.

Traditional fermentation method
Sapindus pericarps were pre-washed in tap water to remove dust and impurities.Then Sapindus pericarps, jaggery, and water with the ratio of 1:1:10 (w/w/w) were added together and thoroughly mixed in a container with a lid.Subsequently, the immersion was covered, placed at sunny places, and fermented in anaerobic conditions.At the beginning of the fermentation process, the mixture should be gently stirred once a day to provide oxygen, boosting the growth of microorganisms.Until a thin layer of microorganisms appeared on top, the immersion can completely be sealed and fermented in anaerobic conditions for three months.The sample solutions were collected weekly and fi ltered with fi lter aid powder to determine the total saponin content.

Determination of total saponins
The total saponins of SW and SWF were determined according to the vanillin-sulfuric acid assay 25 .Oleanolic acid was used as the saponin standard.
Sample measurement: 0.3 mL of vanillin 8% solution and 3 mL of sulfuric acid 72% were respectively added into 0.3 mL of sample diluted several times.The solutions were evenly mixed using a vortex shaker, warmed in a water bath at 60 o C for 10 min then cooled in ice--cold water for 10 min.The absorbance was determined at 538 nm by using a UV-Vis spectrophotometer (model GENESYS™ 10S, ThermoFisher Scientifi c Inc., USA). (1) Where: Yi (%) is the extraction yield of total saponins W s (g) is the weight of total saponins in the extract.W p (g) is the weight of the Sapindus pericarp.

Determination of total sugars
The total sugars were determined according to the phenol -acid sulfuric 26 .The absorbance was measured at 490 nm, D-glucose was used as the sugar standard.The quantitative colorimetric method can determine the content of sugars and their methyl derivatives, oligosaccharides, and polysaccharides.
Sample measurement: 0.5 mL of phenol 5% solution and 2.5 mL of concentrated sulfuric acid were respectively added into 0.5 mL of sample diluted several times.The solutions were allowed to stand for 10 min, then they were evenly mixed and placed for 20 min.The absorbance was determined at 490 nm by using a UV-Vis spectrophotometer (model GENESYS™ 10S, Thermo-Fisher Scientifi c Inc., USA) and the concentration of total sugar (mg Glu/mL) was calculated.

Evaluation of turbidity
The turbidity was evaluated by UV-Vis spectrophotometer (model GENESYS™ 10S, ThermoFisher Scientifi c Inc., USA) according to Batch's report 26 .Principle of the evaluation was based on the amount of light absorbed, scattered, or refl ected by particles in the solution.The absorbance was determined at 860 nm. (2) Where: OD 860 indicated the turbidity of Sapindus solution at 860 nm OD S is the absorbance of a sample OD B is the absorbance of a sample fi ltered by 0.2 μm microfi lter.

Evaluation of color
The sample colors were measured by a CR-400 Chroma Meter.The L axis describes the Lightness of the color, going from absolute black (L = 0) to absolute white (L = 100).Samples were measured color directly without any dilution in a 1 cm glass cuvette.ethanol extraction was slightly higher, water was preferred in Sapindus saponins extraction because it is an inexpensive and safe solvent 9, 12 .Since Sapindus saponins were stable with high temperature 1, 27 , it was evident that increasing the extraction temperature could improve the effi ciency and reduce extraction time without leading to degradation of the compounds.However, extraction of Sapindus saponins was generally conducted at low temperatures (40-60 o C) 12 .It is because water extraction under high temperatures could increase the swelling ability of starch Inoculation amount-based components 28 that fully existed in the Sapindus extract, making the fi lter process impossible.In this study, Sapindus pericarps were kept at their original size, hence the swelling of the material was minimized, and the water extraction of Sapindus saponins has become more possible with higher temperatures.As can be seen, this slight adjustment has made the extraction process of Sapindus saponins more suitable and effi cient on a larger scale.
Factors such as temperature, material-to-solvent ratio, extraction time, and the number of times of extraction were considered to have a signifi cant effect on the extraction yield.The results of the single-factor test are shown in Figure 1.An extraction temperature of 80 o C, a solid-to-liquid ratio of 1/6 (g/mL), an extraction time of 120 min, and extraction two times gave the best results.Under these conditions, the water extraction of saponins yielded 21.4%.

Evaluation of odor
The odor evaluation focused on odor quality and preference.A group of 15 people was invited to evaluate the odor of the samples measured on a 5 -point scale.The samples were presented in random order and marked by each attendant (attendants were required to smell water before starting a new sample).The data were statistical analyses by ANOVA.

Analysis data
The procedure was repeated three times for each sample.The mean and standard deviation of the results were calculated using Microsoft Excel program (Microsoft Inc., Redmond, WA, USA).Experiment data were analyzed using a one-way analysis of variance (ANOVA) test in the SPSS program (IBM Company, USA) with a level of signifi cance at 5%.

Effects of extraction conditions
Sapindus saponins can be extracted effi ciently by water and ethanol.Although the yield of total saponins with

Fermentation condition
To improve the purity as well as sensory properties of SW, further fermentation by S. cerevisiae was investigated.Since the optimal temperature for the growth of S. cerevisieae was 28-32 o C, the fermentation was conducted at room temperature, and factors such as fermentation time and inoculation amount were optimized.

Fermentation time
Each container was fi lled with 150 mL SW inoculated with 1.5% (v/v) activated yeast and incubated at room temperature for 1-7 days.The concentration of total sugar decreased markedly, and saponins were also consumed in small amounts during the fermentation process due to the growth of yeasts (Fig. 2).At the beginning of 4 days, the total sugar content reduced rapidly from 42.86 ± 1.58 mg Glu/mL to 23.04 ± 1.81 mg Glu/mL and then became quite stable.In contrast, the concentration of saponins declined signifi cantly after 4 days of fermentation.The potential explanation was the preference of yeast in using carbohydrates and proteins in the Sapindus extract; hence the amount of sugar in the fi rst four days decreased tremendously, and the saponins content was less affected.Some previous studies also show that Sapindis's fermentation time of 4 days is necessary.At this time, the total sugar content dropped to the lowest level, while the purity of saponin and ethanol content increased signifi cantly 9, 12 .the inoculum amount.Therefore, it was concluded that the appropriate amount of inoculum was 2%.

Effects of fermentation on sensory characteristics of Sapindus extract
The dark brown, intensely sour odor, and high turbidity of the Sapindus extract have limited its application in commercial cleaning products.By purifying the crude extract, fermentation has made signifi cant improvements in the sensory properties of the fi nal product.

Turbidity and color
The SW treated by fermentation showed a remarkable decrease in turbidity over the days (Fig. 4).The decreasing stage of turbidity was most apparent in the In terms of sensory characteristics, SWF also showed a vast improvement in turbidity, color, and smell after 4-5 days of fermentation, which will be presented afterward.Consequently, the optimal fermentation time of SW by S. cerevisiae was 4 days.

Inoculation amount
As can be seen clearly, the inoculation amount showed little effect on saponins but signifi cantly affected the sugar content (Fig. 3).The concentration of saponins only declined less than 1 mg Ole/mL when the inoculum amount changed from 0.5% to 3%.There was a signifi cant drop in total sugar content when increasing the inoculum amount to 2% (from 39.66 ± 0.60 mg Glu/mL to 21.74 ± 0.93 mg Glu/mL).The increased inoculum volume (3%) is related to a chang e in the concentration of total sugar.When considering sensory properties, the SWF also showed evident progress with 2% or 2.5% of fi rst 4-5 days of fermentation, and with the inoculation amount above 1% (improved more than 70%).In terms of color, the SWF showed an increase in the lightness axis (Table 1) and fermentation had changed the dark brown of the SW into a light yellow-brown color (Fig. 5).
The improvements in turbidity and color have made the fi nal product's appearance possible to apply in commercial cleansers.
The activities of S.cerevisiae can explain the improvement in turbidity and color of the SW.The cloudiness of Sapindus crude extract was due to an abundant amount of impurities such as sugars, proteins, and polysaccharides 19 which were consumed by yeast.Additionally, fermentations helped to reduce a large number of sugars that could be responsible for the browning intensity due to caramelization and participation in Maillard reactions during a long time of extraction 13 .

Odor
The Sapindus extract was intensely sour in odor, which might create displeasing experiences for customers.However, after being treated by yeast fermentation, the products showed great changes in aroma, which was more familiar and tolerant to most people.The odor  Table 2. Sensory ratings of Sapindus extract before and after fermentation by S. cerevisiae of samples was evaluated on a 5 -point scale based on odor quality and preferences.
The results showed that the aroma of Sapindus extract after being fermented was given a higher mark, especially after 4-5 days of fermentation and the inoculum amount of 2% or 2.5%.(Table 2).The changes in the odor of SWF can be explained by various volatile compounds produced by S. cerevisiae during a fermentation process.This yeast strain was known to produce signifi cant amounts of alcohols, ethyl, acetate esters, and ethyl esters, which were generally associated with fresh fruit and citrus aromas 29 .Also, the fermented products by S. cerevisiae were characterized as having aromas of fruity apple, bakeries, yeast, dairy, and acidity 30 , which was preferable to most experiencers.

Natural fermentation of Sapindus saponin
Traditional fermentation is applied by many Vietnamese people to obtain the SWF as a dish-or cloth-washing liquid.Although this method is relatively affordable and simple to perform, it is considered time-consuming and produces low-quality products (Fig. 6).
The extraction effi ciency of Sapindus saponins by natural fermentation was illustrated in Figure 7.The extraction process of saponins by natural fermentation was much more than heat and stirring-assisted extraction (yielded 21.4% in 2 hours).After the fi rst week, the extraction yield was only 12.7% and gradually increased to the peak of 20.1% after 5-6 weeks.However, the weight of saponins extracted decreased signifi cantly after 8 weeks of fermentation.The explanation for this tendency is the activities of microorganisms during the natural fermentation process.After the maximum amount of Sanpindus saponins has been obtained, the microorganisms continue to grow and spend the extracted saponins, leading to a gradual decrease ineffi ciency.Generally, the natural fermentation process was fi nished after 3 months to collect the SWF.Therefore, the low content of saponins in Sapindus solution was unavoidable 31 .Besides, the natural fermentation of Sapindus extract is spontaneous and uncontrolled.Consequently, achieving consistent quality in the fi nal product is the major challenge 32 .Naturally fermented products come with a diverse bacterial community and contamination of unwanted which might fail to meet the quality assurance and raise many customer concerns.Therefore, the naturally fermented product of Sapindus extract is not suitable to become a commercial product.In the quest for better quality and process management, yeast fermentation after water extraction is a promising approach to industrial scale.

CONCLUSION
In this study, water extraction and yeast fermentation were optimized to purify the SW and enhance its sensory characteristics, traditional fermentation was also examined.Under 2 hours of extraction at 80 o C with a solid-to-liquid of 1/6 (w/v) and 2 times, the yield of Sapindus saponins reached 21.4%.Fermentation by S. cerevisiae with inoculate amount of 2% within 4 days was the ideal condition to purify Sapindus extract.While the saponins concentration of SWF slightly decreased, the total sugar content reduced approximately by 50%, turbidity decreased by 75.6% and obvious improvements in color and odor were observed.
In conclusion, yeast fermentation was a potential method to purify and improve the poorness in sensory properties of the crude Sapindus water extract (the fermentation had changed the dark brown of the SW into light yellow-brown color and sensory ratings from 1.93±0.7 and 3.6±0.51before and after fermentation by S.cerevisiae).These results could contribute to the development of large-scale production of Sapindus saponins and their applications in commercial products.

Figure 1 .
Figure 1.Effect of extraction conditions (temperature (a); material to solvent ratio (b); time (c) and times (d)) on the yield of total Sapindus saponins

Figure 2 .Figure 3 .Figure 4 .
Figure 2. Effects of fermentation time on saponins and total sugar content

Figure 5 .Figure 6 .
Figure 5. Effects of fermentation time (a) and Inoculation amount (b) on color of Sapindus extract

Figure 7 .
Figure 7. Extraction yield of Sapindus saponins by natural fermentation

Table 1 .
Effects of fermentation on the lightness of Sapindus extract