What Are the Production Methods of Galacto Oligosaccharide (GOS)?

With the development of society and changes in human dietary structure, people's intake of meat and dairy products is increasing, while the intake of cereal foods is gradually decreasing. Changes in dietary structure have directly led to a surge in hypertension, diabetes, and various oral and digestive diseases. Moreover, with the aggravation of an aging society, the demand for functional foods and health foods will continue to increase. Galacto-oligosaccharides (GOS) are a typical functional food additive that is of great scientific research value and market development prospects due to their excellent physical and chemical properties and outstanding physiological effects.

1. Introduction to galacto-oligosaccharides and their physiological effects

1.1. Introduction to galacto-oligosaccharides

Prebiotics are non-digestible food components that can be selectively utilized by bifidobacteria and lactobacilli to promote host health. They play an important role in maintaining the balance of intestinal flora. Having “optimal” intestinal flora can enhance the body's resistance to pathogenic bacteria, reduce the blood ammonia mass fraction, enhance immunity, and reduce the risk of cancer [1-2]. Galactooligosaccharides (GOS) are a functional oligosaccharide and a member of the prebiotic family that has attracted a lot of attention in recent years [3-5]. Galactooligosaccharides consist of 2 to 10 galactose units and a terminal glucose unit, with the structural formula (Galactose)n -Glucose.

In addition, the disaccharide composed of two galactose units is also considered a type of oligosaccharide. Oligosaccharides are found in small quantities in nature, only in breast milk and some fruits and vegetables. They have excellent physical and chemical properties and outstanding physiological effects, making them very suitable for use as additives in the food industry [6]. In addition, the safety of oligosaccharides has been widely recognized. For example, Japan has already regarded oligosaccharides as a specific health food (FOSHU), the United States has recognized them as a generally recognized as safe substance (GRAS) [7], and China has defined them as a nutritional enhancer and new resource food. Therefore, galacto-oligosaccharides have a broad range of applications in many fields such as infant formula, fermented milk, confectionery products, baked goods, livestock feed and pet food, and have a promising market outlook.

1.2 Physicochemical properties and physiological effects of galacto-oligosaccharides 

1.2.1 Physicochemical properties

Commercially available galacto-oligosaccharides are translucent, yellowish to colourless, and have a moderate sweetness, generally 0.3 to 0.6 times that of sucrose. The viscosity is similar to that of high fructose syrup, and the caloric value is lower than that of sucrose, generally less than 50%. Galacto-oligosaccharides contain many hydrophilic groups, which gives them good water solubility and water retention, and strong moisturising properties. In addition, due to its structure, which contains many β-(1→ 3), β-(1→ 4) and β- (1→ 6) glycosidic bonds that are not easily hydrolyzed, it has high stability at high temperatures and over a wide pH range. For example, it can remain stable for several months at a temperature of 37°C and pH 2. It can also remain stable physically and chemically after being treated at a neutral pH of 7 and a temperature of 160°C or a pH of 3 and a temperature of 120 .

1.2.2 Physiological effects

1) Non-digestibility. Non-digestibility is one of the factors that must be present for a component to be defined as a prebiotic[1]. Galacto-oligosaccharides are generally not digested or absorbed by the body's digestive enzymes because they contain many β-galactoside bonds between the sugar units that are not easily hydrolyzed, except for a very small number of disaccharides. Studies have shown[8-9] that oligosaccharides with a mass fraction of more than 90% are not digested or absorbed by the stomach and small intestine, but instead pass directly into the colon. In vitro experiments have also shown that oligosaccharides such as galactooligosaccharides and galactotrioses are not hydrolyzed by human saliva α-amylase, artificial gastric juice, or porcine pancreatic α-amylase. Only a small proportion of disaccharides can be digested by the enzymes in the rat intestine . In addition, the caloric value of galacto-oligosaccharides is very low, at only 5–8 kJ/g. For this reason, galacto-oligosaccharides can be used as a sweetener and bulking agent in foods for diabetics and obese patients.

2) Promotes the proliferation of probiotics. Galacto-oligosaccharides can be selectively utilized by bifidobacteria and lactobacilli, thereby promoting the proliferation of beneficial bacteria, inhibiting the growth of harmful bacteria, and maintaining the balance of intestinal flora. Davis et al. [10] studied the effect of galacto-oligosaccharide dosage on bifidobacteria and found that a daily intake of chewing candies containing 5 g or more of galacto-oligosaccharides for 3 weeks produced a significant effect on bifidobacteria proliferation. Chang Jinjin et al. [11] studied the addition of 2% oligosaccharides to the diet of weaned piglets, which can increase the relative abundance of lactobacilli in the piglet's intestine and improve the intestinal microbial composition.

3) Promote mineral absorption. Probiotics such as Bifidobacterium can use galacto-oligosaccharides to produce weak acids such as short-chain fatty acids (acetic acid, butyric acid, isobutyric acid, etc.) and lactic acid, which can lower the intestinal pH, promote the absorption of calcium and iron ions, and prevent osteoporosis [7, 11-12].

4) Prevention of tooth decay. Galacto-oligosaccharides cannot be utilized by Streptococcus mutans, which reduces the production of Streptococcus mutans and prevents tooth decay. At the same time, galacto-oligosaccharides have a sweet taste, so they can be used as a sweetener in children's food or in the production of anti-caries candy to reduce the incidence of tooth decay in children.

5) Prevention and treatment of constipation. Short-chain fatty acids and gases such as CO2, H2 and CH4 produced by the fermentation and decomposition of galacto-oligosaccharides by bifidobacteria can stimulate bowel movement, increase the moisture content of stools and prevent constipation.

6) Other: Galacto-oligosaccharides also play an important role in regulating the immune system, regulating lipid metabolism and inhibiting the growth of tumor cells [12-14]. Galacto-oligosaccharides have also been found to selectively stimulate the growth of “beneficial” bacteria on human skin. As a cosmetic additive, they have a certain moisturizing effect and can help to clear acne [15]. In addition, research has found that changes in the intestinal flora environment may lead to the occurrence of diseases such as depression, obesity, Alzheimer's disease and Parkinson's disease. Since oligosaccharides are beneficial to the regulation of intestinal flora, their physiological effects have also attracted widespread attention from researchers in the pharmaceutical field.

2 Preparation of oligosaccharides

At present, domestic and foreign studies have shown that the main methods of preparing oligosaccharides are extraction from natural raw materials (such as the acid hydrolysis of natural polysaccharides), chemical synthesis, fermentation and enzymatic methods (biocatalysis). Since the content of oligosaccharides in natural raw materials is very low, such as honey, some fruits and vegetables and animal milk contain trace amounts of oligosaccharides, and only breast milk contains slightly more, it is not realistic to extract large amounts of oligosaccharides from natural raw materials. When natural polysaccharides are hydrolyzed, the yield of oligosaccharides is not high, and the composition of the hydrolyzed products is complex, containing a large amount of other non-functional monosaccharides/oligosaccharides, which makes it difficult to separate and purify, and unsuitable for large-scale industrial production. The chemical synthesis method for preparing galacto-oligosaccharides is highly polluting, costly, and not ecologically or economically efficient, and is also not suitable for industrial production. Among the methods for synthesizing galacto-oligosaccharides, the fermentation method and the enzymatic method have been studied more. The enzymatic (β-galactosidase) method, with its specific transglycosylation activity, has become the main method for industrial production of galacto-oligosaccharides [16-17].

2.1 Mechanism of enzymatic synthesis of galacto-oligosaccharides

β-Galactosidase uses its hydrolytic activity to break down the substrate lactose into galactose and glucose units, and then uses β-galactosidase's transglycosylation activity to transfer the galactose unit to different acceptors. When the acceptor of the galactose unit is water, galactose is formed; when the acceptor of the galactose unit is another sugar acceptor, oligosaccharides are formed. Therefore, the reaction of β-galactosidase synthesizing oligosaccharides is a kinetically controlled reaction accompanied by hydrolysis and synthesis [18].

2.2 β-galactosidase, sources and synthesis of oligosaccharides

2.2.1 β-galactosidase

β-galactosidase (E. C. 3 . 2 . 1 . 23), also known as lactase, is a type of glycosidase that can hydrolyze β-galactoside bonds[4, 19] and has been applied in fields such as food, biosensors and basic research. In the food industry, the hydrolytic activity of β-galactosidase is often used to degrade lactose in dairy products, produce low-lactose dairy products, improve the digestibility, solubility, sweetness and flavor of dairy products, and reduce the risk of lactose intolerance symptoms caused by lactose [4]. Studies have shown that nearly 70% of adults in the world suffer from lactose intolerance[8]. It can also be used to treat whey wastewater to reduce environmental pollution[20-21]. In the field of biosensors, some β-galactosidase enzymes are also used in biosensors to detect lactose in dairy products[22]. In the field of basic research, the coding β-galactosidase gene (lacZ) is often used as a reporter gene to monitor transfection rates [4]. More interestingly, in addition to its hydrolytic activity, some sources of β-galactosidase also have transglycosylation activity, which can be used to synthesize galacto-oligosaccharides as prebiotics, which is another of its main uses in the food industry.

2.2.2 Sources of β-galactosidase

There are a wide range of sources of β-galactosidase. The main sources are: 1) plant sources. such as Arabidopsis, tomatoes, strawberries, sweet peppers, apples, mangoes, and bananas[23]; 2) animal sources. Mainly found in the small intestines of young mammals; 3) microbial sources. Bacteria (e.g. Escherichia coli, Lactobacillus and Bifidobacterium), moulds (e.g. Aspergillus oryzae, Aspergillus niger and Penicillium), yeasts (e.g. Kluyveromyces lactis, Kluyveromyces fragilis and Kluyveromyces marxianus) and actinomycetes (e.g. Streptomyces coelicolor) [5, 8]. mold] [5, 8]. The β-galactosidase from animal and plant sources has a small mass fraction and is difficult to isolate and extract, so it is not suitable for industrial production. However, β-galactosidase from microbial sources has the advantages of high yield, low cost and short cycle, so it has become the  the main source, and Aspergillus niger, Aspergillus oryzae, Kluyveromyces lactis, Bifidobacterium and Bacillus circulans are the main enzyme sources for the industrial production of β-galactosidase [5, 24].

β-galactosidases from different sources differ significantly in terms of their protein sequences, molecular weights, structures, and enzymatic properties. Based on the similarity of β-galactosidase protein sequences, a bioinformatics database search of the CAZy (http://www.c azy. org/) ,   β-galactosidase can be subdivided into multiple glycoside hydrolase families such as GH1, GH2, GH35, GH42, GH59 and GH147. The GH1, GH2, GH35 and GH42 families have been reported to have the potential for industrial application. The β-galactosidase families from different sources and their activity characteristics are shown in Table 1.

2.2.3 β-galactosidase synthesis of oligosaccharides

In recent years, as the beneficial effects of oligosaccharides have gradually become known, research on oligosaccharides has become a hotspot of attention for scholars at home and abroad. At present, there have been a large number of reports on the synthesis of oligosaccharides by β-galactosidase at home and abroad. The main ways of synthesizing oligosaccharides by β-galactosidase are to use crude or pure β-galactosidase from wild bacteria[32-33], recombinant β-galactosidase[34-35], whole cells or permeabilized cells of microorganisms[30- 36], immobilized enzymes or cells [37, 29] to catalyze the hydrolysis of lactose and the transglycosylation process, and achieve the biocatalytic preparation of galacto-oligosaccharides.

The advantage of using free enzymes to synthesize galacto-oligosaccharides is that the free enzymes directly participate in the reaction, and the product has high purity and is easy to purify. The disadvantage is that the amount of enzyme used is large and the stability is not high. In addition, the β-galactosidase from wild bacteria is difficult to isolate and purify, and the cost is high, but the biosafety is high, while the β-galactosidase from recombinant engineered bacteria is more readily available than the wild enzyme. The immobilized enzyme synthesis of oligogalactose is more stable than free enzymes and can be reused, and is the focus of research on the industrial production of oligogalactose.

Since the reaction of β-galactosidase synthesizing oligosaccharides is accompanied by a dynamically controlled reaction of hydrolysis and synthesis, the properties of β-galactosidase (enzyme source) are crucial for the efficient preparation of oligosaccharides by biocatalysis. For example, the yield, degree of polymerization and type of bond of oligosaccharides all depend on the properties of β-galactosidase enzyme properties [5]. Currently, the β-galactosidase used in the commercial synthesis of oligosaccharides is mainly derived from bacteria such as Bacillus circulans, Aspergillus oryzae and Kluyveromyces lactis [5, 20]. The reaction temperature for the synthesis of oligosaccharides by β-galactosidase from Bacillus circulans is 40~60 ℃, the pH is close to 6, and the yield is 40%; the optimal reaction temperature for β-galactosidase from Aspergillus oryzae is 40~60 ℃, the optimal pH is 4.5, and the yield of oligosaccharides is close to 30%, and it is more convenient to prepare the enzyme compared to the β-galactosidase from Bacillus circulans. The reaction temperature of β-galactosidase from Kluyveromyces lactis is 35~40 ℃, the pH is 6.5, and the yield of oligosaccharides is about 30%. In addition, β-galactosidase derived from Lactobacillus kluveri has strong hydrolytic activity and is more suitable for lactose hydrolysis than for the synthesis of oligosaccharides [5].

It has also been reported that the three-dimensional structures and reaction mechanisms of β-galactosidase proteins from different sources are different, and they have different selectivities for water and sugar. The reaction conditions are different, which leads to differences in the yield and structure of galacto-oligosaccharides (i.e., the source of β-galactosidase determines the yield, composition and type of GOS) [38]. For example, Huang et al. [29] heterologously expressed two β-galactosidase genes derived from acid-producing Klebsiella [39-40] and investigated the transglycosylation and hydrolysis activities to obtain a highly active β-galactosidase. The enzyme had a high yield of oligosaccharides at a reaction temperature of 37 °C, an initial lactose mass concentration of 400 g/L, a reaction pH of 7.5, an enzyme addition of 10 U/g lactose, and a reaction time of 48 h.

The reaction pH was 7.5, the amount of enzyme added was 10U/g lactose, and the reaction time was 48h. Under these conditions, the yield of galactose was about 45%, and the mass concentration of the product reached 178 g/L (including isomaltose, oligosaccharides, trisaccharides and tetrasaccharides). Zhu Wuer et al. [41] used β-galactosidase from Salinomonas S62 as the research object. At a reaction temperature of 40 °C, an initial lactose mass concentration of 300 g/L, a reaction pH of 7.0, an enzyme addition of 50 U/mL, and a reaction time of 6 h, the yield of galacto-oligosaccharides was about 4 2%. The products include isomaltose, galactose, two kinds of oligosaccharide galactose, and two kinds of oligosaccharide galactose.

Rodriguez-co-lins et al. [42] used β-galactosidase from Lactobacillus kluveri to obtain a maximum yield of 177 g/L of oligosaccharides with a lactose conversion rate of 76% under the conditions of an initial lactose mass concentration of 400 g/L, a reaction pH of 7.0, a reaction temperature of 40 °C, an enzyme addition amount of 1.2–1.5 U/mL, and a reaction time of 6 h. The products included disaccharides such as isomaltulose, galactose, two types of oligosaccharides with three galactose units, and two types of oligosaccharides with four galactose units. 5U/mL, the reaction time was 6h, the maximum yield of galactooligosaccharides was 177 g/L, the lactose conversion rate was 76%, and the products included the disaccharides 6-galactose and isomaltose and 6-galactose-lactose. Urrutia et al. [43] used β-galactosidase from Aspergillus oryzae to produce GOS.

The maximum concentration of GOS was 107 g/L (26.8% of the total sugar content), which is equivalent to a lactose conversion rate of about 70%. The products include galactose, 3-O-β-galactosylglucose, and 6′-O-β-galactosyl-lactose. Yanahira et al. [44] used β-galactosidase from Bacillus circulans to add the enzyme (275 U) to a solution containing lactose (55 g) (pH 6.0) at a reaction temperature of 60 °C (45 mL). The reaction was carried out for 23 h, and the products were 11 oligosaccharides (including 3 disaccharides and 8 trisaccharides), namely β-D-Galp-(1 6.0). The reaction was carried out for 23 h. The product contained 11 types of oligosaccharides (including 3 disaccharides and 8 trisaccharides), namely β-D-Galp-(1 → 3) -D-Glc, β-D-Galp-(1 → 6) -D-Glc, β-D-Galp-(1 → 2) -D-Glc, β -D-Galp-(1→ 4)-β-D-Galp-(1 → 4) -D-Glc ,β-D-  Galp-(1 → 6) -[β-D-Galp-(1 → 2)] -D-Glc , β-D-Galp-  (1 → 6) -[β-D-Galp-(1 → 4)] -D-Glc , β-D- Galp-(1 → 4)-β-D-Galp-(1 → 3) -D-Glc , β-D-Galp-(1 → 4)-β-D-  Galp-(1 → 2) -D-Glc , β-D-Galp-(1 → 4) -[ β-D-Galp-  (1→ 2)] -D-Glc ,β-D-Galp-(1→ 4)-β-D-Galp-(1 → 6)-D-Glc, β-D-Galp-(1→ 6) [β-D-Galp-(1 → 3)]-D-Glc. The results of the study on the biocatalytic preparation of GOS by β-galactosidase from different sources are shown in Table 2.

In addition to the enzymatic properties of β-galactosidase itself, the catalytic conditions of initial lactose concentration and reaction temperature are also key factors in the biocatalytic preparation of galacto-oligosaccharides. An initial lactose concentration above a mass-to-volume ratio of 30% is conducive to the synthesis reaction, i.e., it is beneficial to increasing the yield of galacto-oligosaccharides [50]. However, the solubility of lactose is weaker than that of other sugars. At a temperature of 30 °C, the solubility is only 25% of water, and at 40 °C, it is only 33% of water. Although a high concentration of lactose solution can be obtained by supersaturation, the supersaturated solution is unstable and lactose is prone to precipitate.

Therefore, increasing the temperature of the conversion system not only allows a higher initial substrate (lactose) mass concentration to be obtained, but also improves the efficiency of oligosaccharide synthesis. concentration, but also helps to improve the efficiency of the synthesis of galacto-oligosaccharides. At the same time, excessively high reaction temperatures can easily denature and inactivate the catalyst β-galactosidase. Screening or molecular directed evolution to obtain a high-temperature-resistant β-galactosidase to improve the efficiency of oligosaccharide synthesis (conversion system substrate feeding ratio, product yield and purity, etc.) is another hot topic in the research of enzymatic synthesis of oligosaccharides. At present, domestic and foreign research [8] has found that glycoside hydrolases from hyperthermophilic microorganisms such as Sulfolobus tokodaii, Pyrococcus furiosus, Thermus thermophilus, Thermus thermophilus, Staphylococcus saccharolyticus and Halothermus marinus have the ability to catalyze transglycosylation reactions at temperatures of 80 °C or higher, which is beneficial to improving the yield of oligosaccharides prepared by the biocatalytic method .

3. Enzyme-synthesized oligosaccharide galactose separation and purification

To date, the low yield remains a shortcoming of the industrial production of oligosaccharides prepared by the biocatalytic method. For the β-galactosidase-mediated enzymatic synthesis route, the yield of oligosaccharides is usually 20% to 45% (corresponding to a substrate lactose conversion rate of 40% to 60%), and attempts to significantly increase the yield of oligosaccharides by optimizing biocatalysts or process engineering techniques have not yet been successful. Therefore, the removal of unreacted lactose and un-polymerized monosaccharides (glucose and galactose) after hydrolysis from the final reaction solution of the enzymatic synthesis route has become the main difficulty and bottleneck in the research on the separation and purification of oligosaccharides [32]. It has been reported that the current purification methods for oligosaccharides include chromatographic separation, membrane separation, enzymatic methods, and selective fermentation [51].

Chromatographic separation is based on the different binding forces between the components of the material to be separated and the stationary phase and mobile phase, and the components are separated sequentially. Ion exchange resins are most commonly used to separate saccharides[52] . Li Liangyu et al. [53] used a homemade simulated moving chromatograph and a sequential simulated moving chromatograph device to purify crude low-molecular-weight galactose, respectively, and obtained good separation results. Through comparative analysis, the results obtained using the sequential simulated moving chromatograph were better. At a feed refractive index of 60%, a column temperature of 60 °C, the experimental conditions were a feed rate of 467 mL/h, an inlet water flow rate of 722.4 mL/h, and a column temperature of 60 °C. The yield of oligosaccharides was 91.3%, and the mass fraction was 95.1%.

Wisniewski 4mL/h, the yield of oligosaccharides was 91.3%, and the mass fraction was 95.1%. Wisniewski et al. [54] reported that oligosaccharides with a mass fraction of 99.9% could be obtained using simulated moving bed (SMB) technology. 9% low-molecular-weight galacto-oligosaccharides. Membrane separation is based on the fact that substances with different molecular weights can pass through the pores of the membrane, while macromolecules are blocked. This allows the separation of components with different molecular sizes. Among them, ultrafiltration and nanofiltration are often used for the separation and purification of functional polysaccharides [55].

Feng et al. [56] used an NF-3 membrane with a cut-off phase of 800–1,000 Da to purify crude low-molecular-weight galactose products. The removal rates of monosaccharides and lactose were 90.5% and 52.5%, respectively, and the oligosaccharide mass fraction was 54.5% (1.5 times that of the crude product).  5%, the oligosaccharide mass fraction was 54.5% (1.5 times that of the crude product), and the oligosaccharide yield was 70%.

Goulas et al. [57] used two asymmetric cellulose acetate membranes (NF-CA-50 and UF-CA-1) to continuously filter and dialyze the crude galacto-oligosaccharide product, and the oligosaccharide mass fraction could reach 98%. Due to the similar molecular mass between galacto-oligosaccharides and contaminants (mainly lactose and monosaccharides), separation by membrane grading is a difficult task. While the effective removal of simple sugars is a reasonable approach, the removal of lactose would require an enzymatic pre-hydrolysis step, which would lead to lower productivity and increased costs. Membrane separation, although selective, non-polluting and energy-efficient, is expensive and requires frequent cleaning and maintenance, which may otherwise cause membrane contamination, thus limiting its application in large-scale industrial production.

The enzymatic method selectively removes the corresponding simple sugars and lactose by adding an enzyme preparation. The enzymes used in this method have the advantages of specificity, good purification results, and high product purity. Maisch-Berger et al. [58] used a highly specific cellobiose dehydrogenase from Lactobacillus roxannae to purify the crude product of galacto-oligosaccharides. Then, a chromatographic step was used to remove ions and monosaccharides to obtain a purer galacto-oligosaccharide with a monosaccharide and lactose mass fraction of less than 0.3%. 3%, and the yield of oligosaccharides reached 60.3%. The enzymatic purification process is costly due to factors such as the poor stability, recovery rate and high price of enzyme preparations. In addition, as the enzymatic reaction occurs, the pH of the reaction system gradually decreases, which also affects the activity of the enzyme, thus limiting its industrial application.

In addition to the above-mentioned separation and purification methods, the fermentation separation method, which is based on the selective fermentation characteristics of microorganisms, can also effectively improve the purification effect of galacto-oligosaccharides. It is also a hot research topic at home and abroad recently. For example, using Kluyveromyces and Saccharomyces cerevisiae strains, the fermentative conversion (bioconversion) of raw oligogalactose can selectively remove metabolizable sugars (glucose, galactose and lactose) from the conversion solution, thereby achieving the goal of oligogalactose purification. Rengarajan et al. [59] studied the continuous production of high-purity isomaltooligosaccharides (IMOS) by selective fermentation of Saccharomyces cerevisiae.

The low-quality fraction of IMOS (67%) was incubated with a separated strain of Saccharomyces cerevisiae (4%) in a 3L bioreactor for 1 h, combined with a microfiltration membrane cycle to obtain high-quality IMOS, product mass fraction >91%, yield 79%, and the highest space-time yield was 198.79 g/(L · h). Yoon et al. [60] studied the selective fermentation characteristics of low-molecular-weight galactose-converted mother liquor using Saccharomyces cerevisiae. The study found that anaerobic sugar fermentation for 24 hours can specifically remove the mass concentration of monosaccharides (glucose and galactose) in the low-molecular-weight galactose-converted mother liquor and improve the product quality fraction. Although this selective fermentation and separation method can obtain a product with a high mass fraction, selective fermentation (bioconversion) also has its drawbacks. The fermentation process requires a high cell mass and the unpurified galacto-oligosaccharides need to be diluted. The production of metabolic by-products such as ethanol, acetic acid and glycerol can also affect the mass fraction and yield of the product [5].

4 Current status of industrial production of oligosaccharides

The industrial production of oligosaccharides originated in Japan[4] , and was later commercialized in Europe and the United States. China's oligosaccharide industry started relatively late. Today, many domestic and foreign companies have set their sights on the oligosaccharide industry. Manufacturers of commercially produced oligosaccharides mainly include Japan's Yakult Honsha Co., Ltd., Japan's Nisshin Sugar Manufacturing Co., Ltd., the Netherlands' Z, the United States' Illinois Corn Products International, China's Baolingbao Biology, and Quantum Hi-Tech Biology Co., Ltd.  .

The main oligosaccharide products on the market are shown in Table 3. Although great progress has been made in the industrial production of oligosaccharides at home and abroad, there are still some urgent problems to be solved in the oligosaccharide industry, such as the difficulty of separating and purifying oligosaccharides (currently, the quality fraction of most domestically produced oligosaccharide products in the domestic market is less than 57%), and the existing commercialized β-galactosidase activity is not high (GOS yield is about 30% to 40%). Therefore, the development of efficient methods for the separation and purification of oligosaccharides and the search for new β-galactosidases with excellent performance will be the main direction of future industrial research on oligosaccharides, which has high research value and development prospects [61].

5 Conclusion

Through the above analysis, oligosaccharides have been widely used in industries such as infant formula, fermented milk, confectionery products, baked goods, livestock feed and pet food due to their excellent physicochemical properties and physiological effects, and they have lasting development potential. With the deepening of research on the preparation of galacto-oligosaccharides by the biocatalytic method, scholars at home and abroad have made many attempts in the development of the source of the enzyme used in the preparation of galacto-oligosaccharides by biocatalysis, the development of the catalytic preparation process, and the research and development of methods for the separation and purification of galacto-oligosaccharides, and have made considerable progress. However, the research results still cannot adequately meet the needs of industrial production and the ever-expanding market demand. At present, from the perspective of the production process, the low yield of oligosaccharides and the difficulty of separation and purification are still important factors restricting the industrial development of oligosaccharides.

In addition, China's oligosaccharide production industry still has problems such as a single source of catalysts, low product purity and a lack of testing methods that need to be improved. It is believed that with the continuous efforts of domestic researchers in the above research directions, breakthroughs will be made in the difficult and bottleneck issues in the preparation of oligosaccharides by the biocatalytic method, and the shortcomings in technological development will gradually be made up. This will enable the efficient preparation of oligosaccharides by the biocatalytic method, so that oligosaccharides can gradually penetrate into the daily lives of the Chinese people as prebiotics, serve China's health industry, and generate corresponding economic benefits and social effects.

Reference:

[1]    MANNING T S ,  GIBSON  G R.   Prebiotics[J] . Best p ractice &research clinical gastroenterology, 2004 , 18(2) :287-298.

[2] Ma Yuechao, Cui Yi, Chen Ning, et al. Research on the nutritional and health-preserving functions of microbial products [J]. Fermentation Science and Technology Newsletter, 2018, 47(4): 240-244.

[3]    MANO M C R ,  NERI-NUMA I A ,  DA-SILVA J B ,  et al.  Ol- igosaccharide biotechnology :  an  approach  of  p rebiotic  revolu- tion on the industry[J] . Applied microbiology and biotechnolo- gy, 2018 , 102(1) : 17-37.

[4]    LU L , GUO L ,  WANG K ,  et al.   Beta-galactosidases :  a great tool for synthesizing galactose-containing carbohydrates[J] . Bi- otechnology advances , 2020 , 39 : 1-15.

[5]    VERA C ,  SUAREZ S ,  ABURTO C ,  et al.  Synthesis and pur- ification of galacto-oligosaccharides :  state of the art [J] . World journal of microbiology and biotechnology, 2016 , 32(12) : 1-20.

[6] Wei Chun, Kong Lingmin, Liu Lifeng. Process optimization for the production of galacto-oligosaccharides by permeabilized cell catalysis of Lactobacillus plantarum [J]. Fermentation Technology Communications, 2016, 45(1): 18-22.

[7]    TORRES D P M ,  GONCALVES M D P F ,  TEIXEIRA J A ,  et al.  Galacto-oligosaccharides :  p roduction ,  properties ,  applications , and  significance  as  p rebiotics[J] .  Comprehensive  re- views in food science and food safety, 2010 , 9(5) :438-454.

[8]    LOO J ,  CUMMINGS  J ,   DELZENNE  N ,   et  al.   Functional food p rop erties of non-digestible oligosaccharides :  a  consensus report from the ENDO p roj ect (DGXII AIRII-CT94-1095)[J] .  British journal of nutrition , 1999 , 81(2) : 121-132.

[9]    CHONAN O ,  SHIBAHARA-SONE H ,  TAKAHASHI R ,  et al. Undigestibility of galactooligosaccharides  [ J ] .   Nippon shokuhin kagaku kogaku kaishi , 2004 , 51(1) :28-33.

[10]    DAVIS L M G ,  MARTINEZ  I ,  HUTKINS  W J ,  et  al.   A dose dep endent impact of p rebiotic galactooligosaccharides on the intestinal  microbiota  of  healthy  adults[J] .  International journal of food microbiology, 2010 , 144(2) :285-292.

[11] Chang Jinjin, Hu Ping, Wang Jue, et al. Effect of oligosaccharides on the morphological digestion and absorption function of the ileum and the structure of the flora in weaned piglets [J]. Animal Husbandry and Veterinary Medicine, 2020, 52(2): 47-53.

[12] Pan Yuning, Liu Chengzhi, Yan Chunrong, et al. Research progress on the physiological functions of oligosaccharides[J]. Journal of Food Safety and Quality, 2019, 10(10): 2849-2855.

[13] WEAVER C M.   Diet ,  gut microbiome ,  and bone health[J] .Current osteoporosis reports , 2015 , 13(2) : 125-130.

[14]    WANG J ,  TIAN S ,  YU H ,  et al.  Response of colonic mucosa-associated microbiota composition ,  mucosal immune home- ostasis ,  and barrier function to early life galactooligosacchar- ides intervention in suckling piglets[J] . Journal of agricultural and food chemistry, 2019 , 67 :578-588.

[15] Li Xinhong, Zhang Jinlong, Bi Yongxian, et al. Research on the application of several oligosaccharides in cosmetics [J]. Fragrance, Flavor and Cosmetics, 2019(3): 70-74.

[16]    PARK  A  R ,   OH  D  K.    Galacto-oligosaccharide  p roduction using microbialβ-galactosidase :  current state and p ersp ectives [J] .  Applied  microbiology  and  biotechnology , 2010 , 85 (5) :  1279-1286.

[17]    ESKANDARLOO H ,  ABBASPOURRAD  A.   Production  ofgalacto-oligosaccharides from whey permeate using β-galacto- sidase  immobilized  on   functionalized  glass   beads [J] .  Food chemistry, 2018 , 251 : 115-124.

[18]    GUERRERO C ,  VERA C ,  CONEJEROS  R ,  et  al.   Transgalactosylation and hydrolytic activities of commercial p repa- rations ofβ-galactosidase for the synthesis of p rebiotic carbo- hydrates[J] .  Enzyme  and  microbial   technology , 2015 ,  70 :  9-17.

[19]    PHAM M L ,  LEISTER T ,  NGUYEN H A ,  et al.  Immobilization ofβ-galactosidases from Lactobacillus on chitin using a chitin-binding domain[J] . Journal  of  agricultural  and  food chemistry, 2017 , 65(14) :2965-2976.

[20]    XAVIER J R , RAMANA K V ,  SHARMA R K.  β-galactosidase :  biotechnological  applications  in  food  p rocessing [J] . Journal of food biochemistry, 2018 , 42(2/3) : 1-15.

[21]    SAQIB S ,  AKRAM A ,  HALIM S A ,  et al.  Sources of beta-galactosidase and  its  applications  in  food  industry[J] .  Bio- tech , 2017 , 7 :79.

[22]    SHARMA S K ,  LEBLANC R M.   Biosensors based on beta galactosidase enzyme :  recent  advances  and  p ersp ectives[J] . Analytical biochemistry, 2017 , 535 : 1-11.

[23] Li Junling, Yan Shuangyong, Zhang Rongxue, et al. Research progress of plant β-galactosidase [J]. Anhui Agricultural Science, 2020, 48(1): 15-18.

[24] Zhang JQ, Wang SW, Tao F, et al. Research status and industrial development trend of oligosaccharide synthesis. Journal of Food Safety and Quality, 2019(10): 2829-2835.

[25]    MORACCI M ,  CIARAMELLA M ,  NUCCI R ,  et al.   Thermostableβ-glycosidase from sulfolobusSolfataricus[J] . Bio- catalysis , 1994 , 11(2) :89-103.

[26]    IQBAL S ,  NGUYEN T H ,  NGUYEN H A ,  et al.   Charac- terization of a heterodimeric GH2 β-galactosidase from Lacto- bacillus sakei Lb790and formation of p rebiotic galacto-oligo- saccharides[J] . Journal  of  agricultural  and  food  chemistry ,  2011 , 59(8) :3803-3811.

[27]    ARREOLA S L ,  INTANON1  M ,  SULJIC J ,  et al.   Two β-galactosidases from the human isolate Bifidobacteriumbreve DSM 20213 :  molecular  cloning  and  expression ,   biochemical characterization and synthesis of galacto-oligosaccharides[J] .  Plos one , 2014 , 9(8) : 1-13.

[28]    LI Y ,  LU L ,  WANG  H ,  et al.   Cell surface engineering of a beta-galactosidase  for  galactooligosaccharide   synthesis [J] .  Applied and environmental microbiology, 2009 , 75(18) : 5938- 5942.

[29]    HUANG J ,  ZHU S , ZHAO L ,  et al.  Anovelβ-galactosidase from Klebsiellaoxytoca ZJUH1705for efficient p roduction of galacto-oligosaccharides from lactose[J] . Applied microbiolo- gy and biotechnology, 2020 , 104:6161-6172.

[30]    KATROLIA P ,  ZHANG M ,  YAN Q ,  et al.   Characterisation of a thermostable family 42 β-galactosidase(BgalC)  family fromThermotogamaritima showing efficient lactose hydroly- sis[J] . Food chemistry, 2011 , 125(2) : 614-621.

[31]    FAN  Y ,   HUA  X ,  ZHANG  Y ,  et  al.   Cloning ,  expression and  structural  stability  of  a  cold-adapted  beta-galactosidase from Rahnella sp.   R3[J] .  Protein  expression  and  purifica- tion , 2015 , 115 : 158-164.

[32]    SCHWAB C ,  LEE V ,  SORENSEN K I ,  et al.  Production of galactooligosaccharides  and  heterooligosaccharides  with  dis- rupted cell extracts and whole cells of lactic acid bacteria and bifidobacteria[J] . International dairy journal , 2011 , 21 (10) :  748-754.

[33]    CHANALIA P ,  GANDHI D ,  ATTRI P ,  et al.   Purification and characterization of β-galactosidase from p robiotic Pedio- coccus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide  synthesis [J] .  Bioorganic   chemistry ,  2018 , 77 : 176-189.

[34]    LIAO X ,  HUANG  J ,   ZHOU  Q ,   et  al.   Designing  of  a  novel β-galactosidase for  production  of  functional  oligosaccharides[J] . European food research and technology, 2017 , 243:979-986.

[35]    SRIVASTAVA A ,  MISHRA  S ,  CHAND  S.   Transgalactosylation  of  lactose  for   synthesis  of  galacto-oligosaccharides using Kluyveromyces marxianus  NCIM  3551[J] .  New  bio- technology, 2015 , 32(4) :412-418.

[36]    GONZALEZ-DELGADO I ,  YOLANDA  S ,  ANTONIO  M ,et al.  β-galactosidase covalent immobilization over large-pore mesoporous silica supports for the p roduction of high galacto- oligosaccharides(GOS)[J] . Microporous and mesoporous ma- terials , 2018 , 257:51-61.

[37]    TAVARES G F ,  XAVIER M R ,  NERI D ,  et al.   Fe3O4@polypyrrole core-shell composites applied as nanoenvironment for  galacto-oligosaccharides   p roduction [J] .  Chemical  engi- neering journal , 2016 , 306 :816-825.

[38]    CAREVIC M ,  COROVIC M ,  MIHAILOVIC M ,  et al.   Galacto-oligosaccharide  synthesis  using  chemically  modified  β- galactosidase from Aspergillus oryzae  immobilized  onto  mi- croporous amino  resin[J] . International dairy journal , 2016 ,  54:50-57.

[39] Huang Jin, Zhu Shengquan, Du Meini, et al. A β-galactosidase, gene, engineered bacteria and its application: 201911335256.7[P]. 2019-12-23.

[40] Huang Jin, Zhu Shengquan, Tang Wan, et al. Acid-producing Klebsiella and its application: 201711112168.1[P] .2017-11-13.

[41] Zhu Wuer, Miao Mingyong, Gu Zhenghua, et al. Research on the synthesis of galactooligosaccharides by the β-galactosidase of the salt-tolerant bacterium S62 [J]. Bioprocesses, 2019, 17(2): 131-137.

[42]    RODRIGUEZ-COLINAS B ,  DE-ABREU  M  A ,   FERNANDEZ-ARROJO L ,  et  al.   Production  of  galacto-oligosaccha- rides b y theβ-galactosidase from Kluyveromyces lactis :  com- parative analysis of p ermeabilized cells versus soluble enzyme [J] . Journal of agricultural and food chemistry, 2011 , 59(19) :  10477-10484.

[43]    URRUTIA P ,  RODRIGUEZ-COLINAS  B ,   FERNANDEZARROJO L ,  et al.   Detailed  analysis  of galactooligosacchar- ides synthesis  with β-galactosidase from Aspergillus oryzae [J] . Journal of agricultural and food chemistry, 2013 , 61(5) :  1081-1087.

[44]    YANAHIRA S ,  KOBAYASHI T ,  SUGURI T ,  et al.  Formation of oligosaccharides from lactose b y Bacillus circulans beta-galactosidase[J] . Bioscience biotechnology and biochem- istry, 1995 , 59(6) : 1021-1026.

[45]    FRENZEL M ,  ZERGE  K ,  CLAWIN-R-DECKER  I ,  et  al. Comparison of the galacto-oligosaccharide forming activity of differentβ-galactosidases[J] . Lwt food science &technology ,  2015 , 60(2) : 1068-1071.

[46]    GAO X ,  WU J , WU D.   Rational design of the beta-galactosidase fromAspergillus oryzae to improve galactooligosaccha- ride p roduction[J] . Food chemistry, 2019 , 286 :362-367.

[47]    OSMAN A , TZORTZIS G ,  RASTALL R A ,  et al.  BbgIV is an important Bifidobacterium β-galactosidase for the synthe- sis of  p rebiotic  galactooligosaccharides  at  high  temperatures [J] . Journal of agricultural and food chemistry, 2012 , 60(3) :  740-748.

[48]    SRIVASTAVA A ,  MISHRA  S ,  CHAND  S.   Transgalacto sylation  of  lactose  for   synthesis  of  galacto-oligosaccharides using Kluyveromyces marxianus  NCIM  3551[J] .  New  bio- technology, 2015 , 32(4) :412-418.

[49]    PLACIER G ,  HILDEGARD W ,  RABILLER C ,  et al.   Evolved beta-galactosidases from Geobacillusstearothermophilus  with imp roved  transgalactosylation  yield  for  galacto-oligosaccha- ride p roduction[J] . Applied and environmental microbiology ,  2009 , 75(19) :6312-6321.

[50]    VERA C ,  GUERRERO C ,  CONEJEROS R ,  et al.  Synthesis of galacto-oligosaccharides b y β-galactosidase from Aspergil- lus oryzae  using partially dissolved and supersaturated solu- tion of lactose[J] . Enzyme and microbial technology, 2012 , 50 (3) : 188-194.

[51] Sun Chun, Zhu Wenxing, Liu Xinli. Research progress of oligosaccharides. Chinese Condiments, 2017, 42(11): 170-174.

[52] Li Suyue, Zhang Mingming, Yan Xiaojuan, et al. Research progress on the isolation and purification of oligosaccharides[J]. China Brewing, 2015, 34(11): 6-9.

[53] Li Liangyu, Jia Pengyu, Li Chaoyang, et al. Simulated moving chromatography for the efficient purification of oligosaccharides [J]. Chinese Journal of Food Science, 2016, 16(3): 138-145.

[54]    WISNIEWSKI L , ANTOSOVA M , POLAKOVIC M.  Simulated moving bed chromatography separation of galacto-oligo- saccharides[J] . Acta chimica slovaca , 2013 , 6(2) :206-210.

[55] Yao Chunxiao. Study on the enzymatic synthesis and purification of low-molecular-weight galactose [D]. Beijing: Beijing Forestry University, 2019.

[56]    FENG Y M ,  CHANG X L ,  WANG W H ,  et al.   Separation of galacto-oligosaccharides mixtureby nanofiltration[J] . Jour- nal of  the  Taiwan  institute  of  chemical  engineers , 2009 , 40 (3) :326-332.

[57]    GOULAS A K ,  KAPASAKALIDIS P G ,  SINCLAIR  H  R ,et al.   Purification  of  oligosaccharides  b y  nanofiltration[J] . Journal of membrane science , 2002 , 209(1) :321-335.

[58]    MAISCHBERGER T ,  NGUYEN T  H ,  SUKYAI P ,  et  al. Production  of  lactose-free  galacto-oligosaccharide  mixtures :  comparison of two cellobiose dehydrogenases for the selective oxidation of lactose to  lactobionic  acid[J] . Carbohydrate  re- search , 2008 , 343(12) :2140-2147.

[59]    RENGARAJAN S ,  RAMESHTHANGAM  P.   High  purity p rebiotic isomalto-oligosaccharides  p roduction  b y  cell  associ- ated transglucosidase of isolated strain Debaryomyces hanse- nii SCY204and selective fermentation b y Saccharomyces cerevisiae SYI065[J] . Process biochemistry, 2020 , 98:93-105.

[60]    YOON S H ,  MUKERJEA  R ,   ROBYT  J  F.   Specificity  of yeast(Saccharomyces cerevisiae)  in  removing  carbohydrates b y fermentation[J] . Carbohydrate  research , 2003 , 338 (10) :  1127-1132.

[61] Ping Liying, Chen Lin, Fang Lina, et al. A brief discussion on the influencing factors of microbial fermentation pilot tests [J]. Fermentation Science and Technology Newsletter, 2017, 46(4): 212-215.