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Abstract Palmitoleic acid (C16:1n7, POA), a 16-carbon ω-7 monounsaturated functional fatty acid, has been demonstrated a variety of beneficial properties to human health and a wide range of applications in nutrition, medical and chemical industries. Lipozyme® RM IM (Rhizomucor Miehei lipase), a Sn-1,3 specific lipase, was used to catalyze the hydrolysis of seabuckthorn fruit oil and produce monoglycates rich in palmitoleic acid. The fruit oil of seabuckthorn was used as the research object, the dosage of enzyme taking 10% wt.% oil, the influence of water/oil ratio, pH value and reaction temperature to hydrolysis rate were investigated, and then the response surface methodology was applied for optimizing the process conditions. Further, the relationship between the hydrolysis rate and the content of monoacylglycerol was studied under the optimal hydrolysis conditions. The result indicated that the optimal combined condition is as follows: the reaction temperature is 55°C, the pH is 7, the water-oil quality ratio is 1:1. The hydrolysis rate achieved 79.59% within 12 h in the condition above. Under the optimal conditions, the hydrolysis rate was positively correlated with the content of monoester, and the correlation coefficient was 0.999 within the first 10 h of the reaction. The hydrolysis rate was 66.76% at 10 h, and the content of monoglyceride reached the highest of 61.11%. Hereafter, the hydrolysis of monoglyceride began if the reaction continued. The content of palmitoleic acid increased from 27.11% to 40.84%, and that of linoleic acid increased from 4.51% to 33.34%, so the total amount of the two functional fatty acids reached 74.18%. Enzymatic hydrolysis can be an effective way to improve the function of seabuckthorn fruit oil. C161n7, C161n7 Cn C16 1n7, 1n7 C n (C16:1n7 POA, POA , POA) 16carbon carbon 16 ω7 ω 7 ω- nutrition industries Lipozyme Rhizomucor lipase lipase) Sn1,3 Sn13 Sn 1,3 1 3 Sn-1, object wt wt. wateroil water investigated conditions Further follows 55C 55 55°C 11. 11 1. 1:1 7959 79 59 79.59 above monoester 0999 0 999 0.99 6676 66 76 66.76 6111 61 61.11% Hereafter continued 2711 27 27.11 4084 40 84 40.84% 451 4 51 4.51 3334 33 34 33.34% 7418 74 18 74.18% C161n C1 1n (C16:1n Sn1 Sn1, 13 1, Sn-1 5 1: 795 79.5 099 99 0.9 667 6 66.7 611 61.11 271 2 27.1 408 8 40.84 45 4.5 333 33.34 741 74.18 Sn- 79. 09 9 0. 66. 61.1 27. 40.8 4. 33.3 74.1 61. 40. 33. 74.